Thyme

The genus Thymas

Medicinal and Aromatic Plants - Industrial Profiles
Individual volzmes in this series provide both industry and acadenzia with zn-depth coverage of one major
medicinal or aromatic plant of industrial inzportance.
Edited by Dr Roland Hardman

Volume 1
Valerian, edited by Peter J. Houghton
Volume 2
Perilla, edited by He-ci Yu, Kenichi Kosuna and Megumi Haga
Volume 3
Poppy, edited by Jeno Bernith
Volume 4
Cannabis, edited by David T. Brown
Volume 5
Neem, edited by H.S. Puri
Volume 6
Ergot, edited by Vladimir KPen and Ladislav Cvak
Volume 7
Caraway, edited by ~ v Nkmeth
a
Volume 8
Saffron, edited by Moshe Negbi
Volume 7
Tea Tree, edited by Ian Southwell and Robert Lowe
Volume 10
Basil, edited by Raimo Hiltunen and Yvonne Holm
Volume 11
Fenzlgreek, edited by Georgios Petropoulos
Volume 12
Gingko biloba, edited by Teris A. Van Beek
Volume 13
Black Pepper, edited by P.N. Ravindran
Volume 14
Sage, edited by Spiridon E. Kintzios
Volume 15
Ginseng, edited by W.E.Court
Volume 16
Mistletoe, edited by Arndt Biissing
(Continued)
Thyme
The genus Thymtls

Edited by

Elisabeth Stahl-Biskup
Institut f i r Pharzazie, Abteilung Pharmazeutische Biologie,
Universitat Hamburg, Germany
and

Francisco S6ez
Facultad de Biologia, Departamento de Biologia Vegetal (Botknica),
Universidad de Murcia, Spain

London and New York

First published 2002
by Taylor & Francis
1 1 New Fetter Lane, London EC4P 4EE
Simultaneously published rn the USA and Canada
by Taylor & Francis Inc,
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Taylor & Frnnczr is an zlizpllnt of the Taylor & Fran~.ijGrozp
O 2002 Taylor & Francis

All rights reserved. N o part of this book may be repr~ntedor reproduced

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in writing from the publishers.
Every effort has been made to ensure that the advice and information
in this book 1s true and accurate at the time of going t o press. However,
neither the publisher nor the authors can accept any legal responsibility
or liabll~tyfor any errors or omissions that may be made. In the case of
drug administration, any medical procedure or the use of technical
equipment mentioned within this book, you are strongly advised t o
consult the manufacturer's guidelines.
Brztish Lzb~aryCatalognzng zn P7~blzcatzonData
A catalogue record for t h ~ sbook is available from the British Library
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ISBN 0 4 1 5-28488-0
For Rainer, Inma, Natalia, Angel and RubCn
Contents

Prefdce to the series

Preface
List of contributors

The history, botany and taxonomy of the genus Thymus

RAMON MORALES

Population structure and the spatial dynamics of genetic

polymorphism in thyme
J O H N D THOMPSON

Essential oil chemistry of the genus Thymus - a global view

ELISABETH STAHL-BISKUP

Essential oil polymorphism in the genus Thymzls

FRANCISCO SAEZ A N D ELISABETH STAHL-BISKUP

Flavonoids and further polyphenols in the genus Thymzls

ROSER VILA

Field culture, in vitro culture and selection of Thymus

CHARLES REY AND FRANCISCO SAEZ

Harvesting and post-harvest handling in the genus Thymus

PETRAS R. VENSKUTONIS

Thyme - processing of raw plant material

PETRAS R. VENSKUTONIS

The Genus Thymus as a source of commercial products

BRIAN M LAWRENCE A N D A R T H U R 0 . TUCKER
10 The medicinal and non-medicinal uses of thyme
A N T O N I O ZARZUELO A N D ESPERANZA CRESPO

11 Thyme as a herbal drug - pharmacopoeias and other product

characteristics
ELISABETH STAHL-BISKUP

Index
Preface to the series

There is increasing interest in industry, academia and the health sciences in medicinal
and aromatic plants. In passing from plant production to the eventual product used by
the public, many sciences are involved. This series brings together information which is
currently scattered through an ever increasing number of journals. Each volume gives
an in-depth look at one plant genus, about which an area specialist has assembled infor-
mation ranging from the production of the plant to market trends and quality control.
Many industries are involved such as forestry, agriculture, chemical food, flavour,
beverage, pharmaceutical, cosmetic and fragrance. The plant raw materials are roots,
rhizomes, bulbs, leaves, stems, barks, wood, flowers, fruits and seeds. These yield gums,
resins, essential (volatile) oils, fixed oils, waxes, juices, extracts and spices for medicinal
and aromatic purposes. All these commodities are traded worldwide. A dealer's market
report for an item mlay say 'Drought in the country of origin has forced up prices'.
Natural products do not mean safe products and account of this has to be taken by
the above industries; which are subject to regulation. For example, a number of plants
which are approved for use in medicine must not be used in cosmetic products.
The assessment of safe to use starts with the harvested plant material which has to
comply with an official monograph. This may require absence of, or prescribed limits
of, radioactive material, heavy metals, aflatoxic, pesticide residue, as well as the
required level of active principle. This analytical control is costly and tends to exclude
small batches of plant material. Large scale contracted mechanised cultivation with
designated seed or plantlets is now preferable.
Today, plant selection is not only for the yield of active principle, but for the plant's
ability to overcome disease, climatic stress and the hazards caused by mankind. Such
methods as in vitro fertilization, meristem cultures and somatic embryogenesis are used.
The transfer of sections of DNA is giving rise to controversy in the case of some end-uses
of the plant material.
Some suppliers of plant raw material are now able to certify that they are supplying
organically-farmed medicinal plants, herbs and spices. The European Union directive
(CVOIEU No. 2092191) details the specifications for the obligatory quality controls to
be carried out at all stages of production and processing of organic products.
Fascinating plant folklore and ethnopharmacology leads to medicinal potential.
Examples are the muscle relaxants based on the arrow poison, curare, from species of
Chondrodendron, and the anti-malarials derived from species of Cinchona and Artenzisia.
The methods of detection of pharmacological activity have become increasingly reliable
and specific, frequently involving enzymes in bioassays and avoiding the use of labora-
tory animals. By using bioassay linked fractionation of crude plant juices or extracts,
x Preface to the seriej

compounds can be specifically targeted which, for example, inhibit blood platelet
aggregation, or have anti-tumour, or anti-viral, or any other required activity. W i t h
the assistance of robone devices all the members of a genus may be readily screened.
However, the plant material must be fully authenticated by a specialist.
The medicinal traditions of ancient civilisations such as those of China and India
have a large armamenoaria of plants in their pharmacopoeias which are used through-
out South-East Asia. A similar situation exists in Africa and South America. Thus, a
very high percentage of the World's population relies on medicinal and aromatic plants
for their medicine. Western medicine is also responding. Already in Germany all
medical practitioners have to pass an examination in phytotherapy before being allowed
to practise. It is noticeable that throughout Europe and the USA, medical, pharmacy
and health related schools are increasingly offering training in phytotherapy.
Multinational pharmaceutical companies have become less enamoured of the single
compound magic bullet cure. The high costs of such ventures and the endless competi-
tion from 'me too' compounds from rival companies often discourage the attempt Inde-
pendent phytomedicine companies have been very strong in Germany. However, by the
end of 1995, eleven (almost all) had been acquired by the multinational pharmaceutical
firms, acknowledging the lay public's growing demand for phytomedicines in the
Western World.
The business of dictary supplements in the Western World has expanded from the
health store to the pharmacy. Alternative medicine includes plant-based products.
Appropriate measures to ensure the quality, safety and efficacy of these either already
exist or are being answered by greater legislative control by such bodies as the Food and
Drug Administration of the USA and the recently created European Agency for the
Evaluation of Medicinal Products, based in London.
In the USA, the Dietary Supplement and Health Education Act of 1994 recognised
the class of phytotherapeutic agents derived from medicinal and aromatic plants.
Furthermore, under public pressure, the US Congress set up an Office of Alternative
Medicine and this office in 1994 assisted the filing of several Investigational New Drug
(IND) applications, required for clinical trials of some Chinese herbal preparations. The
significance of these applications was that each Chinese preparation involved several
plants and yet was handled as a single IND. A demonstration of the contribution to effi-
cacy, of each ingredient of each plans, was not required. This was a major step forward
towards more sensible regulations in regard to phytomedicines.
My thanks are due to the staffs of Harwood Academic Publishers and Taylor & Francis
who have made this series possible and especially to the volume editors and their chapter
contributors for the authoritative information.

Roland Hardman
Preface

When Roland Hardman asked us to edit the book "The Genus Thymas" within his
remarkable series "Medicinal and Aromatic Plants - Industrial Profiles" it was a big
challenge for us because the amount of material concerning the genus Thymw has
increased continuously and immensely up to the present. W e knew that it would not
be an easy job to compile all the results revealed by more than 2000 scientific publica-
tions. Nevertheless, from the beginning we were enthusiastically dedicated to this
task - as editors and authors - always convinced that we were doing a very valuable job
discussing Thymw under different aspects. W e both spent several years with scientific
studies on the botany, chemistry and systematology of Thynzw always being aware of
the urgent need of a compilatory work as a fundamental basis for further research
projects. Therefore with the publication of this book not only the wish of the series
editor but also our own wish for a comprehensive report on the status quo of the genus
Thymus came true.
Everyone who has ever dealt with the genus Thymus knows that Thynzw vulgavzs L.
deserves close attention. Its pleasant aroma and flavour as well as its potent pharmaco-
logical activities give him the predicate of one of the most popular plants widely used
as flavouring agent, culinary herb, herbal medicine, and it is used in perfumes as well as
being a commercial source of the monoterpene thymol. Therefore Thynzus vulgarzs L.
became the central species in quite a few contributions to the book, especially in those
dealing with thyme as a source of commercial products and as an herbal drug on the
one hand and on the other hand in those articles discussing field culture, harvesting,
post-harvest handling and processing of thyme. Nevertheless for us the presentation of
the material covering the whole genus Thymus has been an aim of prime importance.
Therefore we made every endeavour to find authors who were willing- to elucidate the
scientific achievements of the whole genus and discuss the problems of this species-rich
plant genus, of a taxonomically complex group present in all temperate regions of the
northern hemisphere. The result is a review of the history, the botany, and the taxonomy
of the genus Thymus including several aspects of the population structure in thyme as
well as the complete essential oil and flavonoid chemistry of the genus. Following our
own scientific inclinations a separate chapter was dedicated to the problem of essential
oil polymorphism in the genus Thymus.
W e hope we have succeeded in presenting an informative overview of the information
presently available on the genus Thymus: botany, taxonomy, chemistry, and pharmacology
xii Prefdce

which also takes into account several applied aspects such as field culture, harvesting
and processing of thyme. The readers may consider this book to be a reliable basis and
feel stimulated to invest further efforts into the research of this promising genus which
still shows many gaps which are worth filling during the coming decades.

Elisabeth Stahl-Biskup
Francisco SBez
Contributors

Dr Esperanza Crespo Bundesstrasse 45

Departamento de Farmacologia D-20146 Hamburg, Germany
Facultad de Farmacia
Campus de La Cartuja Dr John D. Thompson
Universidad de Granada Centre d'Ecologie Fonctionelle
1807 1-Granada, Spain et Evolutive
CNRS
Dr Brian M. Lawrence 1919 Route de Mende
c/o R.J. Reynolds Tobacco Company 34293 Montpellier Cedex 5
Research and Development France
Bowman Gray Technical Center
950 Reynolds Boulevard Dr Arthur 0.Tucker
Winston-Salem, NC 27 105, USA Department of Agriculture and Natural
Resources
Dr Ram6n Morales Delaware State University
Real Jardin Botinico de Madrid, CSIC Dover DE 19901-2277, USA
Plaza de Murillo, 2
28014-Madrid, Spain Dr Petras R. Venskutonis
Department of Food Technology
Charles Rey Kaunas University of Technology
Station fgdgrale de recherches en Radvilenu pl. 19
production vggetale de Changins Kaunas
Centre des Fougkres LT-3028, Lithuania
CH-1964 Conthey, Switzerland
Dr Roser Vila
Dr Francisco SBez Unitat de Farmacologia i
Departamento de Biologia Vegetal Farmacognhsia
(Botinica) Facultat de Farmacia
Facultad de Biologia Avda. Diagonal, 643
Universidad de Murcia 08028 Barcelona, Spain
30100-Espinardo, Murcia, Spain
Dr Antonio Zarzuelo
Prof. Dr Elisabeth Stahl-Biskup Departamento de Farmacologia
Institut fiir Pharmazie Facultad de Farmacia
Universitat Hamburg Campus de La Cartuja
Abteilung Pharmazeutische Universidad de Granada
Biologie and Mikrobiologie 1807 1-Granada, Spain
1 The history, botany and taxonomy
of the genus Thymus

INTRODUCTION

Within the Labiate family, with about 220 genera, the genus Thymus is one of the
eight most important genera with regard to the number of species included, although
this number varies depending on the taxonomical point of view. If we choose criteria
to minimise variability, available data report 215 species for the genus, a number
on1y exceeded by the genera Salvia, Hyptis, Scatellaria, Stachys, Teucrium, Nepeta, and
Plectranthus.
The common English word 'thyme' has traditionally been used to name both the genus
and its most commercially used species, Thymus vulgaris, sometimes leading to misun-
derstandings. Generally speaking, thyme is an aromatic plant used for medicinal and spice
purposes almost everywhere in the world. The genus Thymw is very frequent in the
Mediterranean region, where some species form a special type of bushy vegetation not
more than 50cm high, well adapted to hot and dry summer weather. The Spanish
name for these vegetation communities, 'tomillares', include other Labiate species such
as Sideritis, Satureja, Salvia or Lavandz~la,with similar climatic and edaphic patterns.
A common feature of these and many orher aromatic plants is the presence of countless
glandular hairs of different forms which contain volatile essential oils that evaporate
when the glandular hairs are damaged. This way they produce an intensive fragrance
that embraces the plant. It is probably due to the strong scent that humans have always
been attracted to these plants and have exploited their essential oils for popular and
industrial purposes.

HISTORICAL BACKGROUND

T h e history of Thymus before Linnk

Several explanations exist concerning the origin of the name 'Thymus'. Some authors assume
that the Latin name Thymus comes from the Greek word thyo (perfume). Another inter-
pretation of its etymology considers the Greek word thymos (courage, strength). Originally
'thymus' described a group of aromatic plants with similar aspects which were used as
stimulants of vital functions. Many popular names in the Romance languages are
derived from the Latin name. The same occurs with the English name.
In his work about medicinal plants and poisons, Dioscorides (First century, translation
of Laguna, 1555) writes about 'Thymo'. Laguna however did not find there any Thymus
species, but a plant corresponding to the genus Satureja. O n page 294 Laguna describes
the Serpol, presenting two varieties, a cultivated and a wild one. The latter, Zygis, resembles
a Thymus species. It is presented as an erect plant, whereas the former shows a creeping
habit.
In his Natural History, Book 21, Chapter 10 (translation of Huerta, 1629), Plinio (First
century) reports on T. vz~lgarisas follows: 'in the Narbonne province, the stony fields
are full of thyme, and thousands of sheep come from very far provinces to feed on it'.
Later (page 289) he speaks about two different varieties of thyme, a white and a black
one, and he comments on their therapeutic attributes. In Chapter 62 of his first book,
Clusius (1576) refers to T . vulgaris with his Thymum durius sive Plinii. The subsequent
chapter 'De Serpyllo silvestri Zygi' includes a description of T . zygis, which is one of
the most common species in Spain; and in Chapter 64 entitled 'De Tragorigano' he
writes: 'Multis Hispaniae locis provenit solo arido petroso cum Stoechade permista',
refering to T . nzastichina, whose Spanish name is 'sarilla'. Some years later, in the book
of icons of Lobelius (1581) five drawings of thyme are presented all being very difficult
to identify.
In the beginning of the seventeenth century, like preceding authors, Dodonaeus
(1616) also described two varieties saying: 'Thymo: unum cephaloton dictum, alterum
durius'. Today we can be sure that with the first he refers to Thynzbra capitata and with
the second to Thymus vulgaris. His Serpyllo vulgari seems to be a Thymus species of the
section Serpyllzlm. Furthermore, in his chapter 'De Serpyllo ex Dioscoride, Theophrasto
et aliis', he comments on the different ideas about Serpyllunz expressed by several
authors. H e describes T . mastichina, the first plant which he treats in his Chapter 1 8
on Tragoriganu?iz,applying the criteria of Dioscorides. W e can find in the work of
Bauhin (1623) a few years later, that he divides Thynzw into four parts: the first
( T . vulgaris folio tenzliore), as well as the second ( T . vulgaris folio latiore) seems to be
T . vulgaris; the third is called Thynzw capitatus (today Thynzbra capitata), and the fourth is
Thyvzum inodorunz. Within his Serpyllum nine different varieties are considered; the
last one, 'Serpyllum folio Thymi', has turned out to be identical with the Zygis of
Dioscorides.
In the eighteenth century Barrelier (1714) presents a book of icons. Icon number 788
represents T . nzorodevi (Martinez, 1936) from 'the kingdom of Valencia'; icon number
780 shows T . hyenzalis (Figure 1. l ) and number 694, entitled Marum hispanicurn, contains
a drawing of T . piperella. In his list of names with short explanations Tournefort (1719)
described six varieties of T . lusitanicus, four of them are T . lotocephalus and another is
T. mwohi (Figure 1.2). Within Thymbra he considers 'ThyvzLva hispanica', with T. mastichina
and T . zygis.

The Linnean Thymus

It is very interesting to observe the changes made by LinnC in his different works
about the Thymus species. Most of his knowledge is based on experiences of former
authors. In Hortus Cliffortianus (1737, pp. 305-306) he describes six species.
Nowadays we know that two of them, the latter ones, do not refer to Thynzw but to
Saturejd and Acinos. His No. 1, T , erectus turned out to be T . vulgaris (Figure 1.3), No. 2
T . repens is a species within the section Serpyllum, No. 3 is Thymbra capitata, and No. 4
T . mastichina (Figure 1.4). In his Hortus Upsalielzsis (1748, pp. 160-161) only
T . vulgaris and T . mastichina are mentioned. The reference work for the binomial
 The hi~tory,botany and taxonomy oftbe genus Thymus 3

Fzgz~re 1.1 Drawings from Barrelier (1714)work, number 780 corresponds to T. hye7zaIis.

system of nomenclature in Botany 'Species Pluntarunz 1st edition' (1753) includes the
following eight species in Thynzus: 1. T. serpyllanz, 2. T , vulgaris, 3. T . zygis, 4. T . acinos
(today Acinos urvensis),5 . T. alpinus (today Acinos a&inus),6. T. cephdlotos (today T. lotocephulus),
7 . T. villosas, and 8. T. pzlegioides (Figure 1.5). Within Saturejd we find 4. Sdt~~reja
nzastichina (today T . ~nastichina).
In Genera Plantarum (1754, p. 257) Linnaeus lists in 646. Thymas: Serpyllzm, Acinos,
and Mustirhina. Species Pluntarz~nz2nd edirion (1762, pp. 825-827) transferred T. nzustichina,
former Sutureju nzastichina, as number 8 into Thynzas. In the 1st edition this number was
 Figure 1.2 Drawings from Barrelier (1714) work, number 788 corresponds to T. moroderi.

established for T. pulegioides. This transfer Linnaeus commented on literally: 'Ambigit

media inter Saturejam et Thymum, sed cum stamina delirescant in fundo corollae,
et stylus corolla longior ad Thymum refero'. In Systema Nutarae 2 (12th edition, 1767,
P. 400) for the first time T. piperella appears.
 The history, botany and taxonomy of the genus Thymus 5

Ftgure 1.3 Typus of T. vulgaris in Linn6 (1737)Hortus Cliffortianus

After Linnaeus
Brotero (1804) described a new species, T. caespititiu~.Also Hoffmannsegg and Link
(1809), in their magnificent and big work about the Flora of Portugal, described
some new species: T. albicans, T. capitellatus, T. camphoratus, and T. sylvestris. It was
Bentham (1834) who, for the first time, divided the genus Thynzw into sections:
Matichina, with T. mastichina and T . tomentosuJ;Serpyllzkm with T, vulgaris, T. piperella,
 Fzgzue 1.4 Typus of T. nzastichzna in Li~zne'( 1 73 7) Hortus Cliffort~anus

T . villosus, T . capitellatus, and T . capitatus; and Pseudothyvzbra, with T . cephalotos (today

T . lotocephalus).

Edmund Boissier (1839-1845), the famous Swiss botanist from Geneva, studied
and described new Thynzus species from the Iberian Peninsula, a result of years of
research travelling through Spain. He also left valuable descriptions of Thymus species
from the north of Africa (Figure 1.6) as well as from Greece and Turkey written down
 DIDYNAMIA GYMNOSPERMIA. f i r
fnymus repem, foliis planis, floribus verticifhto-fpi-
catis. Ilurt. riif. 306. Koy. liu dB. 3zy.
Scrpyli~l!~~
f
Scrpylium \u!v,sre minus. Bax .ptiw. 229,
vtilga~e.Ded. prmpt. 277-
0.Sct t>yllunt vutgarc maluq. B ~ u bpru. . 220.
y.Serpylium vulgsrc mit~us,capituli~fanugiilofis. !7'"xr-
nqf. ief. 19?. lt. goti. 219.
2. Serpytlum nngcr:i~;bliurnhirl'utum. Bauh. pin. 220.
r. Scrpyllum foli;s c'ilr i odore. Banb. piat. 220.
&,thitc-rr i* Europsx: midis apriris. B
2 . THYMUS ereaus, fofiis revolutis ovatis, floribusrrkmk.
vcrticiliatu-Cpd'catis. Hert, citx. 30f.Horz.sp$ 160.
Mat. mrd, zSr, Roy. i q d h . 3ag;. Sawv. m o ~ f148.
i
Thymus vufy;aris, folio tenuiore. Bdxb. pi#. 219.
8.f hptnus vulgaris, folio istiore. Barb. pin. 219.
Tllvmum durius. Ihd. ptmpt. 276.
.
l i d i t a t i s G Narboneniis, HiCpania: metit& flk8-
l;s, 8
3. THYMUS floribus ver~icillato-fpicatis,mule ftaffru-Z&
ticoio, foliis Iiuaribus bati iliatin. Lorfl.
4
Thymo vtilgatiori rigidiori fi 11e. B u d . hip. 2.p. 271.
Thymum atlgufto longhri ue folio. Bart. ic. 777.
Se:pyllarn fylr&rc%yg~s&ofcotidis. C l g . b$. 3 ~ 8 .
Serpyllum folio thymi, B w b . pis. zro?
N d ~ i t 21~ t Hifpnnia.
Facics 7: ztklgari~,at Foiilt baJi ciliata .
4. T ~ Y M UHoribns
S verticillatis, ~eduaculisuniRofs, Rcinrrr,
csul;bus ctetkis fubramofis , foliis acutis ferr-atis,
ki. j b c ~ .475. *
Thgtnus csuliSus vix ramofis, fotiis ovatis scutis, pe-
ciunculis plurilnis uuifloris, Hart. ciilj; 306. Kay.
liigdb. 32s.
Clirlopodium arvenk, ocymi facie. Bash, pir, Z A ~ .
Clitiopodium vulgare. Lob., ic. p5.
Iiabit4t in Europa: glaretJs, crctacris,Jccir. @
,
y. THYMUS verticiitir ferfloris foiiis obtufiufcullr <jismC
conccrvis t~blii.~ratis.
Clii~opodiumvetticillis paucifloris in fpicam congeftis.
f f d l . briv. 653.
t7linopodium n~o~mnum. Barb. pin. 22f. Botc. mxJ-
2 . p. f0. t . 4y.
Acitii pulchrn ipecies. Bawh. kg. 3. p. Gro,
Hh-

Figure 1.5 Page from Linn6 (1753) Specles Plantarum, where T. vulgaris and 2'. zygzs are described
 Figure 1.6 T. broussonetzi (Boissier, 1839-1845, tab. 141).

in his Flora Orientalis (1867-1884). Fortunately various beautiful illustrations are

available. In his Elenchus (1838) he describes T.willdenowii, T. granatensis, T.longiflorus,
and T. membranaceus. In 1845, he created the section Pseudothymbra and later he
described T. carnosus, T. lusitanicus, and T. baeticas to be a variety of T. hirtus.
Willkomm (1868), a German botanist and author of the Prodromw Florue Hispanicae,
together with his Danish colleague Lange, stated that the genus comprises five sections:
Mastichina, Zygis, Piperella, Serpyllum, and Pseudothymbra. The section Serpyllum includes
 The history, botany and taxonomy oJthegenus Thymus 9
two groups: the first one with T. chamaedrys, T. serpyllum, and T . herba-barona and the
second one with T. bracteatus, T . serpylloides, and T. granatensis.
Briquet (1897) edited the Labiatae in Engler's monumental work, and considers
two sections, Pseudothymbra and Serpyllum, the latter with five subsections: Bracteatae
( T . capitellatus, T. villasus, T . algardiensis, T . albicans); Serpylla; Piperellae (T. piperella,
T . caespititiw, T. origdnoi&s, T . bovei);Vulgares (T. vulgaris, T . sabulicola, T. hyemdlis, T. zygis,
T. carnosus, T . hirtus); and Mastichinae (T. mastichina, T . tomentosus, T . welwitschii,
T . fontanesii).
Velenovsky (1906) focused on Thymw writing a monography on it. There he con-
sidered ten sections: Coridothymzts; Vulgares;Orientales; Anonzali (T. antoninae, T. portae);
Mastichina (T. fontanesii); Thymastra ( T . algardiensis, T . aldicans, T. capitellatus);Pseudo-
thynzbra with 2 groups (suffruticosi: T. membranacez~s,T . longiforus, T . funkii, and herbacei:
T. cephalotos, T. villosus, and T . granatensis); Piperella; Micantes; and Serpyllum (includes
T. serpy lloides).
The most important Spanish author is Pau, whose interest in Thymus runs throughout
his whole botanical work. In his important article published in 1929 entitled 'Intro-
ducci6n a1 estudio de 10s tomillos espafioles', he analyzes the previous works of
Linnaeus, Boissier, and Willkomm. In this article, many interesting details can be
found. Further remarkable Spanish authors who worked in this genus were Huguet del
Villar (1934), Vicioso (1974), and Elena-Rossell6 (1976), and in recent years many
contributions from Spanish authors are known.
Although Spain has always been a centre of the systematic, research on thyme, also
outside the Iberian Peninsula several famous botanists were dedicated to Thymus.
They are enumerated here in alphabetical order: Bonnet, Braun, Debray, Klokov,
Lyka, Machule, Negre, Opiz, Podlech, Paulovsky, Ronniger, Roussine, Roux,
Schmidt, Sennen. Two of them shall be emphasized: Ronniger, who left a very
valuable herbarium (today in Vienna) and Jakko Jalas, a Finnish botanist, who edited
the genus Thynzus within the Flora Europaea (1972), Flora Iranica (1982), and the
Flora of Turkey (1982).

Illustrations
A lot of old illustrations of Thymw are available, specially in the works of Hoff-
mannsegg and Link (1809), and Boissier (1838, 1839-1845, 1859). The early depic-
tions were very primitive drawings like those of Laguna's translation (1555) of the
Dioscorides or those of Barrellier (Figures 1.1 and 1.2). The herbariums from the
seventeenth and the first half of the eighteenth century were bound like books and as
we can see in the Linnean herbarium of the Hortus Cliffortianus, the plants were
ornately arranged in vases (Figures 1.3 and 1.4). After Linnaeus, at the end of the 18th
and in the 19th century, the drawings of plants spectacularly improved. Figure 1.7
shows beautifully coloured icons of T . caespititzzls with details of the calyx and the
corolla. It is taken from the Portuguese Flora of Hoffmannsegg and Link. Another
coloured icon showing T. broussonetii of North Africa is taken from Boissier's work
(Figure 1.6). Although plant photography has reached a high standard, we must be
aware that drawings can mediate more information on botanical details of plants than
photographs. Therefore classification of plants can better be performed with drawings
than with photographs.
 Figfi~reI , 7 T. iiae.pititizi.r (Hoffmannsegg and Link, 1809).

BOTANY - THE MORPHOLOGY A N D BIOLOGY OF THYMUS

Thymus plants are morphologically characterised by their habit or life-forms. W e can

differentiate two groups, on the one hand little bushy plants, usually below 50 cm, only
sporadically up to I m , e.g. T. baeticus and T. hyemalis in the south and southeast of
Spain. O n the other hand there are creeping life-forms sometimes with rooting twigs.
The latter is very common among the species belonging to the section Serpyllum or
 The history, botany and taxononzy ofthe gems Thymus 11

Figure 1.8 Stem morphology: (a) alelotrichous (T. praecox), (b) goniotrichous (T. pulegioides),
( c )holotrichous (T. piperella).

Hyphodromi. T . caespititius is an exception with its caespitose habit which can have very
long stems. Like most of the Lamiaceae, Thymw plants have quadrangular stems, the
young being hirsute. The hairs can cover either all four faces of the stem (holotrichous)
or only two faces alternating in each internode (alelotrichous). They also can be found
only on the four ribs of the stems (goniotrichous). The function of the diffetent types of
hairs on the stems are not yet known. Figure 1.8 represents the diffetent types of stems
found within the genus, and Figures 1.17 to 1.23 show the plant morphology for
different species of Thymw.
The leaves can be flat and more or less wide, or with revolute margins and almost
acicular. All intermediates seem to be possible. The indumentum is very variable. Some
species have leaves without hairs. The tector hairs in Thymw are always simple, but rarely
single-celled. Leaves are very frequently ciliate at the margins, either at the whole margin
or only at the base or on the petiole (Figure 1.9). The glandular trichomes are very
important containing the essential oil. There exist two types of glandular trichomes:
pedicellate glands with the upper cells full of essential oils, or big globose glands,
typical of Lamiaceae, with some basal cells (Figure 1.10). Chapter 3 provides additional
information on the anatomy and physiology of these glands.
The flowers grow more or less in clusters in the nodes. Few species have only two
flowers per node (e.g. T . antoninae), but usually there are bigger clusters of flowers. Species
with shorter internodes have globose and capituliform inflorescences. In these cases
both leaves of the inflorescence node usually differentiate from the rest of the plant's
leaves in form and size, and they are called bracts. This goes for T . menzbranaceus, T. carnosus,
and other species belonging to the sections Psegdothymbra and Thymus. In some species
the bracteoles can be extraordinarily long as in T. satureioides.
The calyx of thyme (2.5-8mm) when dry plays an important role in the dispersion
of the small fruits, or nuculas. Therefore its throat is closed by a hairy row and wind
can take it over quite a big distance. The calyces of some species, like that of T . mas-
tichina, have long ciliate teeth and seem to be a flying device like the pappus of the
?igure 1.9 Leaf and bract morphology. Leaves: (a) T. richardzi, (b) T. albicans, (c) T. lacaitae,
(d) T. hyernalis, (e) T. camphoratus, (f) T. longiflorus, ( g ) T. lotocephalus, (h) T. villosus,
(i) T. zygis. Bracts: (j) T. lacaitae, (k) T. villosus, (I) T. lotocephalus, (m) T. canzphoratus,
(n) T. longzj5lorus, (0) T. albicans.
Numbers beside the bars mean the length in mm.
 The history, botany and taxonomy ofthe genus Thymus 13

Flgure 1.10 Morphology of essential oil glands (up) and hairs (down).

Asteraceae. Usually the calyx has five teeth; three upper and two lower, the latter
always being longer and frequently curved upwards. They probably have to keep hold
of the corolla's tube. The three upper teeth are shorter than the lower and sometimes
reduced to one (T. caespititiw). The corolla varies between 4 and 10 mm in length and
finishes in one upper and three lower lobes, a typical structure to be pollinated by
bees or similar insects. The production of pollen in the four stamens is low. Occa-
sionally, the corolla can reach 2 cm like in T. longiflorus. Such long-tubed flowers are
pollinated by insects with long trunks which can pollinate the flowers while they
fly, like the flies of the Bombilidue family or crepuscular butterflies of the Sphyngidae
and Noctuidae families do. Figure 1.11 presents examples of calyx and corolla
morphology.
Thyme commonly presents gynodioecy, meaning that they produce two types of
individuals, some with female flowets without stamens, and others with hermaphro-
dite flowers. It is proven that pollinators can pollinate female flowets faster than the
hermaphrodites. The fruits are nutlets, up to four per flower, but usually some of
them abort during early development. Seeds collected from wild populations germi-
nate usually very easily and the seedlings grow relatively fast. Most of the species
bloom in spring, others in summer like e.g. T. serpyllum or T. praecox. In the Mediter-
ranean area, T. vulgaris subsp. aestivw and T. piperella flower in autumn, while T. hye-
nzalis in winter. The latter inhabits the arid region of the southeast of the Iberian
Peninsula.
 Z nznstichirzn Z piperelln ?: serpyllwn

Fzgure 1.11 Morphology of calyx and corolla.

If we analyse some characteristics from the evolutionary point of view, flat leaves
without hairs seem to be more primitive than leaves with revolute and hairy margins.
The same occurs with spiciform inflorescences that present bracts similar to leaves.
Globose inflorescences with special bracts seem to be more evolved. Woody species
 The history, botany and taxonomy of the genw Thymus 15
with erect life-forms may be phylogenetically older than herbaceous species with only
woody parts at the base. An interpretation of the evolutionary relationships among the
different sections within the genus is shown in Figure 1.12.

ECOLOGICAL ASPECTS

Thymes are heliophylous plants and like the sun, a fact which reflects the ecology of the
genus. Thynzus plants frequently live on rocks or stones and it is very important that the
soils are well drained. But different Thymus species require very different substrata, e.g.

Ftgz~re 1.12 Evolutionary relationships in the genus T ~ Y ~ I Z Z L J . of species in brackets

Number
T. carnosus lives on sand dunes near the sea (Figure 1.13), T . lacaitae on gypsaceous
soils, and T . vulgaris usually on calcareous soils.
Thymes are very resistant plants, which allows them to live under extreme climatic
conditions concerning temperature and water supply. They do not avoid either cold or
aridness. Dense and tomentose hairs as well as acicular leaves enable some species to
support very dry conditions. The high production of essential oils can also be an adap-
tive characteristic for dry climate, because the volatile substances evaporate and pro-
duce a saturated atmosphere around the plant that makes the loss of water more
difficult. Especially some species of the section Serpylhm can live in very cold climate,
like T. glacialis in Siberia or T . praecox in Greenland. From an ecological point of view
we can find the following correlation: bushy, woody, and erect plants are widely
distributed in dry climates, whereas in more fresh and humid climates usually plants
with flat leaves and woody only at the base are more common. The latter usually are
herbaceous with creeping or lying stems. Such species mostly belong to the sections
Hyphodromi and Serpyllzm. The production of essential oils in this group is probably
lower than in the first one.

SYSTEMATIC BOTANY

The genus Thymus is one of the most important genera of the Lamiaceae. It belongs to
the tribe Mentheae within the subfamily Nepetoideae. The most related genera are
Origanum, Satureja, Micromeria and Thymbra. Thymw is considered a well-defined genus,
based on the morphological and chemical features of its species.

Fzgure 1. I ? T. carnosus from Portugal

 T h e history, botany a n d taxonomy of the genus Thymus 17
General description
Perennial, subshrubs or shrubs, sometimes with herbaceous shape, but woody at the base,
aromatic; stem erect to prostrate, sometimes caespititious and radicant, hairy in all the
four sides, only in two alternating or only in the angles; leaves simple, entire or some-
times toothed, frequently revolute, glabrous or hairy, very variable in indumentum;
inflorescence spiciform, interrupted in verticillasters or capituliform, bracts like the leaves
or very different, lanceolate to broad ovate, usually coloured; flowers pedicellate or not,
usually with little bracteoles (very small bracts at the pedicels' base); calyx two-lipped,
sometimes nearly regular, more or less campanulate or cylindrical, ten-nerved; upper lip
with three triangular teeth sometimes reduced to one, lower lip with two long triangular
teeth curved upwards or widespread, throat barbate; corolla bilabiate, sometimes nearly
regular, more or less tubular, sometimes with a very long tube, up to 20 mm, four-lobed,
white, cream, pink or violet, frequently with clear spots in the throat or lower lobe; upper
lobe more or less rounded, emarginate, straight, lower and lateral lobes rectangular to
suborbicular, rounded, perpendicular to the tube; four stamens, sometimes reduced or not
present (gynodioecy), inserted in the upper half of the rube, exserted or not; anthers with
two parallel thecae; style apex branched; nutlets ovoid, smooth.

Biogeography
Thymw is widely distributed in the Old World (Figure 1.14).The Mediterranean region
can be described as the centre of the genus - strictly speaking the West Mediterranean
region. Only species of two sections occur outside the Mediterranean area. Seven sections
are spread over the Iberian Peninsula and northwest Africa, five of them are endemic.
In the Iberian Peninsula 35 species can be found, 24 of them endemic to the area. Two

Figure 1.14 Distribution of the genus T h y m u in the world. Dotted line represents all sections except
sect. Serpyllum and sect. Hyphodromi subsect. Serpyllastrum.
18 Ramh Morales

species can be found in the Macaronesian region, one on the Canary Islands (T. origanoides)
growing only at Riscos de Famara and surroundings, and the other one (T. caespititius) on
Madeira and the Azores; the latter grows also in the western part of the Iberian Peninsula.
Fifteen species (12 endemic) grow in northwest Africa, north of the Sahara desert
(Morocco, Algeria, Tunis, and Libya), with only three of them also occurring in the
Iberian Peninsula. Two species are common in the mountains of Ethiopia (T. serrulutus,
T. schimnperi) and one occurs in the southwest of the Arabian mountains (T. laevigatus).
In Greece 18 species are recorded, 36 in Turkey and 17 in the Flora Iranica. Further east-
wards Thymus can be found on the Sinai Peninsula (T. bovei and T. decussatus) and in the
arid regions of West Asia up to the Himalayas reaching the limits of the tropical region
up to East Asia and Japan. In China 11 species have been recorded. In the north it occurs
in Siberia and northern Europe, the coasts of Greenland can be described as the most
northern occurrence of Thynzw (T. praecox). Introduced populations now growing wild
are known to exist in regions as distant as Canada (T. serpyllum and T. pulegioides), Chile
(T. vulgaris) or New Zealand (T. pulegioides and T. vulgaris).
W e can suggest the origin of some taxa of the genus to be in the Mediterranean area,
seeing that the sections Serpylhm and Micantes have been present there since the Paleocene.
In the Miocene, some species of section Thymus and Hyphodromi developed. During the
Quaternary the ancestors of the section Serpyllum and, to some extent, those within the
section Hyphodromi have produced new speciation processes, colonizing all the ice-free
land after the last Ice Age. These processes are not yet finished and may be the reason why
all these species are difficult to be distinguished. W e can assume that they are halfway
in a process of speciation to produce clear species (Morales, 1989).

Pollen
The pollen grains of this genus have a very homogeneous morphology, both within the
same species and among different taxa. According to Wunderlich (1967), it can be
ascribed to the Satuveja type. It has a radial isopolar symmetry and is usually hexacol-
pate (NPC 643) and three-celled. Octocolpate and tetracolpate grains are also known.
The colpi are regularly disposed, and the mesocolpi usually are of the same width with
one exception: the mesocolpi of T. cuespititius are of varying wideness alternating a
wider and a more narrow one. The pollen grains are more or less spheroidal and the
index of Polar distanceIEquatoria1 distance (PIE) varies between 0.9 and 1.3 (from pro-
late-spheroidal to oblate-spheroidal). The sizes of the pollen grains vary from 21 to
46 pm depending on the species and a correlation between ploidy level and size can be
assumed. The ornamentation usually is suprareticulate, less frequently semitectate or
reticulate. In the case of suprareticulate ornamentation, thick walls delimit in a lower level
a net of narrower walls and pores. The wideness of walls and pores varies from one species
to another, but it is homogeneous within each species. As an exception, pollen grains
with cerebroid ornamentation can be found, which seems to be usual for tetracolpate
pollen grains. Figure 1.15 illustrates the morphology of pollen grains from T. hyemalis.

Chromosomes
In the genus Thymus the chromosomes are very small. With 1-2 pm they appear like
dots under the optical microscope. The following chromosome numbers are known:
2n=24, 26, 28, 30, 3 2 , 4 2 , 4 8 , 50, 52, 54, 56, 58,60, 84 and 90, corresponding to the
 y taxonomy ofthe g e 7 z ~Thymus
The history, b o t ~ z ~and 19

Fzgz~re 1. I S Pollen grains of T. hyevzalir from Murcia (Spain). Images 1-5 are vlews from an optical
rnlcroscope. Images 6 and 7 were obtained with a scanning electron rnlcroscope with
a magnification of 1600x and 7000x respectively.

diploid, tetraploid and hexaploid levels. The secondary basic numbers x = 14 and x = 15
probably originate from a basic number x = 7 . The most frequent numbers are 2n=28,
3 0 , 56 and 6 0 . Aneuploidy has been an important phenomenon during the evolution of
this genus and is responsible for the other numbers. There are a lot of interesting cases
of different levels within the same species. This is true for T . mastichina with 2n= 30,
60; T . vulgaris 2n=28, 58; T . zygis, T . leptophylhs, T . glabrescens, T . longicaulis, T . praecox
2 n = 2 8 , 56; T . algeriensis 2n=30, 5 6 ; T . conzptus 2 n = 2 6 , 52; T. zygioides 2 n = 6 0 , 9 0 ;
T. longedentatzls 2n=30, 90; T. striatus and T. herba-barona 2n=28, 56, 84. The latter is
most remarkable because the chromosome numbers studied in the West Mediterranean
populations resulted to be 2n=28 in Majorca, 2n=56 in Corsica, and 2n=84 in
Sardinia. Chromosomes from different Thymus species are shown in Figure 1.16.

Other features
In Thynzus hybridization is very common where two or more species live together.
U p to date 6 0 hybrids have been detected among the 35 species living in the Iberian
20 Ram& Morales

Figure 1.16 Chromosomes of some species of Thynzus. (1): T. 77zastigophorus, 2 n = 28 (Zaragoza, Spain).
(2): T. cupitellatus, n = 1 5 (Algarve, Portugal). (3): T . canzphoratw, 2n= 30 (Alentejo, Por-
tugal). (4): T. canzphoratw, 2 n = 3 0 (Algarve, Portugal). (5): T. camphoratz~s,2 n = 3 0
(Algarve, Portugal). (6): T. cumoiw, 2n = 56 (Algarve, Portugal).

Peninsula, as we can see in the appendix (Morales, 1995). Some chemical studies show
the genus to be homogeneous, in the comparison with others such as Teucrizlm or Siderztzs
both chemically heterogeneous (Morales, 1986). These two features are the evidence to
consider Thymus to be a good taxonomical genus, probably monophyletic. Within the
genus genetic incompatibility between species does not seem to exist, which makes
taxonomic studies in this genus very difficult, especially in some taxonomical groups
e.g. in the section Hyphodrami and particularly in the section Serpylhm, where the concept
of species is more difficult to apply. If we impose synoptocal criteria, probably a lot
of forms, sometimes ecological forms, would be included as simple populations into
a given taxon. But when using analytical criteria we risk overlooking existing species
considered as natural units. In case of doubt I recommend synoptical criteria. At the species
level, there are a lot of names, more than 1 000, many of them of course are synonyms.

Popular names
In the whole area of distribution, Thynzzls is usually well known and used by the popu-
lation as spice, medicinal plant or source of essential oils. Therefore a big variety of
 The history, botany and tuxononzy ofthe genus Thymus 21

Fignve 1.17 Plant habitus. ( a ) T. piperella, ( b )?: zygis, ( c ) T. gvanatensz.r.

popular and vernacular names are known for the different species. If we begin in the
west of its habitat T . cuespititius, from the Azores, Madeira and the western part of the
Iberian Peninsula has the Portuguese names 'tomentelo' or 'tormentelo', and in Galicia
'tomelo do pais', 'tomentelo do pais' or 'tomillo'. The only species grown on the Canary
Islands from this genus is T . origunoides, in the Riscos de Famara of Lanzarote island. An
old name of this plant is 'tajosC'. 'Tomillo' is the popular name in other islands for Thy-
mus-looking species of Micronzeriu. In continental Africa species of Thynzus are found in
Morocco, Algeria, Tunis and Libya. T. ulgeriensis is the most common in the four
countries, and its popular names in arabic and berber languages are: 'azoukni', 'djertil',
'djoushshen', 'hamriya', 'hamzousha', 'khieta', 'mezoukesh', 'rebba', 'toushna'. T . broussonetii
is named there 'zatar', 'za'atar el-hmir', 'za'ter el hmir', 'ze'itra', 'z'itra'. The Moroccan
T. maroccunw has the name 'azukenni'. In the mountains of Ethiopia two Thynzus species
grow, T. serrulutus and T. schinzperi, with the Abyssinian names for the first one: 'tausi',
Fig~lre 1.18 T. 7tznstichinn (Spanish marjoram) very common in the Iberian Peninsula, from Central Spain.

'tazi.', 'tenni', 'teschin', 'tessni', 'tesni.', 'thasne', and 'tessni', 'tosign', 'tosigne', 'tossign'
or 'tossine' for the latter.
In Asia, in the Arabic Peninsula, the mountainous areas of Yemen are the southern-
most localities in this continent, where T.luevigutz~slives. It is named 'za'tar' or 'sa'tar'.
In the far east in China the popular name of several species of thymes are 'bai li xiang'
and the most used species, T. quinquecostutus, is called 'di jiao' or 'bian zhong'.
In the North of Europe T. serpyllz~nzand other species of this group are widespread.
The vernacular names in the nordic languages are 'timian' or 'timjan', 'stortimian' or
'backtimian'. And in Central Europe they are called 'Thymian', 'Feldthymian', 'Quendel',
'Kudelkraut', 'Kuttelkraut' in Germany; 'serpolet', 'piolet', 'piliolet', 'pignolet', 'pClevouC',
'pCnCvouet' in France; 'pepolino', 'sermollino selvatico' in Italy; 'erba pevarina', 'siisCmbar'
in Slavic (Puschlav); 'timian', 'masar6n salvatg', 'pavradel', 'pavradella' in Ratorom. In
English, the following names are known: 'thyme', 'wild thyme', 'penny mountain',
'hillwort', 'brotherwort', 'shepherds thyme', and in dutch 'tijm'.
In the different languages of the Iberian Peninsula, a lot of names are in use for the
multitude of species of Thynzus or 'tomillo' (Morales eta/., 1996):

T. bueticus: tomillo, tomillo basto, tornillo fino, tornillo gris, tomillo limonero.
T . g~unutensis:hierba luna, serpillo, serpol, tomillo, tomillo colorao, tomillo serpol.
T. hyemalis: tomillo, tomillo de invierno, tomillo fino, tomillo macho, tomillo morado,
tomillo rojo.
T . lucuitae: tomillo lagartijero, tomillo de Aranjuez.
T . longzflorus: tomillo, tomillo real.
 The history, botany and taxonomy ofthe genus Thymus 23

Ftgzwe 1.19 T. pzpef*ella from Valencia, S p a n

T. loscosii: ajedrea, tomillo sanjuanero.

T. lotocephalas: tomilho-cabepdo.
T. nzastichina: ajedrea de monte, almoradux, alrnorafi, amgraco, bela-luz, cantueso,
escombrilla, marahri, marduix silvestre, mejorana, mejorana de monte, mejorana silvestre,
rnendaro, rnendaroa, rnoraduix bord, salpurro, sarilla, tomilho-alvadio, tomillo, tomillo
blanco, tomillo de las aceitunas, tomillo macho, tomillo salsero.
T. vmzbranaceas:cantueso, escombrilla, mejorana, tornillo blanco, tomillo macho, tomillo terrero.
T. nzoroderi: cantahueso, cantueso, mejorana.
Figzure 1.20 T. nzernbrunaceu from Murcia, Spain (Morales, 1986).

T.orospedanus: tomillo.
T. piperella L.: peberela, peberella, pebrella, pebrinella, piperesa, tim6.
T. praecox: erva-ursa, farigola, farigoleta, folc6, herba de pastor, hierba luna, salia de
pastor, samarilla, sarpoil, serpEo, serpil, serpildo, serpilho, serpol, serpol, serpolio, timd
negre, tornillo de puerto, tomillo rastrero.
T. pulegioides: apiua, charpota, serpEo glabro, serpol, te fino, te morado, t6 morau,
tomelo, tomentelo, tomillo.
T.richardii: farigola de muntanya, farigola mascle, farigoleta, hierba luna, salsa de pastor,
serfull, serpol, si.rpol, serpoll, tim6 negre, tomillo rojo.
T.serpylloides: sarnarilla, tomillo, tomillo de la sierra, tomillo de Sierra Nevada.
T.serpylloides subsp. gadorensis: samarilla, tomillo rojo, verbena.
T. villosw:azeitoneira, erva-azeitoneira, erva-das-azeitonas, tomilho-peludo, tomillo ansero.
T. vulgarzs L.: ar@, arqanha, axedrea, boja, bojas, elar, elharr, ellbor, entremunsell, erle-bedarr,
estremoncello, estremoncillo, estremonzillo, estremunsell, estrernunzillo, ezkai, ezkaia,
farigola, farigoleta, fariguala, forigola, frigola, frigola, friula, ispillu, lo tim6, morquera,
sajolida, senyorida, tem, tirn6, tim6 femella, tim6 mascle, tim6 normal, tim6n, timoncillo,
tirnonet, timons, tomani, tomelo, tomello, tomello vulgar, tomentelo, tomilho, tomilho-
ordingrio, tomilho-vulgar, tomilo, tomillo, tomillo ansero, tomillo blanco, tornillo horde,
tomillo com6n, tomillo limonero, tomillo negrillo, tomillo royo, tomillo salsero, tomillo
vulgar, tornillua, tomizo, trernoncillo, tremonsillo, tremontillo, trernonzillo, tumillo.
T. zygis: farigola salsera, morquera, paticas de mona, salsero salseta de past6, serpiio-do-
monte, tomilhinha, tomillo, tomillo aceitunero, tomillo aceytunero, tomillo albar, tomillo
ansero, tomillo blanco, tomillo del campo, tomillo espafiol, tomillo fino, tornillo negrillo,
tomillo risquero, tomillo rojo, tomillo salsero, tomillo sansero, tomillo sansero fino.
 The history, botany and taxonomy ofthe genus Thymus 25

Figure 1.21 T. vulgaris (Hallier, 1884: 188, Tafel 1796)

T, zygis subsp. graczlzs: tomillo, tomillo aceitunero, tomillo blanco, tomillo fino, tomillo
rojo, tomillo salsero.

Sections of the genus Thymus

According to Jalas (197 I), Thymzls is divided into eight sections: Micantes, Mastichina,
Piperella, Teucrioides, Pseudothymbra, Thymw, Hyphodronzi, and Serpyllum. The sequence
used here is that established by Jalas, but other ordinations would perhaps be more
Fzgzlre 1.22 T. Iacaitae from Central Spain

logical considering phylogenetic or evolutionary criteria. For distribution patterns of

the sections and subsections see Morales (1997).

I. Sect. Micantes
11. Sect. Mastichina
111. Sect. Piperella
IV. Sect. Teztcvioides
V. Sect. Psez~doth~mbru
1. Subsect. Pseztdothynzbva
2. Subsect. Anomalae
VI. Sect. Tlqtmzts
1. Subsect. Thyrrzastra
2.Subsect. Thynzw
VII. Sect. Hyphodvonzi
1. Subsect. Sztbbvacteati
2.Subsect. Serpyllastrztm
3. Subsect. Thyrrzbropsis
VII I. Sect. Serpyllztnz
1. Subsect. Insulares
2. Subsect. Kotschyani
3. Subsect. Pseudopiperellae
4. Subsect. Isolepides
5 . Subsect. Alternantes
 The history, botany and taxononzy ofthe genus Thymus 27

Ftgz~re 1.23 T. nzaroccan~ds(Bot.J . Lznn. Soc., 16: pl. 27).

6. Subsect. Psezdomarginati
7 . Subsect. Serpyllunz

I. Sect. Micantes Velen., Bei. Bot. Centr. 19(B2): 278 (1906)

Typus: T. caespititius Brot.
Erect plants (North-African species) or caespitose; stems holotrichous; leaves flat, glabrous,
long oblong-obovate; inflorescence spiciform, sometimes dense; lateral upper teeth of
calyx very short or reduced.
 It comprises three species, two of them are North African woody species, that occur
in Morocco: T. satzlreioides and T. riatarum. The former inhabits the High Atlas region,
while T. riatarzlm is a prostrate plant and lives in the Rif mountains. The Ibero-Macaro-
nesian species T. caespititizls occurs in the northwest of the Iberian Peninsula and also in
Madeira and Azores. If we take into account their plesiomorphic features, like flat,
non-revolute and glabrous leaves, and their geographical distribution, this section
seems to be very old.

11. Sect. Mastichina (Miller) Bentham, Lab. Gen. Sp.: 340 (1834)
Mastichina Miller, Gard. Dict. ed. 4 (2) (1754).
Typus: T. mastichina (L.) L.
Erect plants with holotrichous stems, leaves flat, lanceolate to obovate; inflorescence
capituliform; calyx very hairy, teeth similar and subulate, with long cilia.
This section is endemic to the Iberian Peninsula, and comprises T. mastichina, with
two subspecies, and T. albicans. T. mastichina subsp. mastichina is a very common plant
in Spain and Portugal. The subspecies donyanae occurs only in the southwest of the
Iberian Peninsula around the 'Coto de Dofiana' and in some locations in the 'Algarve'.
The other species, T. albicans, is also living in the southwestern pinewoods of Pinzls
pinea. T. mastichina subsp. donyanae and T. albicans, with 2 n = 3 0 chromosomes, are
probably the origin of the tetraploid apoendemic T.mastichina subsp. mastichina, a
modern taxon that has spread throughout the entire Iberian Peninsula.

111. Sect. Piperella Willk., Prodr. F1. Hisp. 2: 404 (1868).

Typus: T.piperella L.
Erect or decumbent plants, with holotrichous stems and leaves obovate, flat and glabrous;
flowers growing in lax verticillasters.
T. piperella is found at Valencia province and surroundings, and it is the unique species
of this monotypic section, endemic to this region.

IV. Sect. Teucrioides Jalas, Bot. J. Linn. Soc. 64(2): 201 (197 1).
Typus: T. teucrioides Boiss. and Spruner.
Plants usually decumbent with leaves revolute, ovate or triangular-ovate; flowers in
verticillasters. Endemic to the Balkan Peninsula. It inhabits the mountains of Greece
and Albania. Three species can be recognised within this section: T. teucrioides,
T.hartvigi, and T. lezlcospermzls, that have been studied by Hartvig (1987). Chromosome
numbers of these species are not yet known.

V . Sect. Pseudothymbra Bentham, Lab. Gen. Sp.: 341 (1834).

Typus: T. lotocephalzls G. L6pez and R. Morales (T. cephalotos auct. non L.)
Erect plants with holotrichous stems and linear revolute leaves, usually hairy and with
cilia at the base; inflorescence capitulifotm with broad bracts; corolla very long.
In this section are included nine Iberian-North-African species, usually with long
corollas, up to 2 cm, and bracts rather different from the leaves and subglobose inflorescence,
except in subsection Anomalae. The North-African species are T.munbyanw, common and
very variable, extending from the Middle Atlas and the Rif Mountains as far as the Alge-
rian mountains. A difficult species with two subspecies and hybrids with T. algeriensis
and T. willdenowii. T. bleicherianus is only known from three locations, one in Algeria and
two more in the north of Morocco. The other species of this section are all Iberian.
 The history, botany and taxononzy ofthe genw Thymus 29
W e recognize two subsections:

V1. Subsect. Pseudothymbra (Bentham) R. Morales, Ruizia 3: 146 (1986)

Inflorescence capitulifotm and bracts are very different from the leaves.
V2. Subsect. Anomalae (Rouy) R. Morales, Rzlizia 3: 146 (1986).
T. sect. Anomalae Rouy, Bull. Soc. Bot. France 37: 166 (1890).
Typus: T.antoninae Rouy and Coincy
Flowers in verticillasters, bracts with similar appearance as the leaves.

VI. Sect. Thymzts

Erect or radicant plants with holotrichous stems, revolute leaves, usually hairy; flowers
in spiciform or globose inflorescences.
Western Mediterranean section, with three most important species: T . vulgaris,
T.zygis and T. willdenowii. The first usually occurs on basic soils and is distributed in
northern Italy, south of France and east of Spain. T. zygis is a very common species in all
the Iberian Peninsula and T. willdenowii is common in North Africa (Morocco and
Algeria) and also grows only in Gibraltar area in the Iberian Peninsula.
W e recognize two subsections:

V I 1. Subsect. Thymastra (Nyman ex Velen.) R. Morales, Raiziu 3: 146 (1986).

T. sect. Thymastra Velen., Bei. Bot. Centr. 19(B2): 276 (1906).
Typus: T.cupitellatzls Hoffmanns. and Link.
Erect plants with triangular-ovate or lanceolate-ovate leaves, without cilia at the base;
inflorescence more or less globose with bracts different from the leaves.
V12. Subsect. Thymus
Erect or subtended plants with leaves usually hairy, and ciliate or not at the base, with
revolute margins and more or less linear, bracts broader than the leaves, but not very
different.

VII. Sect. Hyphodromi (A. Kerner) Halksy, Denkschr. Akad. Wiss. Wien 61: 252 (1894).
Typus: T.bracteosus Vis. ex Bentham.
Plants usually subtended and rooting; stems holotrichous; leaves flat or revolute, usually
not hairy; inflorescence frequently capituliform with bracts different from the leaves.
This section extends throughout the Mediterranean area and comprises around
60 species. From the three subsections, Subbructeati is characterized by more or less revo-
lute or convolute leaves and seems to be Oriental. Only one species occurs in North
Africa, from Morocco to Libya: T.algeriensis. Another species occurs in Central Spain:
T.nzastigophorzls. T. spinzllosw occurs in Sicily and Italy, and T. strzdtzls in the Italian and
Balkan Peninsulas. Both species are very variable. T. argueus, T.brachychilas, T.cappa-
docicus, T . cherlerioides, T . convolz~tus,T.pulvinutzts, and T. revolatz~soccur in Turkey;
T. boissieri, T.comptus, T.dolopicas, and T. plusonii in the Balkan Peninsula; T.atticas,
T.purnassiczls, and T. leucotrichus inhabit Turkey and the Balkan Peninsula. The last
species also grows in Syria and in the Lebanon. T. integer is only found on the island of
Cyprus. This species is probably not different from T . leacotrichas. T.samizls occurs in
the Aegean islands. T.borysthenicas and T.pallasiunas occur north of the Black Sea,
T. persicus south of the Caucasus, but only one location for this species is known.
W e recognize three subsections:

VIIl. Subsect. Subbructeuti (Klokov) Jalas, Bot. J. Linn. Soc. 64(2): 205 (1971), emend.
T. sect. Subbracteati Klokov, Not. Syst. (Leningrad) 16: 315 (1954) pro parte.
Typus: T. pallasianus H. Braun.
VII2. Subsect. Serpyllustrum Huguet del Villar, Cavanillesia 6: 124 (1934).
Lectotypus: T. bracteosw Vis. ex Bentham.
VII3. Subsect. Thymbropsis Jalas ex R. Morales, Anales Jard. Bot. Madrid 45(2): 562
(1989).
Typus: T. nzaroccanus Ball.

Subsection SerpylZustrum is a group of species characterized by the presence of

prostrate stems and flat leaves more or less wide. Five species from this section are
living in Spain: T. bracteatus, T. leptophyllzts, T.fontqueri, T. grandtensis and T. lacaitae. It
is also well represented in the East, but no species occur in Italy and North Africa.
T.aznavourii and T. bracteosus occur in the Balkan Peninsula; T. canoviridis, T. haz~ssknechtii,
T . pectinatus and T.spathulifolius are found in Turkey. T. zygioides extends from the
Balkan Peninsula as far as the Crimean Peninsula and also in Turkey. This species and
the Spanish endemic T. lacaitae are morphologically very similar. There is also a group
of species that occur only in the Caucasus: T . dagestanicus, T. hadzhievii, T.helendzhicus,
T. Rarjagnii, T.ladjanuricz~s,T. lipskyi, T. majkopiensis, and T. sosnowskyi. Seven more
species from Central Asia are considered inside this subsection: T. cuneatus, T. eremita,
T. incertus, T.irtyschensis, T . kirgisorunz, T.nerczensis, T. petraeus.

Subsection Thymbropsis includes the North African T. broussonetii, T.nzaroccanus,

T. lanceolatus, T. numidicus, T. pallescens, and the two endemic species from Greece
T. laconicus and T. holosericeus. Five more species from this section are found in Turkey:
T. cariensis, T. ciliciczls, T. ezgii, T.leucostonzus, and T. sipyleus. T. syriacus occurs in
Lebanon, Syria and a location in northern Irak; T. bovei lives in the Sinai Peninsula,
Israel, Jordan, Irak and Saudi Arabia; and T. decuu-atus in Sinai and Saudi Arabia. This
group has predominantly North-African and East-Asian species.

VIII. Sect. Serpyllz~nz(Miller) Bentham, Lab. Gen. Sp.: 340 (1834).

Serpyllunz Miller, Gard. Dict. ed. 4 (3) (1754).
Woody plants or only woody at the base, but with herbaceous appearance, usually sub-
tended and rooting, with holotrichous stems or hairy only in two opposite sides or in
the angles (goniotrichous or alelotrichous), leaves flat and usually ciliate at the base,
with distinct lateral veins; inflorescence spiciform or mote or less globose.
In this section there are around 120 species. They occur throughout the area of the
genus, except in Madeira and the Azores. W e find in the species of Serpylluvz the widest
chromosomal variation. There are also woody species that grow in the mountains in
arid areas like T. origanoides on Lanzarote (Canary Islands); T. serrulatw and T. schivzperi
in Ethiopia, T. laevigatus in the southwest of the Arabian Peninsula. Another group of
species are more or less herbaceous and occur in the Mediterranean mountains, and all
of Eurasia and also along the coasts of Greenland. The species of the last group seem to
be younger in evolutionary terms and have probably been actively evolving since the
last glaciation when this group colonized the new lands free of ice. This group is also
 The histo~y,botany and taxononzy ofthe genus Thymus 31
very difficult taxonomically and corresponds to the last three subsections. Few species
of these subsections are present in the Mediterranean area. According to Jalas (197 I),
we divided this section into seven subsections.

Subsection Znsulares comprises T.willkonzmii, an endemic species that occurs in the

mountains of the provinces of Castelldn and Tarragona (eastern Spain); T.richardii,
with three subspecies: subsp. richardii from Majorca and Yugoslavia, subsp. ebusitanus
from Ibiza and subsp. nitidus from Marettimo island near Sicily; the North-African
T. dredtensis and T.guyonii, the Canary Island endemic T.origanoides and the endemic
species to northwest Turkey T.bornmuelleri.
Subsection Kotschyani includes a lot of Asian species, but only T.fallax and
T.transcaucasicus occur in Turkey. Other interesting species occurring outside the
Mediterranean area are T.laevigatus from the mountains of Yemen or T.schimperi and
T. serrulatus from the Ethiopian mountains.
Subsection Pseudopiperellae comprises T. herba-barona from Majotca, Corsica, and
Sardinia (Mayol et al., 1990) and T. nitens from the south of France.
Five species inhabiting the Balkan Peninsula belong to the subsection Zsolepides:
T. bulgaY.icus,T. glabrescens, T.longedentatus, T. pannonicusm, and T. sibthorpii.
Subsection Alternantes includes T.linearis from the Himalaya mountains; the Euro-
pean T. pulegioides, T.Jroelichzanus, T. alpestris, T. oehmianw, T.bihoriensis, and T. covzosw.
Subsection Pseudomarginati includes the species T.longicaulis and T.praecox, very
common in Europe and also in Turkey; T.nervosw, an endemic of the Pyrenees and the
French Massif Central; T.ocheus, T.stojanovii, and T. thracicus from the Balkan Peninsula
and the East Mediterranean region.
Subsection Serpyllum includes T. quinquecostatz~s from Japan, the European
T. serpyllum and T.talijevii and other Russian species.

LIST OF THYMUS SPECIES OF T H E WORLD

I propose at the moment the following list of species. There are 214 species and 36
subspecies more: 250 taxa. When known, the chromosome numbers and the countries
are given (Ag =Algeria, A1 =Albania, An =Asian Turkey, Az = Azores, B1= Balearic
Islands, Bu= Bulgaria, Co= Corsica, Cy = Cyprus, E = East Aegean Islands, G a = France,
Gr = Greece, Hs = Spain, It = Italy, Ju=former Jugoslavia, Li = Libya, LS =Lebanon and
Syria, Lu= Portugal, Ma=Morocco, Ru= Romania, Sa= Sardinia, Si =Sicily, Tn=Tunis,
T u = European Turkey, URSS = former Soviet Union).

I. Sect. Micantes Velen.

T.caespititiw Brot. 2n= 30 Hs Lu Az Madeira
T. satureioides Cosson subsp. satz~reioidesMa
subsp. conznzutatus Batt. 2n = 30 Ma
T. riataram Humbert and Maire Ma

11. Sect. Mastichina (Miller) Bentham

T. mastichina (L.) L. subsp. ??zastichina2n= 56, 58, 6 0 Hs Lu
 subsp. donyanae R. Morales 2n= 30 Hs Lu
T. albicans Hoffmanns. and Link 2n = 30 Hs Lu

111. Sect. Piperella Willk.

T. piperella L. 2n = 28 Hs

IV. Sect. Teucrioides Jalas

T. teucrioides Boiss. and Spruner subsp. teucrzoides Gr A1
subsp. alpinw Hartvig G r
subsp. candilicus (Beauverd) Hartvig Gr
T. hartvigi R. Morales subsp. hartvigi Gr
subsp. macrocalyx (Hartvig) R. Morales Gr
T. leucospermus Hartvig Gr

V. Sect. Pseudothymbra Bentham

V1. Subsect. Pseudothymbra (Bentham) R. Morales
T. lotocephalus G . Ldpez and R. Morales 2 n = 30 Lu
T. villoszls L. subsp. villosus Lu
subsp. lusitanicus (Boiss.) Coutinho 2n= 54 Lu H s
subsp. oretanicus Hs
T. longiforus Boiss. 2n= 28 Hs
T. membranaceus Boiss. 2n = 28 Hs
T. moroderi Pau ex Martinez 2n = 28 32 Hs
T.munbyanus Boiss. and Reuter subsp. munbyanus Ma Ag
subsp. coloratus (Boiss. and Reuter) Greuter and Burdet Ma Ag
T. bleicherianus Pomel Ma Ag
T. funkii Cosson 2n = 28 Hs
V2. Subsect. Anomalae (Rouy) R. Morales
T. antoninae Rouy and Coincy 2 n = 56 Hs

VI. Sect. Thymus

VI 1. Subsect. Thymastra (Nyman ex Velen.) R. Morales
T.capitellatus Hoffmanns. and Link 2n = 30 Lu
T. camphoratus Hoffmanns. and Link 2n = 30 Lu
V12. Subsect. Thymus
T. curnosus Boiss. 2n= 56 Lu Hs
T. vulgaris L. subsp. vulgaris 2n = 28, 30 Hs Ga It
subsp. aestivus (Willk.) 0. Bol6s and A. Bol6s 2n= 58, 6 0 Hs B1
T. orospedanw Huguet del Villar 2n= 28 Hs
T. hyemalis Lange subsp. hyemalis 2n = 58 Hs
subsp. millefloris (Rivera and al.) R. Morales 2n= 58 Hs
subsp.fumanzfoliw (Pau) R. Morales Ma Ag
T.zygis Loefl. ex L. subsp. zygis 2n = 28 Hs Lu
subsp. gracilis (Boiss.) R. Morales 2n= 28 Hs Ma
subsp. sylvestris (Hoffmanns. and Link) Coutinho 2n= 56, 58 Hs Lu
T. baeticus Boiss. ex Lacaita 2 n = 58 Hs
T.willdenowii Boiss. 2n = 30 Hs Ma Ag
 The hzstory, botany and taxonomy ofthe genus Thymus 33
T. loscosii Willk. 2 n = 54 Hs
T. serpylloides Bory subsp. serpylloides 2 n = 58 Hs
subsp. gadorensis (Pau) Jalas 2n= 56, 58 Hs

VII. Sect. Hyphodromi (A. Kerner) Halkcsy

VII1. Subsect. Sizbbracteati (Klokov) Jalas
T. algeriensis Boiss. and Reuter 2n = 30, 56 Ma Ag Tn Li
T. argaeus Boiss. and Bal. An
T.atticus Celak. An Bu Gr T u
T. boissieri Hal. A1 Gr Ju
T. borysthenicus Klokov and Shost.
T. brachychilus Jalas An
T. cappadocicus Boiss. An
T. cherlerioides Vis. 2n = 28 An
T,comptus Friv. 2n= 26, 28, 52 Gr T u
T. convolutus Klokov An
T. dolopicus Form. Gr
T.integer Griseb. Cy
T. leucotrichus Halksy An Gr Ju LS
T. mastigophorus Lacaita 2n = 28 Hs
T.pallasianus H . Braun subsp. pallastdnus north to Black Sea
subsp. brachyodon (Borbks) Jalas
T. parnassicus Hal6csy An Gr Ju
T. persicus (Ronniger ex Rech. fil.) Jalas
T. plasonii Adamovic G r
T. pulvinatus Celak. An
T.revolutus Celak. An
T. spinulosus Ten. 2n= 56 It Si
T. striatz~sVahl 2n=26, 2 8 , 4 2 , 54, 56, 84 A1 Bu It Gr J u T u
VII2. Subsect. Serpyllastrum Huguet del Villar
T,aznavourii Velen. Tu
T. bracteatus Lange ex Cutanda 2n = 56, 58 Hs
T. bracteosus Vis. ex Bentham Ju
T. canoviridis Jalas An
T. cuneatus Klokov Central Asia
T. dagestanicus Klokov and Shost. 2 n = 2 8 Caucasus
T. eremita Klokov Central Asia
T. fontqueri (Jalas) Molero and Rovira 2 n = 56 Hs
T . granatensis Boiss. subsp. granatensis 2n = 28 Hs
subsp. micranthus (Willk.) 0.B0l6s and Vigo Hs
T. hadzhievii Grossh. 2n = 28 Caucasus
T.haussknechtii Velen. An
T. helendzhicus Klokov and Shost. Caucasus
T. incertus Klokov Central Asia
T. irtyschensis Klokov Altai
T. karjaginii Grossh. Caucasus
T. kirgisorunz Dubjanski 2n= 26 South of Russia and wide area until Siberia
T. lacaitae Pau 2n = 28 Hs
T. landjanuricus Kern. Caucasus
T. leptophyllus Lange subsp. leptophylhs 2n = 28 H s
subsp. paui R. Morales 2n= 56 Hs
subsp. izcoi (Rivas Martinez and al.) R. Morales H s
T. lipskyi Klokov and Shost. Caucasus
T. majkopiensis Klokov and Shost. 2n= 28 Caucasus
T. nerczensis Klokov N Mongolia
T. pectinatus Fisch. and Meyer An
T. petraezs Serg. Central Asia
T. samiw Ronniger and Rech. fil. AE
T. sosnowskyi Grossh. 2n = 60 Caucasus
T. sphatulifolius Hausskn. and Velen. An
T. zygioides Griseb. 2n= 56, 60, 62, 90 An Bu Gr Ru Tu Crimea
VII3. Subsect. Thymbropsis Jalas ex R. Morales
T. bovei Bentham Sinai IJ Irak Saudi Arabia
T. broussonetii Boiss. subsp. broussonetii Ag Ma Tn
subsp. hannonis (Maire) R. Morales Ma
T. cariensis Hub.-Mor. and Jalas An
T. ciliczcz~sBoiss. and Bal. An AE
T. decussatus Bentham Sinai Saudi Arabia
T. eigii (Zohary and Davis) Jalas An
T. holosericeus Celak. 2n = 28 Gr
T. laconicus Jalas Gr
T. lanceolatw Desf. Ag
T.leucostonzus Hausskn. and Velen. An
T. maroccanus Ball. subsp. nzaroccanw Ma
subsp. rhombicz~sHuguet del Villar Ma
T. numidicus Poiret Ag
T. pallescens de No6 (T. fontanesii) Ag
T, sipyleus Boiss. subsp. sipyleus An AE
subsp. rosulans (Borbis) Jalas
T. syriacus Boiss. An LS Iraq
VIII. Sect. Serpyllum (Miller) Bentham
VIII 1. Subsect. lnsulares Jalas
T. bornnzuelleri Velen. An
T. dredtensis Batt. Ma Ag
T. guyonii De Noe Ag
T. origanoides Webb and Berthelot 2n= 28 Canary Islands
T. richardii Pers. subsp. richardii 2n = 28, 30 Bl J u
subsp. ebusitanus (Font Quer) Jalas 2n= 30 B1
subsp. nitidus (Guss.) Jalas 2n = 28 Si
T. willkommii Ronniger 2n = 5 6 H s
VII12. Subsect. Kotschyani (Klokov) Jalas
T. ararati-minoris Klokov and Shost.
T. armeniacus Klokov
T.murkhotensis Malejev
T.punnonicus All. 2 n = 28, 35 URSS, China
T. podolicus Klokov and Shost.
T.przewulskii Kom.
T.sibthorpii Bentham 2n= 28
T. tfisienszs Klokov and Shost.
T. turczuninovii Serg.
VIII5. Subsect. Alternantes Klokov
T. ulpestris Tausch ex A. Kerner 2n = 28
T.alternuns Klokov
T.bihoriensis Jalas
T. bwchiunus Klokov and Shost.
T.caucusicus Willd. ex Ronniger
T.comosw Heuffel ex Griseb. and Schenk 2 n = 2 8 , 58
T. disjunctus Klokov URSS, China
T.froelichiunus Opiz 2n = 56
T.komarovii Serg. 2n = 24, 26
T. nummulurius M. Bieb.
T.oehmiunus Ronniger and Soska
T.pseudonummulariw Klokov and Shost.
T. pseudopulegioides Klokov and Shost.
T.pulchellus C. A. Meyer
T.pulegioides L. 2n = 28, 30
T. semigluber Klokov
VIII6. Subsect. Pseudomarginati (Braun ex BorbBs) Jalas
T. lineuris Bentham subsp. lineuris
subsp. hedgei Jalas
T.longicuulis C. Presl. subsp. longicuulis 2n=26, 28, 30, 50, 56, 58
subsp. chaubardii (Boiss. and Heldr. ex Reichenb. fil.) Jalas
T,nervosw Gay ex Willk. 2 n = 2 8 Ga H s
T.ocheus Heldr. and Sart. ex Boiss. An Bu G r Ju
T. pruecox Opiz subsp. praecox 2 n = 2 4 , 50, 54, 56, 58
subsp. skorpilii (Velen.) Jalas 2 n = 2 8 , 56
subsp.polytrichus (A. Kerner ex Borbas) Jalas 2n=28, 50, 54, 55, 56
subsp. britunnicus (Ronniger) Holub 2n = 28, 50, 54, 5 6
subsp. zygifor~nis(H. Braun) Jalas
subsp. grossheimii (Ronniger) Jalas
T.pulcherrimus Schur subsp. pulcherrimus 2n = 28, 56
subsp. curputhicus (Celak.) MBrtonfi
T. stojanovii Degen. Bu G r Ju
T.thrucicus Velen. 2 n = 28, 56, 58 A1 An Bu G r J u T u
VIII7. Subsect. Serpyllum
T.alatuuensis (Klokov and Shost.) Klokov
T. altuicus Klokov and Shost. URSS, China
T.amurensis Klokov URSS, China
T. arsenijevii Klokov
T.aschurbujevii Klokov
 The history, botany and taxonorrzy ofthe genus Thymus 37
T. asiaticus Serg . 2n = 26
T. bitzlnzinosus Klokov
T. bucharicus Klokov
T.cerebrzfoliw Klokov
T. chancoanw Klokov
T. crenulatus Klokov
T. curtus Klokov URSS, China
T.diminatus Klokov
T. diversifolius Klokov
T. erawinensis Serg.
T.eubajcalensis Klokov
T. extremus Klokov
T. flexilis Klokov
T. glacialis Klokov
T. iljinii Klokov and Shost.
T. inaequalis Klokov URSS, China
T.jenisseensis I1j in
T. mandschuricus Ronniger 2n= 24 China
T. minussinensis Serg.
T.mongoliczls Klokov URSS, China
T. narymensis Serg.
T. nervulosw Klokov URSS, China
T.ochotensis Klokov
T. oxyodontus Klokov 2n= 24, 28
T. phyllopodus Klokov 2n = 24
T.proxinzus Serg. URSS, China
T. quinquecostatus Celak. 2n = 24, 26 China, Japan
T. reverddttoanw Serg.
T. schischkinii Serg.
T. seravshanicus Klokov
T. serpylhm L. subsp. serpylhm 2 n = 24, 26
subsp. tanaensis (Hyl.) Jalas 2n = 24
T. sibiricus (Serg.) Klokov and Shost.
T. sokolovii Klokov
T. talijevii Klokov and Shost.
T. tonsilis Klokov
T. usszlriensis Klokov

Appendix
List of hybrids in the Iberian Peninsula. Hybridization occurs frequently in the Iberian
Peninsula, where 60 hybrids have been detected and some of them described.
T. baeticzls Boiss. ex Lacaita x T. hyemalis Lange
T. x indalicus Blanca, Cueto, GutiCrrez and Martinez, Folia Geobot. Phytotax 28: 138
fig. 1 (VIII-1993)
T. x garcia-martinoi Sinchez G6mez and Siez in SaCz, Sinchez G6mez and Morales,
Anales Jard. Bot. Madrid 51(1): 158 (XII-1993)
T. baeticus Boiss. ex Lacaita x T. nzastichina (L.) L. subsp. mastichina
T. x arundanus Willk., Oesterr. Bot. Z. 41: 52 (1891), pro sp.
T.xfontquerianus Pau, Mem. Mus. Ci. Nat. Barcelona, Ser. Bot. 1(1): 61 (1922)
T. baeticus Boiss. ex Lacaita x T. zygis subsp. gracilis (Boiss.) R. Morales
T. xarcanus G. L6pez and R. Morales, Anales Jard. Bot. Madrid 41(1): 94 (1984)
bracteatus Lange ex Cutanda x T. nzastichina (L.) L. subsp. mastichina
x bractichina R. Morales, Anales Jard. Bot. Madrid 43(1): 37 (1986)
xpectinatus R. Morales, Anales Jard. Bot. Madrid 41(1): 94 (1984) non Fischer and
Meyer, nom. illeg.
T. x rivas-molinae Mateo and M. B. Crespo, Rivasgodaya 7: 130 (1993)
T.xsennenii Pau var. leucodonthus Pau, Bol. Soc. Aragonesa Ci. Nat. 15: 160 (1916), nom. inval.
T. x sennenii auct. ilon Pau
T. bracteatus Lange ex Cutanda x T. pulegioides L.
T. bracteatus Lange ex Cutanda x T. zygis Loefl. ex L. subsp. zygis
T. x borzygis Mateo and M. B. Crespo, Thaiszia, Kosice 3(1): 7 fig. 2 (1993)
T. caespititius Brot. x T. nzastichina (L.) L. subsp. nzastichina
T. x henriquesii Pau, BrotCria, SCr. Bot. 22: 121 (1926)
T.canzphoratus Hoffmanns. and Link x T. 77zastichina (L.) L. subsp. vzastichina
T, x ranzonianw Paiva and Salgueiro, Anales Jard. Bot. Madrid 52(1): 114 fig. 2 (1994)
T. carnosus Boiss. x T. nzastichina (L.) L. subsp. nzastichina
T.x welwitschii Boiss., Diagn. PI. Orient. 3(4): 9 (1859), pro sp.
T. noeanus Rouy, Bull. Soc. Bot. France 52: 507 (1905)
T. funkii Cosson x T. vulgaris L. subsp. vulgaris
T. x lainzii SBnchez G6mez, FernBndez JimCnez and SBez in SBnchez G6mez and
FernBndez JimCnez, Anales Jard. Bot. Madrid 54
T. funkii Cosson x T. zygis subsp. gracilis (Boiss.) R. Morales
T. xparadoxus Rouy, Bull. Soc. Bot. France 20: 78 (1883)
T. granatensis Boiss. subsp. granatensis x T.longfirus Boiss.
T. x alnzijajrdrensis Ruiz de la Torre and Ruiz del Castillo, Ecologia 6: 103 fig. 2(1992), pro sp.
T.granatensis Boiss. subsp. granatensis x T.serpylloides subsp. gadorensis (Pau) Jalas
T.granatensis Boiss. subsp. granatensis x T. orospedanw Huguet del Villar
T. x lnariae Socorro, ArrCbola and Espinar, Lagascalia 16(1): 121 (1991)
T. hyemalis Lange x T. nzastichina (L.) L. subsp. mastichina
T,x nzastichinalis SBnchez Gdmez and SBez in SBez, Sinchez G6mez and Morales, Anales
Jard. Bot. Madrid 51(1): 158 (1993)
T. hyenzalis Lange x T. nzoroderi Pau ex Martinez
T. x diazii Alcaraz, Rivas Martinez and SBnchez G6mez, Itinera Geobot. 2: 118 (1989)
T. hyemalis Lange x T. vulgaris subsp. aestivus (Reuter ex Willk.) 0. Bol6s and A. Bol6s
T, hyenzalis Lange x T. zygis subsp. gracilis (Boiss.) R. Morales
T.x enicensis Blanca, Cueto, Gutikrrez and Martinez, Folia Geobot. Phytotax. 28(2):
138 fig. 2 (VIII-1993)
 The history, botany and taxononzy ofthe genus Thymus 39
T. x sorianoi SBez and SBnchez G6mez in SBez, Sinchez G6mez and Morales, Anales Jard.
Bot. Madrid 5 l(1): 158 (XII-1993)
T.lacaitae Pau x T . vulgaris L. subsp. vulgaris
T.x arnzuniae R. Morales, Anales Jard. Bot. Madrid 41(1): 94 (1984)
T. lacaitae Pau x T.zygis subsp. sylvestris (Hoffmanns. and Link) Coutinho
T. x arcuatus R. Morales, Anales Jard. Bot. Madrid 41(1): 93 (1984)
T. leptophyllus subsp. izcoi (Rivas Martinez, Molina and Navarro) R. Morales x T. nzas-
tichina (L.) L. subsp. nzastichina
T. x celtibericus Pau, Mem. Real. Soc. Esp. Hist Nat. 15: 7 1 (1929)
T. leptophyllus subsp. izcoi (Rivas Martinez, Molina and Navarro) R. Morales x T.vulgaris L.
subsp. vulgaris
T. x moralesii nothosubsp. navarroi (Mateo and M. B. Crespo) R. Morales, Anales Jard.
Bot. Madrid 53(2): 208 (1995)
T. x navarroi Mateo and M. B. Crespo, Rivasgodaya 7: 132 (1993)
T, leptophyllus Lange subsp. leptophyllus x T. mastichina (L.) L. subsp. mastichina
T. x celtibericus nothosubsp. bonichensis (Mateo and M. B. Crespo) R. Morales, Anales
Jard. Bot. Madrid 53(2): 202 (1995)
T. x bonichensis Mateo and M. B. Crespo, Thaiszia, Kosice ?(I): 5 fig. 1 (1993)
T. leptophyllus Lange subsp. leptophyllus x T. vulgaris L. subsp. vulgaris
T. x nzoralesii nothosubsp. cistetorunz Mateo and M. B. Crespo, Anales Jard. Bot. Madrid
49(2): 288 (1992)
T. leptophyllus Lange subsp. leptophyllus x T.zygis Loefl. ex L. subsp. zygis
T. x xilocae Mateo and M. B. Crespo, Anales Jard. Bot. Madrid 49(2): 289 (1992)
T. leptophyllus subsp. paui R. Morales x T. pulegioides L.
T. x benitoi Mateo, Mercadal and Pisco, Bot. Complutensis 20: 70 fig. 1 (1996)
T. leptophyllus subsp. paui R. Morales x T. vulgdris L. subsp. vulgaris
T.x nzoralesii Mateo and M. B. Crespo in Mateo, Cat. F1. Teruel: 234 (1990)
T. longzflorus Boiss. x T. zygis subsp. gracilis (Boiss.) R. Morales
T. x rz~iz-latorveiC. Vicioso in Ruiz del Castillo, Anales Inst. Nac. Invest. Agrar., Ser.
Rec. Nat. 1: 31 lam. 16 (1974), pro sp.
T. loscosii Willk. x T. nzastichina (L.) L. subsp. nzastichina
T. x riojanus Uribe-Echebarria, Est. Mus. Ci. Nat. Alava 5: 67 fig. 1 (1990)
T. loscosii Willk. x T. vulgaris L. subsp. vz~lgaris
T. x rubioi Font Quer, Treb. Mus. Ci. Nat. Barcelona, Ser. Bot. 3: 2 15 (1920)
T. lotocephalus G . L6pez and R. Morales x T.nzastichina subsp. donyanae R. Morales
T. x nzourae Paiva and Salgueiro, Anales Jard. Bot. Madrid 52(1): 114 fig. 1 (1994)
T. nzastichina (L.) L. subsp. nzastichina x T. mastigophorus Lacaita
T. x ibericu Sennen and Pau in Sennen, Bull. Acad. Int. Gkogr. Bot. 18 (229): 461 (1908)
T. nzastichina (L.) L. subsp. mastichina x T. orospedanw Huguet del Villar
T.x mzxtzls Pau, Carta Bot. 3: 7 (1906)
40 Ram6nMomles

T. mastichina (L.) L. subsp. mastichina x T. praecox subsp. britannicus (Ronniger) Holub

T. x genesianus Galin Cela, Anales Jard. Bot. Madrid 45(2): 562 fig. 1 (1989)
T. mastichina (L.) L. subsp. mastichina x T. pulegioides L.
T. x sennenii Pau, Bol. Soc. Aragonesa Ci. Nat. 6: 29 (1907)
T. jovinieni Sennen and Pau in Pau, op. cit.
T. mastichina (L.) L. subsp. mastichina x T. serpylloides subsp. gadorensis (Pau) Jalas
T. x hieronymi Sennen, Diagn. Nouv. P1. Espagne Maroc: 92 (1936)
T. mastichina (L.) L. subsp. mastichina x T. serpylloides Bory subsp. serpylloides
T. x hieronymi nothosubsp. hurtadoi (Socorro, Molero Mesa, Casares and Perez Raya)
R. Morales, Anales Jard. Bot. Madrid 43(1): 39 (1986)
T. x hurtadoi Socorro, Molero Mesa, Casares and PCrez Raya, Trab. Dep. Bor. Univ.
Granada 6: 109 (1981)
T. mastichina (L.) L. subsp. mastichina x T. villosus subsp. lusitanzcus (Boiss.) Coutinho
T. x toletanus Ladero, Anales Inst. Bot. Cavanilles 27: 97 fig. 6 (1970)
T. mastichina (L.) L. subsp. mastichina x T. vulgaris L. subsp. vulgaris
T. x eliasii Sennen and Pau in Sennen, Bol. Soc. Iberica Ci. Nar. 32: 79 (1933); in Pau,
Cavanillesia 4: 55 (193 I), nom. inval.
T. mastichina (L.) L. subsp. mastichina x T. zygis subsp. sylvestris (Hoffmanns. and Link)
Coutinho
T. x brachychaetus (Willk.) Coutinho, Bol. Soc. Brot. 23: 7 9 (1907), pro var.
T. mastichina var. brachychaetus Willk. in Willk. and Lange, Prodr. F1. Hispan. 2: 400 (1968)
T,x nzixtus var. toletanus Pau, Bol. Soc. Aragonesa Ci. Nat. 15: 160 (1916)
T,mastichina (L.) L. subsp. mastichina x T, zygis Loefl. ex L. subsp. zygis
T. mastigophorus Lacaita x T. vulgaris L. subsp. vulgaris
T. x severZdnoi Uribe-Echebarria, Est. Mus. Ci. Nat. Alava 5: 69 figs. 3a y 4 b (1990)
T. nzastigophorus Lacaita x T. zygis Loefl. ex L. subsp. zygis
T. x zygophorus R. Morales, Anales Jard. Bot. Madrid 41(1): 93 (1984)
T. membranaceus Boiss. x T. moroderi Pau ex Martinez
T. membranaceus Boiss. x T. orospedanus Huguet del Villar
T. x beltranii Socorro, Espinar and ArrCbola, Lagascalia 17(1): 186 (1993)
T. membranaceus Boiss. x T.vulgaris L. subsp. vulgaris
T.x guerrae SBez and Siinchez G6mez in SBez, SBnchez G6mez and Morales, Anales Jard.
Bot. Madrid 51(1): 157 (1993)
T. membranaceus Boiss. x T.zygis subsp. gracilis (Boiss.) R. Morales
T. x almeriensis G . L6pez and R. Morales, Anales Jard. Bot. Madrid 41(1): 94 (1984)
T. moroderi Pau ex Martinez x T. vulgaris L. subp. vulgaris
T, x carrionii SBez and SBnchez G6mez in Siiez, Siinchez G6mez and Morales, Anales
Jard. Bot. Madrid 51(1): 157 (1993)
T. moroderi Pau ex Martinez x T. zygis subsp. gracilis (Boiss.) R. Morales
T. x rizdrtinezii Pau ex Martinez, Mem. Real Soc. Esp. Hist. Nat. 14: 467 fig. 7 (1934), pro sp.
 The history, botany and taxonomy ojthe genus Thymus 41
T. funkii var. martinezii (Pau ex Martinez) C. Vicioso, Anales Inst. Nac. Invest. Agrar.,
Ser. Rec. Nat. 1: 19 (1974)
T. capitatas Lag., Elench. PI.: 1 8 (1816), non (L.) Hoffmanns. and Link (typus: MA
106457)
T.villosus sensu Willk., Suppl. Prodr. F1. Hispan.: 146 (1893)
T.orospedanw Huguet del Villar x T. zygis subsp. gracilis (Boiss.) R. Morales
T. x jimenezii Socorro, ArrCbola and Espinar, Lagascalia 16(1): 122 (1991)
T. piperella L. x T. vulguris subsp. uestivus (Reuter ex Willk.) 0.Bol6s and A. Bolds
T. x josephi-ungeli Mansanet and Aguilella, Mediterrinea, Ser. Biol. 8: 84 (1985)
T. piperella L. x T.vulgaris L. subsp. vulgaris
T. x josephi-angeli nothosubsp. edetanus Mateo, M. B. Crespo and Laguna, Anales Jard.
Bot. Madrid 49(1): 140 fig. 1 (1991)
T.pulegioides L. x T. vzllguris L. subsp. vulgaris
T. x carolipaui Mateo and M. B. Crespo in Mateo, Cat. F1. Teruel: 232 (1990)
T. pulegioides L. x T. zygis subsp. grucilis (Boiss.) R. Morales
T. pulegioides L. x T, zygis Loefl. ex L. subsp. zygis
T. x viciosoi Pau ex R. Morales, Anales Jard. Bot. Madrid 53(2): 210 (1995)
T. x viciosoi (Pau) R. Morales, Anales Jard. Bot. Madrid 43(1): 4 1 (1986), comb. inval.
T. bracteatus f. viciosoi Pau, Bol. Soc. Aragonesa Ci. Nat. 15: 159 (1916), nom. inval.
T.serpylloides subsp. gadorensis x T.vulgaris subsp. aestivus
T. x uitunue nothosubsp. dominguezii (Socorro and ArrCbola) R. Morales, Anales Jard.
Bot. Madrid 53(2): 200 (1995)
T. x dominguezii Socorro and ArrCbola, Lagascalia 17(2): 35 5 (1995)
T. serpylloides subsp. gadorensis x T.vulguris subsp. vulgaris
T. xaitanae Mateo, M. B. Crespo and Laguna, Anales Jard. Bot. Madrid 49(1): 142 fig. 3 (1991)
T. serpylloides subsp. gadorensis (Pau) Jalas x T. zygis subsp. gracilis (Boiss.) R. Morales
T. x pustoris Socorro and Arrebola, Lagascalia 17(2): 3 5 3 (1995)
T. valgaris L. subsp. vulgaris x T. zygzs Loefl. ex L. subsp. zygis
T. x monrealensis Pau ex R. Morales, Anales Jard. Bot. Madrid 41(1): 93 (1984)
T. xmonrealensis Pau, Mem. Real Soc. Esp. Hist. Nat. 15: 71 (1929), nom. inval. sine descr.
T. vulgaris L. subsp. vulgaris x T,zygzs subsp. gracilis (Boiss.) R. Morales
T. x nzonrealensis nothosubsp. garcia-vallejoi Sinchez G6mez, Alcaraz and Siez, Anales Jard.
Bot. Madrid 49(2): 289 (1992)

ACKNOWLEDGEMENTS

Thanks are given to Juan Castillo and Leopoldo Medina for their drawings. This work
was made in part under the financial support of the Project Flora Iberica V PB96-0849
of the DGICyT, Spain, that has transferred some unpublished drawings. Part of this
work has been possible thanks to the 'Acciones integradas hispano-austriacas HU96-13
and HU1997-34' from Subdireccidn General de Cooperacidn International, Spain.
42 Rambn Morales
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Bauhin, C. (1623) Pinax tkeatri botanici, Basel.
Bentham, G . (1834) Lab. Gen. Sp.: Thymus, London.
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Boissier, E. (1839-1845) Voyage botanique duns le Midi de I'Espagne, Paris.
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2 Population structure and the spatial
dynamics of genetic polymorphism
in thyme
John D. Thompson

THE SPATIAL STRUCTURE OF GENETIC DIVERSITY I N THYME

Ecological and evolutionary significance of the population structure

Many plant species occur as a mosaic of local populations in discrete patches dispersed
across the landscape. In many populations, pollen and seed dispersal are highly local-
ised, increasing the tendency for reproduction to occur within spatially localised groups.
Spatial structure is thus a characteristic of plant populations. Where natural selection
favours particular genotypes in particular sites or spatial isolation permits random
genetic drift, genetic differentiation can be marked and highly localised.
Evolution at the level of the local population can also be influenced by regional pro-
cesses associated with the arrangement of populations in the landscape and the rates of
colonisation and extinction of such populations. In some cases, the colonisation of new
populations may involve a small number of genetically related individuals that are only
a subset of the genetic variation contained by source populations. Such founder events
can have a marked effect on the genetic variation within populations and also the
spatial organisation of the genetic variation in the landscape. A critical point here is
that such founder events may produce spatial variation in phenotypic traits which cause
their evolution to follow new directions in colonist populations.

Polymorphic variation i n thyme

The genus Thymus provides a particularly interesting situation to study the ecological
and evolutionary significance of the spatial population structure. Since the early 1960s,
one species, Thyrnw vulgaris has been at the heart of ecological and genetic research on
the evolutionary dynamics of not just one but two genetic polymorphisms (see review by
Thompson etal.,1998). First, like most labiates, thyme is an aromatic plant: glandular
trichomes on the leaves and floral parts contain monoterpenoid essential oils. Thyme plants
vary in the monoterpene composition of their essential oils, one monoterpene being
present in a high percentage for a particular plant. In the south of France, one of six
different monoterpenes may dominate the essential oil produced by a plant species, and
thus six different chemical forms can be detected. This secondary compound variation
has a genetic basis and the presence of six distinct genetically-based forms has thus
provided an attractive system to explore the ecological genetics of secondary compound
variation, in particular the role of this variation in mediating inter-specific interactions
and the determinants of variation in relative abundance across the landscape.
 Population structure and the spatial dynanzics in thynze 45
The second of the two polymorphisms is gynodioecy, a sexual polymorphism in which
natural populations contain two types of plants: females and hermaphrodites. Herma-
phrodites bear only perfect flowers whilst females bear smaller flowers that lack anthers
or have only rudimentary anthers that do not bear pollen. All flowers on a given plant
are either female or hermaphrodite. Darwin (1877) first remarked on the occurrence of
gynodioecy in the genus Thynzw, and since 1963, when Professor Valdeyron first started
counting and observing females in the garrigues landscape around Montpellier in
southern France, the functional significance and evolutionary dynamics of gynodioecy
in wild thyme has greatly intrigued researchers. The purpose of this research has been
to elucidate why females are so abundant in many populations and what causes variation
in their abundance among populations.
The purpose of this chapter is to review work on the ecology and evolution of the
chemical and sexual polymorphisms, with focus on the importance of the spatial popu-
lation structure in T. vz~lgdrzs,the species which has received by far the most attention.

THE ESSENTIAL OIL OF THYME: THE ECOLOGICAL GENETICS

OF A CHEMICAL POLYMORPHISM

The Mediterranean is full of aromatic plants. Data on the ecological role of the mono-
terpenes which characterise the essential oils of Mediterranean aromatic plants remains
however rather thin on the ground. Thymw vulgaris shows genetic variation in the pro-
duction of monoterpenes, providing a fascinating opportunity to study the ecological
role and evolutionary significance of monoterpene production.

The genetic basis of thyme monoterpenes

In southern France, natural populations of T.vulgurzs contain one or several of six
genetically distinct chemical forms (hereafter chemotypes) that can be distinguished on
the basis of the dominant monoterpene produced in glandular trichomes on the surface
of the leaves and calyces (Passet, 1971; Vernet etal., 1986). Each of the six chemotypes,
geraniol (G), a-terpineol (A), tr-sabinene hydrate or thuyanol-4 (U), linalool (L),
carvacrol (C) and thymol (T),is named after the dominant monoterpene in the essential
oil of a plant. Each monoterpene is at the end of a branch in a common reaction chain
that has as precursor, geranyl pyrophosphate (Figure 2.1). The six monoterpenes have
different molecular structures (Figure 2.1), with an important difference being the
phenolic nature of carvacrol and thymol and the non-phenolic nature of the other four
monoterpenes. In Spanish populations the geraniol chemotype has not been observed,
whereas a seventh chemotype, 1,8-cineole is present (Adzet etal., 1977). Some of the
chemotypes are discernible to the human nose in the field. This is particularly so for
geraniol which often has a lemon smell and the two phenolics which have the charac-
teristic thyme odour which makes them readily distinguishable from the four non-
phenolic types.
The first description of the essential oil variation in T. vulgaris occurred in the early
1960s (Granger etal., 1963) with a more comprehensive study arriving a few years later
in the form what was to be the first of a long line of PhD theses on this species in
Montpellier (Passet, 197 1). The construction of a chromatograph capable of determin-
ing the chemical phenotype of a plant from a small sample (3-4 leaves), permitted the
46 John D. Tho~npson

Monoterpene Dominant
synthesis monoterpene

Geranyl
pyrophosphate
/ Geraniol

Linalool

Neryl Tespinyl-8
pyrophosphate
alpha-terpineol

Thymol gg/nn/un/ll/cc

Figure 2.1 The biosynthetic synthesis and genetic control of the dominant monoterpenes in T, vzllgarir
in southern France (based on Passer, 1971; Vernet etal. 1986).

rapid determination of the chemotype for a large number of individuals (Passet, 1971;
Gouyon etal., 1981). This method of analysis made it possible to study the genetic
control and spatial distribution of the chemical forms, work which required an extensive
program of controlled crosses and natural population sampling, i.e. thousands of plants
(Gouyon etal., 1986; Vernet etal., 1986).
 Population stmrture and the spatial dynavzics in thyme 47
The presence of the dominant monoterpene in T. vulgaris is controlled by an epistatic
series of five biosynthetic loci that has the following sequence: G+ A + U +L +C + T
(Vernet etal., 1986). As can be seen in Figure 2.1, a plant with a dominant G allele will
have the G phenotype, regardless of whether it has dominant or recessive alleles at the
other loci (Vernet etal., 1986). Two loci probably code for the G phenotype, otherwise
a single pair of alleles at each locus codes for the remaining chemotypes. If a plant is
homozygous recessive at the G loci (i.e. gg) and has a dominant A allele then it will
have the A phenotype, again regardless of whether it has dominant or recessive alleles
at the other loci. If the plant is homozygous recessive at the G and A loci (i.e. gg, aa),
but has a dominant U allele then it will have the U phenotype, and so on down the
chain (Figure 2.1). A plant homozygous for recessive alleles at all five loci has the
T phenotype. Figure 2.1 also illustrates (see Passet, 1971 for details) that the metabolic
pathway leading to the production of the two phenolic chemotypes is much longer than
that of the non-phenolic and that there is an almost perfect fit between the genetic
chain and the metabolic chain, only linalool is "out of place". The basis of this non-
concordance remains unknown.
The most plausible explanation for the relation between genetic constitution and
dominant monoterpene (i.e. genotype and phenotype) is that there is a series of
regulatory proteins coded by alleles at the G, A, U, L and C loci, each of which can
interrupt, at different stages, the sequence of reactions that would normally lead to the
synthesis of the T phenotype (i.e. homozygous recessive for all loci). An alternative
possibility is that enzyme G may consume all the substrate for a particular reaction
causing chemotype G to be produced; likewise for the other chemotypes down the
chain.

Spatial structure a n d t h e adaptive value of thyme monoterpenes

The ecological role of secondary compounds in thyme can be addressed under three
headings: adaptation to the abiotic environment; competitive interactions with other
plants; and chemical defence against herbivores and pathogens. Clearly, these factors
are unlikely to act in isolation from one another, indeed the dynamics of the secondary
compound polymorphism are most likely influenced by the combined and interactive
effects of the different features of the abiotic and biotic environment.

Spatial distribution of chemotypes: an adaptation to the abiotic environment?

There are several pieces of evidence which indicate that the monoterpene variation may
represent an adaptive strategy in relation to environmental variation. The first piece of
evidence for adaptive variation concerns the geographic and localised distribution of
chemotypes in T. valgaris in southern France. Based on bulk samples of plants, it is clear
that phenolic chemotypes (C and T) dominate thyme populations in hot dry sites close
to the Mediterranean sea, whereas the non-phenolic (G, A, U and L) chemotypes dom-
inate sites further inland, particularly above 4 0 0 m elevation, i.e. in wetter, cooler
climates (Passet, 1971; Granger and Passet, 1973). This trend has been confirmed by
a recent study which compared the actual frequency of plants of each chemotype in
approximately 12 sites at low altitude (< 300 m) close to the Mediterranean with a similar
number of sites above 4 0 0 m in the Drome valley (J. Thompson and J.-C. Chalchat,
unpublished data). Above 400 m no phenolic plants were found.
48 John D. Thonzpson
Spatial differentiation in the distribution of the different chemotypes in T. vz~lgaris
also occurs on a very localised scale of 8 x l 0 k m in and around the St Martin-de-
Londres valley roughly 25 km north of Montpellier in southern France (see Vernet etal.,
1977a, b; Gouyon etal., 1986). This valley is reputed for the temperature inversion that
can occur in winter due to the accumulation of cold moist air in the valley. In winter,
temperatures are often several degrees colder than in the hills that surround the valley.
In the valley, soils are less stony, deeper and moister than in the surrounding hills.
Again based on bulk samples of plants, the climatic and soil gradient has been found to
be correlated with differences in the distribution of different chemotypes (Figure 2.2a and
b). Phenolic chemotypes predominate over large areas of hot, dry limestone plateau
areas on shallow, stony iron-rich soils around the valley above 250 m elevation (Figure
2.2b). Below 2 0 0 m elevation inside the valley, where populations are often fragmented
by agricultural land-use, there is a mosaic of smaller thyme populations where one or
two non-phenolic chemotypes are most abundant (Figure 2.2). This differentiation
occurs in about 1 km where altitude drops from 250 to 200 m.
In the 336 populations analysed by Gouyon etal. (1986) roughly 20 per cent (72
populations) only contained a single chemotype and 50 per cent (165) contained a mix-
ture of two chemotypes (Figure 2.3). For the 165 populations with two chemotypes, 72
had the two non-phenolic chemotypes, 69 had two phenolic chemotypes and only 24
had one non-phenolic and one phenolic chemotype. So, although populations with two
chemotypes are the most common (Figure 2.3), those with mixtures of phenolic and
non-phenolic chemotypes are relatively rare. In fact, populations containing both
phenolic and non-phenolic chemotypes (i.e. a combination of those with 2, 3, 4 , or 5
chemotypes) are rare (Figure 2.2b). All populations with appreciable percentages of
both phenolic and non-phenolic chemotypes occur either inside the elevation transition
between 2 0 0 m and 2 5 0 m (Figure 2.2b) or adjacent to it. Most mixed populations
out-side this transition zone tend to be dominated by non-phenolic chemotypes only
and are inside the bassin (Figure 2.2a). Selection or drift must be driving the evolution
of chemotype frequencies such that populations tend to have either phenolic or non-
phenolic forms, but not both. This differentiation between populations has also been
reported over a very small spatial scale of several metres (Mazzoni and Gouyon, 1985).
What is the cause of this sharp pattern of differentiation? Two main patterns of
correlation were commented on by Gouyon etal. (1986). First, marked variation in soil
type across the study region is correlated with the shift from phenolic to non-phenolic
types. Phenolic populations occur on drier, more stony fersiallitic soils than non-phenolic
chemotypes which predominate on deeper, moist soils. Within the non-phenolic types,
it is the a-terpineol chemotype which is most abundant on the wettest soils. For the
two phenolic types, carvacrol is limited to the driest conditions, whereas thymol is less
specialised and can occur on moist soils.
A second clear-cut correlation concerns the absence of phenolic types, particularly
carvacrol, from sites which experience sub-freezing temperatures, i.e. inside the valley
or as mentioned above, at elevations above 400 m further inland. As one drops into the
valley the shift is towards non-phenolic types, as one moves northwards around the
valley, carvacrol is replaced by thymol, with the shift occurring as one passes the Pic
St Loup where freezing temperatures increase in frequency and intensity in winter.
Although Varinard (1983) found no effect of freezing on the survival of seedlings of the
C, T and U chemotypes in controlled conditions, it is still possible that the non-phenolics
suffer less effects due to temperatures several degrees below zero than do phenolic types.
Ftgure 2.2 The spatial structure of T. vulgarzs chemotypes in and around the St Martin-de-Londres valley
in southern France. Data are from the original records compiled by P.-H. Gouyon and used in
Vernet etal. (1977a,b) and Gouyon etal. (1986). In (a) all six chemotypes are shown, black -
geraniol, blue - a-terpineol, green - thuyanol-4, white - linalool, red - carvacrol and yellow -
thymol. In (b) the data are simplified to contrast the distribution of the two phenolic
chemotypes (black circles) to that of the four non-phenolics (yellow circles). Each circle repre-
sents a population in which a bulk sample of ca. 3 0 plants was obtained (see the above cited
authors for details). Three contour lines, 1 5 0 m , 2 0 0 m and 2 5 0 m are shown to illustrate the
altitudinal segregation of the phenolic and non-phenolic types. (See Color Plate 1)
50 John D. Thonzpson

"
1 2 3 4 5 6
Number of chemotypes present

F i g t m 2.3 The frequency of populations with one or more chemotypes in and around the St Martin-
de-Londres valley in the south of France. Data are from Gouyon etal. (1986) and are the
same as those in F ~ g u r e2.2.

In TrzJalizlrn repens the genes responsible for the cyanogenesis polymorphism show a
latitudinal and altitudinal cline and one hypothesis proposed to explain this cline is
that freezing temperatures may, via the rupture of cell membranes, allow the release of
hydrogen cyanide (HCN) that is toxic to the plant (reviewed by Briggs and Walters,
1997). The possibility that phenolic chemotypes of thyme may be excluded from areas
with severe cold temperatures in winter due to the greater toxicity of the phenolic
molecules that may, following freezing and the rupture of cell membranes, cause mor-
tality during harsh winters is currently being investigated. Why plants with a carvacrol
phenotype suffer such effects more than those with a thymol chemotype is not known.
A comparative study of plant species in Mediterranean-type ecosystems has shown
that roughly 49 per cent of the species produce aromatic volatile oils and that these are
predominantly the evergreen, xeromorphic woody shrubs (like thyme) and not the
drought-avoiding annuals and deciduous species (Ross and Sombrero, 1991). Hence the
presence of essential oils is correlated with the persistence during the Mediterranean summer
"drought". In T. vzllgaris, data obtained in controlled conditions by Couvet (1982) sug-
gest that the non-phenolic A chemotype is significantly less resistant to drought and
hot temperature stress than the C, T and L chemotypes. Phenolic types may thus be
better adapted to dry (and hot) conditions. Non-phenolic essential oils ate vapourised
at lower temperatures than the phenolic oils (Couvet, 1982), hence it is possible that
they are less able to withstand high temperatures due to a toxic effect of vapourised oil
in the summer. However, it is difficult to conceive of an ecological role of the oils in
relation to drought stress. There is no evidence that the vapours of such essential oils
permit the regulation of leaf temperature or transpiration rate (Audus and Cheetham,
1940). What is more, the actual compounds that are vapourised may not be in the same
proportions as those detected in the plant (Seufert etal., 1995).
The spatial pattern of chemotype distribution in southern France primarily concerns
the identity of the two most abundant chemotypes at a given site and whether the site
is dominated by either phenolic or non-phenolic chemotypes (Vernet etal., 1977a,b;
Gouyon etal., 1986; Figure 2.2). The presence of one of the six chemotypes has only a
weak contribution to the spatial structure. This is not surprising, in a population dom-
inated by non-phenolic chemotypes, the genes which cause the carvacrol phenotype (C-)
and the thymol phenotype (cc) can be present even though their phenotype is not
 Population strzlctzlre and the sputiul dynumics in thyme 51
detected since their expression is masked by dominant alleles at the non-phenolic loci.
Crosses among plants heterozygous at the non-phenolic loci will produce offspring
with a phenolic chemotype. This may be why one may sometimes observe phenolic
plants at very low frequency (< 5 per cent) in populations dominated by non-phenolic
plants, but not vice versa (J.D. Thompson and J.-C. Chalchat, unpublished data).
Geraniol is the rarest of the six chemotypes in southern France (Granger and Passet,
1973; Gouyon etul., 1986), perhaps in part because it is the only gene that cannot "hide"
behind other phenotypes hence may be more frequently "lost" during episodes of coloni-
sation and extinction. In the study by Gouyon etul. (1986), none of the populations
contained all six chemotypes, three populations contained five chemotypes and 24 popu-
lations were represented by four chemotypes (Figure 2.3). All three populations with five
chemotypes lacked the geraniol chemotype. Of the 24 populations with four chemotypes,
22 also lacked the geraniol chemotype. In fact, when one distinguishes phenolic chemo-
types from non-phenolic chemotypes the frequency of absence of particular chemotypes
depends on their position in the epistatic chain: those chemotypes whose dominant genes
prevent the expression of genes at subsequent loci in the chain are the chemotypes most
frequently absent. For example, when one examines which non-phenolic chernotypes are
absent from populations with four chemotypes, geraniol is absent from 22 of the 24
populations, a-terpineol from six and thuyanol from five of the 24 populations.
In sharp contrast, linalool, the last of the non-phenolic chemotypes in the genetic
chain, is present in all 24 of these populations. A similar pattern is observed for the two
phenolic chemotypes, carvacrol is absent from 13 of these 24 populations and thymol is
absent from only 2 of the 24 populations. The combination of two chemotypes which
are the most often jointly absent from populations with four chernotypes is geraniol
and carvacrol (i.e. the most dominant gene for non-phenolic and phenolic chemotypes),
which are concomitantly absent from 11 of the 24 populations. Finally, for the 72
monomorphic populations, 16 were linalool, 16 carvacrol and 28 thymol, the three
chemotypes which are the most abundant in southern France (Gouyon etul., 1986;
Granger and Passet, 1973; J. Thompson and J.-C. Chalchat, unpublished data).
The data suggest that a combination of natural selection, which also acts at the level
of phenolic-non-phenolic distinction, and chance effects within the two groups of
chemotypes linked to the epistatic mechanism of genetic determination jointly
impinge on the abundance of the six chemotypes. The challenge will be to demonstrate
where and how natural selection acts on chemotype frequency by carefully replicating
transplantation in the field (see also Boursot and Gouyon, 1983) and controlled experi-
mentation of particular factors. It is also possible, as will be discussed in the rest of this
section, that interactions with the biotic environment also contribute to the spatial and
evolutionary dynamics of the chemical polymorphism.

Interactions on a single trophic level

In the context of potential interactions with other species, volatile oils may have a
negative "allelopathic" effect on the germination and growth of associated plant species
and in this way reduce competition with other species. The potential allelopathic
effects of monoterpenes and their role in structuring plant communities have been the
centre of much interest and critical discussion (Muller, 1969; Harper, 1977; Rice, 1979;
Williamson, 1990; Fisher, 1991). Bare zones under and around aromatic shrubs have
been remarked in different aromatic plant species (Muller etul., 1964; Katz etul.,
5 2 John D. Thompson
1987) and much work on the effects of monoterpenes on the germination and growth
of associated species has involved labiates in Mediterranean communities, e.g. Salvia
(Muller etal., 1964), Coridothymus (Vokou and Margaris, 1982) and Calaminth (Tanrisever
etal., 1988).
In the genus Thymus, there have been several investigations of the potential effects of
monoterpene exudates on seed germination and plant growth. In T. serpyllum, Paul
(1970) found that an aqueous extract of leaves and litter differentially inhibits the
germination and growth of different species. For four studied species, the extract from
T. serpyllum leaves had the strongest effect on Plantago ramosa which had significantly
reduced germination in the presence of the extract and which also was least abundant
where T. serpyllzlm occurred. Tarayre etal. (1995) tested the hypothesis that the different
essential oils produced by T. vulgaris, have different effects on the germination of the
grass Bromus phoenicoides, a common grass species in southern France. In a series of
controlled germination trials in petri dishes, these authors found that the two phenolic
chemotypes (C and T), when present in the form of leaves or as pure essence, have a
significantly greater inhibitory effect on the germination of the grass than do the non-
phenolic chemotypes. In the presence of phenolic leaves, the percentage germination of
the grass was around 75 per cent, and in the presence of non-phenolic leaves from 85-
90 per cent. Germination in the absence of thyme leaves was 90-95 per cent. Phenolic
chemotypes may thus be able to better resist competition from associated grasses than
non-phenolic chemotypes.
Using soil collected under thyme plants and away from thyme plants in phenolic and
non-phenolic populations Y. Linhart, P. Gauthier and J. Thompson (unpublished data)
studied the germination and growth of several different species that also occur with
thyme with and without a cover of thyme leaves from the same sites. In general, germin-
ation and growth tended to be lower on soil collected from under phenolic thyme
plants or in the presence of phenolic leaf litter. However, the effects of different chemo-
types on germination and growth varied across the range of associated species (one
Nigella, one Medicago, two Bromus, one Crepis and one Daucus species) used in the study.
So there clearly exists a potential for inhibitory effects on associated species germin-
ation and growth and for variation in such effects depending on the identity of the asso-
ciated species. In this context, it is interesting to note that in the study by Tarayre etal.
(1995) the percentage germination of the grass in the presence of thyme leaves always
exceeded 75 per cent, i.e. even in the presence of phenolic leaves this grass germinates
well. It would thus be most interesting to compare populations or species that occur in
association with thyme with others that do not occur in association with thyme, to
examine whether the latter shows greater inhibition in the presence of thyme leaf litter.
The different monoterpenes may also affect the germination of thyme seeds and sub-
sequent plant growth. Although the term "auto-allelopathy" has been used to describe
the effect of thyme monoterpenes on seed germination one should refrain from using
this term since there is some evidence that a temporal inhibition of germination may in
fact be an adaptive response to irregular germination cues in the form of a short-term
dormancy mechanism and not a toxicity phenomenon. It has been reported that aqueous
extracts from T. vulgaris (Tarayre etal., 1995) and Thymbra capitata (Coridothynzw capitatus)
(once Thynzw) (Vokou and Margaris, 1986; Thanos etal., 1995) can significantly delay
their own seed germination.
In Coridathynzw, germination was significantly slower in the presence of calyces (which
are the unit of dispersal and which contain the essential oil) than when seeds were
 Population structure and the spatial dynamics in thyme 51
germinated alone, an effect relieved by leaching of the essential oil over time (Thanos
etal., 1995). In T. vulgaris all six of the oils cause an inhibition of seed germination to
roughly 50 per cent that of seeds germinated in the absence of thyme monoterpenes
(Tatayre etal., 1995). At the end of the experiment (which was stopped due to fungal
growth in the petri dishes) germination in controls had finished whereas germination
in the presence of the different chemotypes continued (Tarayre etal., 1995). Hence the
inhibition effects may gradually wear off. This delayed rather than completely inhibited
germination has been suggested (see Tarayre etal., 1995; Thanos etal., 1995) to repre-
sent an adaptive response to the irregular germination cues experienced by mature
seeds of such Mediterranean shrubs in late summer when rainfall may be particularly
erratic and interspersed by extreme drought stress for small plants and seedlings. Such
inhibition could represent an evolved response to variation in cues for seed germination
(see Angevine and Chabot, 1979; Fenner, 1985).

Interactions across trophic levels: escape in space and thyme?

As a defence against the strong pressure imposed on them by herbivores, parasites and
pathogens, plants have evolved an immense diversity of chemical defences (Jones,
1962; Ehrlich and Raven, 1964; Bryant etal., 1991). The more diverse, the partners at
different trophic levels, the more important it may be to have a diverse, defence system.
Herein lies a clue to the reason why there may be so many chemotypes in T. valgaris:
spatio-temporal variation in the abundance of different potential herbivores, parasites,
etc. may lead to disruptive selection on chemical phenotype and thus contribute to the
maintenance of several forms.
The first piece of work which investigated the possible role of thyme monoterpenes
as a chemical defence against herbivores was that of Gouyon etal. (1983). These authors
found marked variation between chemotypes in their palatability to slugs: U was the
least palatable and A and C were the most palatable. Experiments with all six chemo-
types by Linhart and Thompson (1995) showed that snails (Helix aspersa) have a prefer-
ence for non-phenolics, particularly the L chemotype, and a marked distaste for the two
phenolics C and T (Table 2.1). What is more, the most deterrent monoterpenes to
snails, the phenolic C chemotype, caused snails fed on a diet of exclusively thyme plants
of this chemotype to lose weight. Interestingly, when L genotypes are at the seedling
stage (1-3 months old) their leaves do not have an L phenotype, they have a phenolic
(C or T) phenotype, and they only develop their "true" phenotype after this very young
seedling stage (Vernet etal., 1986). As suggested by Linhart and Thompson (1995), the
chemotype most preferred by snails (L) may thus "hide" behind a less palatable pheno-
type (C or T ) during early seedling development - a stage in the life cycle that is likely
to be critical for survival in the face of snail herbivory.
Investigation of feeding preferences in a range of herbivores have shown marked dif-
ferences in the rank order of feeding preferences in T. vulgaris, i.e. different chemotypes
vary in their ability to deter herbivore feeding, and different herbivores respond differ-
ently to the different chemotypes (Table 2.1). If one compares the palatability of the
chemotypes to molluscs and grasshoppers (experiments done almost simultaneously in
similar conditions) the deterrence ranks are completely reversed. What is tasty for a
snail is unpalatable for a grasshopper and vice versa. For micro-organisms, Simeon de
Bouchberg (1976) observed a similar reversal of deterrence; whereas the T chemotype
had the most severe effects on bacterial population growth, it was the G chemotype
54 John D. Thonzpson

Table 2.1 The rank order of deterrent effects of the six chemotypes of Thy7?zusvulgaris on
different potential herbivores and inhibitory effects on microbial population
growth and seed germination of an associated grass species. Chemotypes are
ranked from ( I ) least to (6) most deterrent; for chernotype codes see text

Herbivore Rank order of deterrence

Helm (snail)'
Derocerds (slug12
Leptophyes (grasshopper)'
Arima (chrysomelid bee-
t~e)~
Capra &oat)'
Ovic (sheep)'
Agriolimax (slug?
~un~i*
~acteria"
Brachypodiurn (grass)'

Notes
1 Llnhart and Thompson (1995);
2 L~nhartand Thompson (1999);
3 Gouyon et al. (1983) for four chemotypes;
4 Slrneon de Bouchberg (1976);
5 Tarayre etal. (1995).

that had the greatest impact on fungal growth. Elsewhere, the closely related Thymbra
capitata, which has similar monoterpene oils, has been reported to significantly influ-
ence soil microorganism activity in the soil (Vokou and Margaris, 1984). It would thus
be most worthwhile to examine how different chemotypes interact with soil organisms
and the potential feedback effects on plant growth.
A glance down Table (2.1) indicates that every chemotype can be the preferred chemo-
type depending on the component of the environment studied and that all but the
a-terpineol chemotype can be the most deterrent. A key point is that no one chemotype
provides the best defence across the spectrum of potential herbivores, pathogens, etc.
although in general the phenolic chemotypes do tend to be more deterrent than non-
phenolic chemotypes. There are differences in the abundance of different snail species
in phenolic and non-phenolic populations (Linhart and Thompson, 1995). Hence vari-
ation in the deterrence effects of the different chemotypes combined with variation in
the spatio-temporal abundance of different herbivores, parasites and pathogens could
influence the maintenance of the polymorphism in secondary compounds and contribute
to the spatial variation in their relative abundance (Linhart and Thompson, 1999), as
has been illustrated for secondary compounds in other species (Linhart, 1989).
In fact, the different facets of the biotic environment may act in concert with spatial
variation in the abiotic environment (see above) to influence the maintenance of the
spatial structure in the chemotype polymorphism. In one of the 12 doctoral theses on
thyme done in Montpellier, Pomente (1987) reported that phenolic (T and C) plants
had a better tolerance of drought stress, that the U chemotype was particularly favoured
in humid conditions, and that the presence of an associated grass was correlated with
a decrease in the survival of thyme plants in conditions of drought stress. This effect of
grass presence was not an effect of competition, but rather because the grass maintained
 Population structure and the spatzal dynamics in thynze 55
a more humid environment in which slugs sheltered and subsequently caused a greater
mortality of thyme plants. The U chemotype grew best in humid conditions (Pomente,
1987) and is also the chemotype most deterrent to slugs (Gouyon etal., 1983).

Gene Flow Versus Selection

If one wishes to study the spatial structure of plant populations it is necessary to inves-
tigate, and distinguish between, potential gene flow, i.e. the dispersal of pollen and
seeds, and effective gene flow, which depends on the fertilisation of seeds and seed
establishment (Levin and Kerster, 1974). When effective gene flow is significant, the
development of the spatial structure of the genetic variation is limited, populations
will show more genetic homogeneity. This is unless natural selection is strong enough
to overcome the effects of such gene flow.
What do we know about gene flow among thyme populations dominated by different
monoterpenes? To examine this question Tarayre and Thompson (1997) conducted a
study of the spatial structure of several polymorphic isozyme loci by protein electro-
phoresis of 25-50 individuals in each of 23 populations of T. vulgaris representing
a range of the phenolic and non-phenolic populations in Figure 2.2. Despite the signifi-
cant differentiation of chemotype abundance across populations, isozymes showed
relatively low levels of population differentiation. A mean F,, value of 0.038 indicated
that less than 4 per cent of the genetic variation for the loci studied was due to population
differentiation. Even along three transects which ran from 100 per cent non-phenolic to
100 per cent phenolic populations, F,, values did not exceed 0.06.
Pollen transfer distances are likely to be very small in the study region, most insect
visitation is by honey bees which fly almost exclusively between adjacent plants
(Brabant etal., 1980; Rolland, 1999). However, many butterfly species (Lepidoptera)
visit thyme in the study region and since they travel larger distances between plants
than do bees, the potential for pollen flow among populations exists. Indeed, Tarayre
etal. (1997) show that gene flow in pollen is much greater than that via seed, attesting
to at least occasionally important pollinator movements.
The data of Tarayre and Thompson (1997) provide strong support for previous work
(Gouyon etal., 1987) which illustrated that, although limited migration can occur
between phenolic and non-phenolic populations, there is little "effective" gene flow for
the genes coding for the monoterpenes. When gene flow occurs, the chemical genes will
be transported in either the pollen and seed that moves among populations. If genetic
drift were responsible for the patterns of chemotype distribution, then we would have
expected isozymes to show higher levels of genetic differentiation among populations,
since the random nature of genetic drift is likely to act equally on all genes. Selection,
in contrast, only acts on those genes of adaptive significance (although it may secondarily
cause variation in the frequency of other genes linked to those under selection). The
data, at present, strongly suggest that natural selection on the chemical phenotypes
maintains the spatial pattern observed in the study region. What is more, selection
appears to be particularly strong.

Future directions
There are clearly several avenues of work that are urgently needed for a clear under-
standing of the ecological significance of the chemical polymorphism in T. vulgaris.
56 John D. Thonzpson
First, the different chemotypes show marked variation in how they interact with the
complexity of factors that determine the biotic and abiotic environment in which
plants grow. What may be crucial to the dynamics of the chemical polymorphism is
that the interaction of thyme monoterpenes with the biotic environment (herbivores,
parasites, competitors) varies depending on the feature of the environment studied
(Linhart and Thompson, 1999). Thus there is the potential for spatial (and temporal)
variation in such biotic interactions to contribute to the maintenance of this genetic
polymorphism. Documenting the extent to which such interactions occur and vary in
the field represents a major challenge for future work. Not only could such interactions
play a key role in the dynamics of the chemical polymorphism, they could also translate
into effects on the structure and diversity of garrigues plant communities. The study of
the chemical polymorphism in the field is thus a model system to do both population
biology and community ecology, illustrating the often under-appreciated link between
these two fields.
Second, several potentially important factors remain completely unstudied. One that
comes immediately to mind is that the different monoterpenes may not have the same
energy requirements for their production. The evolution of many polymorphisms that
involve resistance to a particular environmental feature can be greatly influenced by
what is known as the "cost of resistance". In the absence of the selective feature of the
environment, a resistant genotype incurs a fitness cost associated with the presence of
the resistance gene it carries. In the absence of the particular feature of the environment
that favours resistance genes, non-resistant types will be favoured. In thyme, although
phenolic chemotypes may be favoured because of a more generalised toxicity to herbi-
vores, the phenolic molecules may be more costly to produce since they are further down
the biosynthetic chain of production and thus requires more enzyme and precursor
synthesis.
In fact, plants with a phenolic phenotype have only 50-70 per cent of their oil dom-
inated by thymol or carvacrol, whereas the non-phenolic chemotypes G, L and A regularly
have >80 per cent of their oil composed of their dominant monoterpene (Passet, 1971;
J. Thompson and J.-C. Chalchat, unpublished data). The cost of production could thus
be greater for the two phenolic molecules, causing a fitness cost to phenolic-based
chemotypes where their selective agents are absent. Alternatively, the resistance cost
incurred by phenolic plants may as mentioned above involve a lack of freezing tolerance
imposed by the toxicity of the phenolic molecules. Future research should thus consider
the possible importance of a cost to monoterpene production and the variation in this
cost among chemotypes.
Third, since aromatic plants are such an essential component of the current day
Mediterranean flora, a feature which would well merit attention in future research is
the possible role of the essential oil variation in speciation and adaptation of new spe-
cies to different environments. The dominant monoterpenes in Thymw vary (a) among
the populations of individual species in different environments and (b) among different
species across the range of the genus. In T. vulgaris it is even possible to observe vari-
ation on this theme of six chemotypes, a seventh form, based on 1,8-cineole occurs in
Spain, where the geraniol chemotype has not been observed (Passet, 1971). Whether
this pattern is due to hybridisation with other species containing this molecule in Spain
or to a selective elimination of the geraniol chemotype in Spain and the 1,8-cineole
chemotype in France merits close attention, as does the position of 1,8-cineole in the
genetic and metabolic pathways.
 Population structure and the spatial dynamics in thyme 57
Finally, in addition to the dominant monoterpene which characterises their essential
oil, thyme plants may also contain a second monoterpene in a non-negligeable propor-
tion. There are several examples of this phenomenon. The oil of carvacrol plants may
contain up to 15 per cent thymol where plants are heterozygous at the C locus (Vernet
etal., 1986). In a recent survey, of approximately 100 plants having a linalool or
geraniol phenotype, four were found to have equal amounts of the two compounds,
while three out of twenty a-terpineol plants also contained 15-30 per cent thymol
(J. Thompson and J.-C. Chalchat, unpublished data). The oil of both carvacrol and
thymol chemotypes frequently contains up to 30 per cent of their two precursors,
a-terpinene andp-cymene (Passet, 1971;J. Thompson and J.-C. Chalchat, unpublished
data). Finally, the thuyanol-4 chemotype is actually a mixture of several compounds
(Passet, 197 1).
Such variation is not just background noise, it no doubt reflects developmental
variation and adjustments, the study of which could provide a more concrete basis
for understanding the precise link between genes and physiology. What triggers the
co-occurrence of different molecules in some plants compared to a more pure oil in
others? What is the relative resource cost of producing different molecules? Where and
how do genes switch on and off the different elements of the monoterpene chain of
synthesis? The answer to such questions may not only provide more precise information
on the genetic control of chemotype in relation to the metabolic pathway of mono-
terpene synthesis, but could, as more species are studied, provide interesting insights
into the genetics of adaptation and diversification in aromatic plants.

GYNODIOECY I N THYME: THE POPULATION GENETICS

OF A SEXUAL POLYMORPHISM

Introduction:high and variable sex-ratios

In his book entitled "The different forms of flowers on plants of the same species" Charles
Darwin (1877) remarked on the occurrence of two types of plants in a species of the
genus Thymus in southern England. One type of plant bore only perfect flowers, with
both male and female functions. The second type had smaller flowers that had no or
completely reduced anthers and were thus completely male-sterile. This co-existence of
hermaphrodite and female plants in a single population is called gynodioecy.
The presence of gynodioecy in the genus Thynzw is associated with two intriguing
observations: first, there is enormous variation in female frequency; and second, mean
female frequency is very high, considerably higher than in populations of most other
gynodioecious species. In T . vulgarzs, populations contain 5-95 per cent females
(Assouad etul., 1978; Dommke etal., 1978; Manicacci etal., 1998) with a mean around
6 3 per cent. The frequency of females is extremely variable and on an average very high
in T. zygis (mean: 5 1 per cent, range 17-87 per cent) and T. mastichinu (mean 72 per
cent, range 34-88 per cent) in central Spain (Manicacci etal., 1998). In the clonal
T. serpyllum a mean of 67 per cent females and a range of 30-95 per cent has been
observed in six populations in south-central France (J.D. Thompson, unpublished
data). Female frequencies above 50 per cent have also been observed in T . sibthorpii in
Greece ( K . Kateradi pers. comm.), T. albicans in South-West Spain (Valdgs etul., 1998)
and T. x arundanw in South-East Spain (J. Arroyo, pers. comm.). So in several different
58 John D. Thompson
species, which occur in different ecological conditions, in different parts of the geo-
graphic range of the genus and in different taxonomic sections of the genus (Motales,
1986), mean female frequency is very high (above 50 per cent) and can be extremely
variable. . . why?

Why so many females: the importance of the spatial

population structure
In a gynodioecious population, females do not produce pollen and therefore do not
transmit genes to the next generation via male function. In thyme, as in many gynodi-
oecious species, sexual phenotype is governed by a complex interaction between nuclear
and cytoplasmic (mitochondrial) genes. The maternally inherited cytoplasmic genes inhibit
male function, causing the sexual phenotype to be female. The effect of these cytoplasmic
genes can be repressed by nuclear alleles at particular loci which restore male function,
causing the individual to be hermaphrodite. The sexual phenotype of an individual will
thus depend on the combination of its nuclear and cytoplasmic genome. In T. vulgaris,
the genetic determination of sexual phenotype is extremely complex and involves several
cytoplasmic types restored by a range of dominant and recessive restorer alleles which
may show epistatic interactions (Belhassen etal., 1991; Charlesworth and Laporte, 1998).
Theoretical models predict the maintenance of a nucleo-cytoplasmic polymorphism
at loci involved in sex determination as a result of either a cost of restoration caused by
negative pleiotropic effects of restorer genes in cytoplasmic backgrounds they do not
restore (Gouyon etal., 1991) or, in the absence of such a cost, due to the spatial structure
of cytoplasmic and restorer genes (Couvet etal., 1998). In the extreme case of spatial
structure, a cytoplasmic male-sterility type may occur in a population where the nuclear
alleles that restore its male-fertility are absent. This situation causes sex determination
to be cytoplasmic and all the offspring
- - of the female will be female. As the classic models
of ~ e w i (1941)
s predict, this cytoplasmic sex determination will allow female frequency
to rise as soon as females produce slightly more offspring than hermaphrodites. Couvet
etal. (1986, 1998) propose (see scenario in Figure 2.4) that nucleo-cytoplasmic sex
determination may vary in space and time. Sex determination may be locally cytoplasmic
due to founder events during colonisation of new sites causing the absence of restorer
alleles for the cytoplasmic types that are present. Subsequent arrival of nuclear restorer
genes, via pollen or seeds, will later permit a decline in female frequency (Figure 2.4).
Strong evidence supporting this hypothesis came with the observation that female
frequencies often attain very high values in young populations that are actively colonising
new sites after either disturbance (e.g. forest fire) or the abandon of agricultural practice
(DommCe etal., 1983; Belhassen etal., 1989). Seeds can resist up to 7 5 OC, hence popu-
lations can rapidly establish after a fire (Belhassen etal., 1987). After this period of
population establishment (usually around 10-1 5 years), the frequency of females appears
to decline (Belhassen etal., 1989). In young populations actively colonising new sites,
the presence of dense patches (3-4 m in diameter) each composed of exclusively female
plants are often observed (Manicacci etal., 1996). This development of female patches
causes female frequency to be locally very high, attaining 95 per cent in extreme cases.
The individuals within a patch tend to have a single cytoplasmic type, both for
mitochondrial (Manicacci etal., 1996) and chloroplast (Tarayre etal., 1997) DNA, but
patches less than 1 0 m apart can have different cytoplasmic DNA profiles. It is thus
probable that each patch originates from a single female or sibling females from a
 Population strz~ctzlreand the spatial dyna7nics ilz thyme 59

"mismatch" of cytoplasmic sterility genes and

nuclear restorer aleles = cytoplasmic sex determination

\ arrival of nuclear restorer genes via pollen

0 4 8 12 16
Population age

Figure 2.4 A schematic illustration of how a shift in the genetic control of sex may be related to
female frequency variation In thyme populations.

common mother plant. The hermaphrodite that pollinated these mother plants must
have lacked the restorer alleles for the maternal cytoplasm, allowing female patches to
develop around the original mother plant (note that there is no mechanism for long-
distance seed dispersal in thyme). Females produce 2-3(5) times more seed than herm-
aphrodites in natural populations (Assouad etul., 1978; Couvet etul., 1986) allowing
the rapid development of such patches in young populations. So there is evidence that
founder events may occur and cause reduced cytoplasmic diversity, which in turn permits
high female frequency in colonist populations.
A comparison of population differentiation for cytoplasmic and nuclear genes has
shown that pollen migration is many times more frequent than seed migration among
thyme populations (Tarayre etul., 1997), hence the possibility that nuclear restorer
genes may arrive via pollen and thus cause female frequency to decline as populations
become older. The number of migrants per generation (Nm) among populations
(estimated from Fs, values) was 1.6 and 11.65 for the cytoplasmic D N A (cpDNA) and
allozyme markers respectively, indicating that gene flow through pollen is roughly
1 4 times that for genes dispersed in seeds among the studied T. vulguris populations.
Within a single population where female patches are monomorphic for their cpDNA
haplotype, N m values for gene flow among patches and the surrounding (roughly 10 m
away) more continuous cover of thyme are 0.42 and 12.91 for cpDNA and allozymes
respectively. Hence, even at the scale of several metres, pollen migration greatly exceeds
seed migration. It is therefore reasonable to assume that restorer genes will arrive via
migration in pollen whilst the founder effect on cytoplasmic genes may persist longer
during the life of a population.
There are several lines of evidence for spatial variation in the frequency of nuclear
restorer genes. First, experimental pollination of plants in an insect-free glasshouse by
60 John D.Thompson
Couvet etal. (1985a) and Belhassen etal. (1991) have shown that male fertility restor-
ation is greatest when females are pollinated with pollen from a hermaphrodite present
in their original population compared to when the pollen source is a hermaphrodite
from a different population. This result suggests that the restorer gene frequency is
variable among populations and that hermaphrodites carry restorer genes adapted to
local cytoplasmic male-sterility types.
Second, Manicacci etal. (1997) cloned five different females that had previously been
found to show different rates of male fertility restoration when pollinated with a range
of hermaphrodites, i.e. they have functionally distinct cytoplasmic male-sterilities.
These authors placed the clonal replicates back into the five original populations from
which the cytoplasmic types had originally been sampled. The females were allowed to
flower and were then returned to the experimental garden where the seeds produced by
pollination in each population were collected and sown. The sex ratio of the offspring of
each female in each population (25 sex ratios in total) was determined the following
flowering season. Marked variation in percentage restoration between populations was
observed for three of the females (Figure 2.5), suggestive of spatial variation in the
abundance of different restorer genes. Two of these three females had a percentage
restoration that was greatest when transplanted into their original population, in agree-
ment with the results of Couvet etal. (1985a) and Belhassen etal. (1991) who suggest
that restorer genes are selected in populations that contain the associated male-sterility.
Interestingly, femaleph was relatively well restored in her home population PH and,
to a lesser extent, in the PB population, the closest other population (ca. 1 km away).
In the three other populations distant by more than 10 km the restorer genes for this
cytoplasm were virtually absent, and thus appear to have a very localised spatial occurrence

Maternal female

Figure 2.5 The percentage of male fertility restoration in the offspring of five females reciprocally
transplanted among five original populations in southern France (redrawn with permis-
sion from Manicacci etal. 1997). Values in parentheses represent the mean percentage of
hermaphrodites in the offspring of either each maternal female (pooled across the five
populations) or for each population (pooled across the five females).
 Popzllation strzlctzlre and the spatial dynafnics in thyme 61
in and around the P H population. A different pattern was however observed in two of
the females (pb and f ) which showed a high percentage restoration in all five populations
(Figure 2.5). There are several potential causes for the lack of variation in the restoration
rates for these two females. First, the cytoplasmic male-sterility carried by these females
may be restored by different restorer alleles which are present in different populations.
Second, some restorer alleles may be very common across populations in the absence of
their associated male-sterility cytoplasm.
Third, the cytoplasmic male-sterility represented by these two females may be present
in the other populations (note that only one cytoplasm was investigated per population)
and as a result their nuclear restorer alleles are also present. Fourth, some cytoplasmic
male-sterilities may be restored by generalist restorer alleles. A fifth interpretation of this
result is particularly appealing. Sex expression is generally observed as a qualitative
phenomenon in thyme, there being females and hermaphrodites. However, the restor-
ation of male fertility may also have a quantitative component since several restorer
genes are probably involved in the determination of male function. If this is the case,
plants of each sex may be more or less close to a threshold which determines the func-
tional sexual phenotype. The two females in the study by Manicacci etal. (1997) that
show consistently high restoration rate across populations may be closer to this threshold
than the three other females.
It has long been known in thyme that some female flowers bear reduced male struc-
tures and that it is possible to classify females according to the phenotypic expression of
stamen reduction. Some females have no visible stamens (type D); some have a small
swelling on the corolla with a very short filament (type C). A third group has a more or
less well-developed filament and anther (type B) (Assouad, 1972, Thompson et al.,
2002). All flowers on a given plant are the same, none of the females produce pollen,
and female offspring are dominated by females with the same type of male fertility
reduction as the mother plant after controlled crosses and in a field population (Dommge,
1973).
What is more, there is a gradient in flower size and rate of restoration in the off-
spring of the different types of females from those with the most reduced male structures
which have the smallest flowers and produce a more female-biased offspring than those
with the most well-developed stamens that have flowers similar in size to those of
hermaphrodites and which produce much less female-biased progenies in the field and in
controlled pollination (J. Thompson and B. Domme'e, unpublished data, Thompson
et al., 2002). The two females in the study by Manicacci etal. (1997) with a high and
stable rate of restoration could thus be females of type B while those with variable rates
of restoration in their offspring may be females of types C or D.
The idea that female frequency is high in young populations due to founder events
causing a mis-match of the cytoplasmic sterility genes and nuclear restorer genes plus
high levels of seed set on females, but declines as populations age following the immi-
gration of nuclear restorer alleles that restore the male fertility of the cytoplasmic types
present in the founding population thus has some support. Other factors may however
also contribute to the pattern of sex ratio variation in natural populations of T. vulgaris.
First, at high female frequency females may suffer pollen limitation and reduced seed
set due to the low numbers of hermaphrodites present, this causing their frequency to
subsequently decline. Second, if colonist females form patches of females via the estab-
lishment of their exclusively female offspring, then their offspring, are likely to be
pollinated by the same hermaphrodite which pollinated the colonist female, i.e. their
6 2 John D. Thompson
father. Such mating among related individuals could cause female offspring to suffer
inbreeding depression.
A study of rates of biparental inbreeding in females that occur in four populations
with very different sex ratios has not however found any evidence that biparental
inbreeding actually occurs on females (M. Tarayre and J. Thompson, unpublished manu-
script). However, Thompson and Tarayre (2000) found a negative effect of biparental
inbreeding on female seed set, hence this hypothesis cannot be completely dismissed.
Third, since females produce many more seeds than hermaphrodites (see below),
their abundance may decline if they suffer a reproductive cost that causes them to have
shorter life span than the hermaphrodites that are also present during colonisation. W e
are currently quantifying the survival of related females and hermaphrodites to test this
possibility. The issue of what causes variation in female frequency is thus complex,
with local variation in sex determination being of paramount importance. The observed
sex ratio variation may nevertheless be greatly enhanced by differences in the produc-
tion of viable offspring by females and hermaphrodites. This so-called female fertility
advantage is the subject of the next section.

The advantages of being female

The natztve of female fitness advantuge

In thyme, as in most gynodioecious species, females produce more viable seeds than
hermaphrodites, 2-3(5) times than that of hermaphrodites, although this female advan-
tage may vary among populations and years (Assouad etul., 1978; Couver etul., 1986).
Female offspring can also be more vigorous than that of hermaphrodites (Assouad etul.,
1978). Survival of the two forms appears to be equal (Assouad, 1972), although this
needs verification over the full life cycle of thyme plants in natural populations. Differ-
ences in the production of viable offspring by females and hermaphrodites may result
from an "outcrossing advantage" due to the fact that females cannot self andlor a "resource
compensation" advantage due to the fact that females may be able to re-allocate
resources (not spent on producing pollen) to seed production.

The outcrossing advantage

Females cannot self, hence their offspring will not suffer inbreeding depression due to
selfing. Hermaphrodites are self-compatible, hence, although they bear protandrous
flowers in which the anthers dehisce before the stigmas are receptive (Assouad, 1972),
may self-pollinate due to pollen movement between flowers on a given plant. An indi-
vidual thyme plant can bear many hundreds of open flowers at a given moment and
since the most important pollinator of T, vzllgaris, the honey bee Apis nzelliferu, tends
to visit many flowers on a plant during each visit (Brabant etul., 1980; Rolland,
1999), high levels of selfing may occur. In fact, selfing rates vary among popula-
tions (Valdeyron etul., 1977; M. Tarayre and J. Thompson, unpublished manuscript)
with the highest rates of selfing in populations with the highest female frequencies
(Table 2.2).
Such a positive correlation between female frequency and hermaphrodite selfing rate
could, as suggested by Sun and Ganders (1986) be interpreted as evidence that gyno-
dioecy may be maintained due to its positive effect on outcrossing. However, as we
 Population structure and the spatial dynanzics in thyme 63

Table 2.2 Variation in hermaphrodite selfing rate in relation to

female frequency observed in two studies of natural
populations of T. uulga~2Jin southern France

Sex ratio Selfing rate

(% females)

Valdeyron et al. (19.77)

Le Vigan
Les Chenes
Pic St Loup
Viols le Fort
Thompson et dl. (2002)

point out elsewhere (M. Tarayre and J. Thompson, unpublished manuscript), it is also
possible that hermaphrodites show higher rates of selfing when females are abundant
simply because of problems of pollen transfer resulting from the reduced abundance
and spatial isolation of hermaphrodite plants in such populations. An important point
here is that the selfing rate on hermaphrodites is very low (< 10 per cent) in populations
with female frequencies of up to 6 0 per cent. Only above 60 per cent females does the
selfing rate on hermaphrodites become significant (Table 2.2). Female frequencies thus
reach their mean value in the study region (ca. 60 per cent) in the absence of significant
rates of selfing on hermaphrodites. Problems associated with selfing would thus not
appear to be driving the evolution of female frequencies in thyme populations, in sharp
contrast to what many authors have argued for other gynodioecious species. Neverthe-
less, once female frequencies do attain high levels, selfing on hermaphrodites may con-
tribute to the maintenance of high female frequencies, if selfing is followed by inbreeding
depression.
Where selfing does occur on hermaphrodites in thyme, females may have a fitness
advantage due to inbreeding depression (the reduced fitness of selfed progeny relative
to outcrossed progeny). Assouad etal. (1978), Bonnemaison etal. (1979), Perrot etal.
(1982) and Thompson and Tarayre (2000) have all found that inbreeding depression
negatively affects the performance of selfed offspring in thyme. A re-analysis of previ-
ous data (Thompson etal., 1998) showed that, when quantified over several stages of
the life-cycle, i.e. seed production, seed germination and seedling size, inbreeding
depression can be extremely high (0.818). This would suggest that selfed progeny con-
tributes little to the next adult generation. In the Tourrii.re population where we have
recently detected significant selfing on hermaphrodites (Table 2.2), the inbreeding
coefficient (Fi,=O. 05) is not significantly different from zero (Tarayre and Thompson,
1997), indicating that selfing contributes few offspring to the mature adult generation.
This result has important bearings on the heterozygosity levels and on the spatial
structure of natural populations. The theoretical models of Gouyon and Couvet (1987)
predict that, for a constant hermaphrodite selfing rate and (at least some degree of)
cytoplasmic inheritance of sex, as female frequency increases, the heterozygosity of local
populations should increase. A study of isozyme variation for one enzyme system along
64 John D. Thompson

Proportion of females

Figure 2.6 Heterozygosity values (1 - FIs) for 23 populations of T. vulgarir in and around the St Martin-
de-Londres valley in southern France. The populations occur across the area shown in
Figure 2.2. Redrawn with perm~ssionfrom Tarayre and Thompson (1997).

a series of adjacent successional populations of thyme has illustrated that heterozygosity

levels do decline as female frequency declines along the succession (Dommee etal.
1983). Using the chemotype genes as markers, Gouyon and Vernet (1982) found that
females are more frequently heterozygous than hermaphrodites at the C locus in a sin-
gle population.
However, Bonnemaison (1980) found that this result was not common to three studied
populations and that females and hermaphrodites had similar heterozygosity values for
the L locus in all three populations. In a more recent study by Tarayre and Thompson
(1997) of several isozyme loci in 23 populations, no evidence for a correlation between
heterozygosity and sex ratio was found (Figure 2.6), and in general adult females and
hermaphrodites had similar levels of heterozygosity. The lack of a positive correlation
between female frequency and population heterozygosity in this study was primarily
due to high levels of heterozygosity in two populations with very low ( < 2 0 per cent)
female frequencies (Figure 2.6), probably due to low levels of selfing on hermaphrodites
(Table 2.2).
In populations with many hermaphrodites, females produce high percentages of
hermaphrodites (Manicacci etal., 1997), hence most hermaphrodites will be produced
by females and thus may have heterozygosity values similar to those of females. So, as a
result of variation among populations in both the rate of selfing and male fertility
restoration, heterozygosity values do not decline at low female frequencies. Furthermore,
any selfing that does occur can be followed by inbreeding depression, allowing heterozy-
gosity levels of adult hermaphrodites to remain high and similar to those for females.
It must also not be forgotten that although females cannot self, they may incur
biparental inbreeding, at levels similar to hermaphrodites, due to mating with related
individuals. Since pollination is primarily by honey bees which fly among adjacent
plants and since seed dispersal is low (Belhassen etal., 1987; Tarayre etal., 1997), pollin-
ation in natural populations is often likely to be among related plants. In a recent
study, females in three populations out of four showed biparental inbreeding depres-
sion on viable seed production when crossed with hermaphrodites of the same family
(Thompson and Tarayre, 2000). This only occurred for hermaphrodites from one of the
populations, hence female seed production may be more negatively influenced by bipa-
rental inbreeding than that of hermaphrodites. In other words, the effects of inbreeding
 Population structure and the spatial dynamics in thyme 65
may not simply be to produce a female outcrossing advantage, but in fact may reduce
the female seed fertility advantage in population contexts where biparental inbreeding
occurs.
So although outcrossing is likely to be important in the ecological adaptation of
thyme to different environments around the Mediterranean basin (Bonnemaison, 1980;
Gouyon and Vernet, 1982) and may contribute to the female fitness advantage in some
populations, it is clearly not the principal cause of female fertility advantage. What is
more, the evolution of high female frequencies in thyme populations is not likely to be
driven by an outcrossing advantage of females, since hermaphrodites only self at sig-
nificant rates when female frequencies are already at very high levels. This provides
further support for the hypothesis that stochastic effects on sex determination are behind
the pattern of sex ratio variation observed in thyme populations.

Resource compensation and sexual specialisation

A second possible cause of female seed fertility advantage is that females may re-allocate
resources (otherwise used to produce pollen) to seed production and provisioning. In
T. vulgaris, Atlan etal. (1992) reported negative correlations between the male (full
pollen grains per flower) and female (germinating seeds per fruit) fertility of hermaph-
rodites grown in uniform garden conditions. In the plants sampled from one popula-
tion, hermaphrodites that were the progeny of females produced small amounts of
pollen but relatively high numbers of seed, whilst hermaphrodites that were the progeny
of hermaphrodites produced more pollen but fewer seeds. Pomente (1987) has docu-
mented genetic variation in pollen production by hermaphrodites, hence the potential
for the evolution of male fertility exists.
Based on a comparison of seed number per fruit in open-pollinated plants ad
controlled crosses, Couvet etal. (1985b) illustrate that hermaphrodites abort 45 per
cent more seeds than females, and that less than 20 per cent of seed abortion can be
attributed to selective embryo maturation. Low fruit set in hermaphrodites would
appear to be primarily determined by sexual selection and subsequent specialisation in
male function. What is more there is a genetically-based variation in the degree of
sexual specialisation and relative female fertility for a range of families from four popu-
lations (Thompson and Tarayre, 2000).
Selection on the functional gender of hermaphrodites may also be imposed via the
female frequency in a population. If female frequency in a population remains stable
long enough to act as a selective force on hermaphrodite resource investment; resource
allocation theory (Lloyd, 1976; Charnov, 1982) makes two predictions. First, for herm-
aphrodites, relative allocation to male function (e.g. number of viable pollen grains as a
function of seed-set) would be positively correlated with female frequency in a popula-
tion. As there are more females, hermaphrodites with greater male function would be
favoured. Second - and following the previous prediction - viable seed production of
females relative to hermaphrodites should also increase with female frequency.
In a study of three Thymus species, T. vulgaris, T. mastichina and T. zygis, Manicacci
etal. (1998) found evidence for a correlation between female frequency and either
hermaphrodite male function or the relative fecundity of females across six populations
of each species. However, on an average hermaphrodites were better males, and females
better females, in the species with the highest female frequency. In orher words, among
the three species, an increase in sex ratio was correlated with both an increase in seed-set
66 John D. Thompson
on females and an increase in the relative allocation of resources to pollen (compared to
seeds) in hermaphrodites.
The lack of any correlation across populations may have several explanations. First,
relative seed set on females may not increase with their frequency because of frequency-
dependent effects on seed-set, notably pollen limitation. In a recent study of female
fertility advantage of females and hermaphrodites from four populations of T. vulgaris
grown and pollinated under controlled conditions, no evidence for the predicted evolu-
tion of gender at high female frequencies was observed (Thompson and Tarayre, 2000),
confirming the data for natural populations in Manicacci etul. (1998). The lack of
greater female fertility advantage at high female frequency is thus not likely due to a
problem associated with pollen limitation in the wild. Second, Manicacci etul. (1998),
propose that thyme populations are often subject to disturbances which cause extinction
and re-colonisation to be particularly frequent. Selection may thus not have time to
precisely adjust the functional gender of hermaphrodites at the population level.
Third, Manicacci etul. (1998) also suggest that where sex ratios are female-biased
this may in fact cause hermaphrodites with a female-biased gender to be maintained.
The argument for this is that since hermaphrodites with a male-biased gender will
pollinate more females they will not contribute to the maintenance of hermaphrodites
in the population since they are unlikely to restore male function on these females
(otherwise female frequency would already be low!). Hence the only hermaphrodites
contributing to the next generation of hermaphrodites will be those with a female-biased
gender that set seed and in doing so produce the next generation of hermaphrodites.
Finally, the results of Gigord etul. (1999) which suggest a positive correlation
between the frequency of hermaphrodites in a given progeny and the male (and female)
function of hermaphrodites of that progeny may contribute to the lack of increased
male function for hermaphrodites in populations with a female-biased sex ratio. These
authors report that hermaphrodites in families with many hermaphrodites may produce
more pollen per flower than hermaphrodites in families with a high female frequency.
Selection due to a high female frequency would thus be constrained by lower male per-
formance of hermaphrodites in progeny with a high female frequency. Important to
note here is that progeny sex ratios ate positively correlated with population sex ratios
(Manicacci etal., 1997). If this genetic constraint on gender variation is real, it could
also contribute to prevent the evolution of dioecy from gynodioecy in Thymus, a genus
where there are many gynodioecious but no dioecious species.
Inherent differences in seed fertility exist between females and hermaphrodites. The
differences do not concern inflorescence and flower production which is similar for the
two sexes (Assouad, 1972; Bonnemaison, 1980). The differences do not vary in relation
to sex ratio and are not just due to inbreeding depression on the seed fertility of
hermaphrodites. Assoauad (1972) first observed that female seed fertility exceeded that
of hermaphrodites even on outcrossing. He also observed that pollen adherence on female
stigmas was greater than that on hermaphrodite stigmas, suggesting that the difference
is not just a resource compensation effect but more the result of specialisation of sex
functions in the two morphs.
In a recent study of females and hermaphrodites from four populations of T. vulgurzs
that vary in sex ratio from 11-80 per cent females, it has also been found that even
when plants are outcrossed with pollen from hermaphrodites of a different population,
female seed fertilty is at least twice that of hermaphrodites (Thompson and Tarayre,
2000). Given that selfing rates on hermaphrodites in three of these populations are
 Population structare and the spatzal dynanzics zn thyme 67
close to zero (Table 2.2). It would appear that the seed fertility advantage of females has
little to do with inbreeding depression and is more likely the result of sexual specialisa-
tion andlor resource compensation. An interesting feature of the results of this study is
that the seed fertility advantage of females relative to hermaphrodites showed variation
across families. In some families this seed fertility advantage was fairly high, while in
others much lower. The precise combination of cytoplasmic and nuclear genes responsible
for sex determination may thus also impinge on quantitative variation in sexual function
within each of the two sexual phenotypes.

Flower size dimorphism: f r o m pollination biology t o t h e genetics

of sex determination
As in most gynodioecious species (see review by Delph, 1996), female flowers of
T. vulgaris are smaller than hermaphrodite flowers (Assouad, 1972). This flower size
dimorphism shows a variation which has implications for both the pollination ecology
of thyme and out understanding of the genetics of sex determination in this species.
In natural populations, flower size varies significantly between the two sexual pheno-
types, among populations and also as a result of an interaction between population and
sexual phenotype (Thompson et al. 2002). In other words, the difference in flower size
between the sexual morphs varies in extent across populations, primarily because
female flower size varies to a greater extent across populations than does hermaphrodite
flower size. For the same populations, the relative proportion of bees and butterflies
visiting thyme also varies markedly suggesting that pollinator-mediated selection may
contribute to differences among populations (Rolland, 1999). A study of the F, progeny
of three populations by Thompson et al. (2002) confirmed that there is a population-
sex interaction in homogeneous conditions, due to greater genetic variation among
females in different populations. Within populations, these authors also detected
a family-maternal sex interaction, due to greater genetic variation of females among
families than among hermaphrodites of different families.
Although Rolland (1999) found that hermaphrodites are more attractive to bee
pollinators than females, she did not detect any effect of flower size within females on
bee attraction. Although a range of populations and different pollinators should be
studied, it would appear reasonable to conclude that the functional significance of
variation in female flower size is not related to pollinator-mediated selection.
An alternative explanation for the variation in female flower size among families and
populations is that this variation is related to the genetic determination of sex, in
particular to differences in the degree of restoration of females in different families and
populations. As mentioned above, several types of female phenotypes can be observed
depending on the degree of stamen reduction. Thompson et al. (2002) found that
females with larger flowers have a more or less well-developed filament and anther
(type B females), whilst females with the smallest flowers have no visible trace of
stamens (type D females). Females which have a small swelling on the corolla where the
filament would normally be fixed (type C females) have intermediate flower size. These
are the different female phenotypes previously described above which give different sex
ratios in their offspring. It is thus possible that populations or families with large-
flowered females may contain predominantly females of type B whilst populations and
families with small-flowered females are dominated by females of type D.
68 John D. Thompson
Perspectives: t h e importance of variation within sexes
Gynodioecy in thyme is controlled by a complex interaction of nuclear and cytoplasmic
genes. The difference in inheritance of cytoplasmic and nuclear genes causes the selective
pressures acting on the genes they contain to fundamentally differ: selection for femi-
nising genes in the maternally-inherited cytoplasm is in conflict with selection for
restorer genes in the nucleus (Couvet etal., 1990). A key point here is that the precise
determination of sex in a local population may vary in space and time causing the sex
ratio to show high levels of variation and allowing for unusually high female frequen-
cies. In thyme populations, the random impact of founder events on the relative distri-
bution of cytoplasmic and nuclear genes and differences in gene flow via pollen and
seeds among established populations may greatly contribute to this pattern of sex ratio
variation.
The seed fertility advantage of females is primarily due to sexual specialisation and
has very little to do with any outcrossing advantage. This female seed fertility advantage
may contribute to the local population structure by allowing the rapid development of
high female frequencies in colonist situations. Gynodioecy may thus be a key parameter
in the colonising ability of T. valgaris, a species which rapidly establishes in early succes-
sional habitats.
W e do not know how many functionally different male-sterilities exist in natural
populations nor whether nuclear restorer genes are highly specific to particular cyto-
plasms or whether they can restore different cytoplasmic male-sterility types. Particularly
an interesting point concerns whether or not male fertility restoration involves a quan-
titative effect of restorer genes on male function. The presence of a range of different
female phenotypes that vary in the degree of stamen reduction, flower size and rate of
offspring restoration represents particularly an intriguing aspect of variation in sex
expression in this species. The study of these different female types will provide a useful
tool for advancing our knowledge of the genetics of sex expression and the spatial
dynamics of gynodioecy in thyme.
Unfortunately, we only have a faint inkling of the relationship between spatial vari-
ation in restorer gene frequency and how selection may act on such genes. The variation
in the frequency of some restorer genes among thyme populations (Figure 2.5) is higher
than what one would predict given that population differentiation for nuclear isozyme
variation among populations in the same zone (F,,=O. 038) is relatively low (Tarayre
and Thompson, 1997). The greater population differentiation for some restorer alleles
may be due to selection on particular restorer alleles in certain environments. The
marked population structure for cytoplamsic genes (Tarayre etal., 1997) could provide
the selective context for particular restorer genes once they arrive in a population (Couvet
etal., 1998).
Why then do some restorer genes (for example those that restore the Jandpb females
in Figure 2.5) have a more widespread distribution than others (i.e. those that restore
the ph, m and I j females in Figure 2.5)? Is it because that the latter three cytoplasmic
types are in fact present in the different populations or because these cytoplasmic types
are close to the threshold necessary for the restoration of male fertility? This is a distinct
possibility: females that have well-developed anthers do tend to produce more hermaph-
rodites than females that have no visual male structures. The former may thus incur
a fitness cost since the latter are producing (female) offspring that will set at least twice
as many seeds as the (hermaphrodite) offspring of the former. In fact females with no
 Population structure and the spatiaI dynamics in thyme 69
trace of any anther development are the most common in natural populations (Assouad,
1972; J.D. Thompson, unpublished data).
One would also predict that females that bear reduced anthers would set less viable
seed than females with no trace of male organs. Furthermore, females with no trace of
male organs are the most abundant female type, such populations should have the
highest female frequency and a smaller mean flower size than populations with a low
female frequency (and a higher frequency of type B females). Whether or not the differ-
ent female types are caused by the expression of different cytoplasmic male-sterility
types, the expression of a gradient of male fertility restoration, or the result of an inter-
action between the cytoplasmic type and the nuclear gene is the subject of ongoing
experimental work.
Hermaphrodites also vary in their sex expression, both among each other (Thompson
and Tarayre, 2000) and over time, plants having a male-biased pattern of resource
allocation early in flowering and a more female-biased pattern of resource allocation
later in flowering (Manicacci, 1993). The functional significance of such genetic and
environmental (developmental) variation will be interesting to study. So a shift in
emphasis, from studying differences between females and hermaphrodites, towards an
appreciation of the relevance of sex variation in sexual phenotype and functional gender
will be a key component of the future work necessary for us to make further advances in
our understanding of the population structure and genetics of gynodioecy in thyme.

CONCLUDING REMARKS: SEX, MONOTERPENES A N D THYME

In the early 1960s, chemical variation in T. vulgaris began to attract the attention of
ecologists and geneticists with the report that this species had a variety of different
chemical forms (Granger etal., 1963). At roughly the same time, a discussion between
L. Emberger and G . Valdeyron led the latter to start counting the frequency of female
plants in natural populations around Montpellier. Since then, as this chapter attests,
the two kinds of polymorphism have been the focus of continued research on the spatial
dynamics of polymorphic variation in thyme.
A question that is often asked when one talks about the two kinds of polymorphism
in thyme concerns whether or not there is a link between sexual and chemical pheno-
types or more subtly between the patterns of variation observed for each polymorphism.
There is in fact evidence for a subtle relationship. Gouyon etal. (1986) found that
although 61 per cent of populations with more than 50 per cent hermaphrodites are
predominantly phenolic populations, only 34 per cent of populations with less than 50
pet cent hermaphrodites are populations dominated by phenolics.
What may cause populations with many hermaphrodites to tend to be dominated by
phenolic chemotypes and populations with high female frequencies to be predominantly
of the non-phenolic type? Gouyon and Vernet (1982) provide data which suggests
that hermaphrodites are more homozygous than females and that the correlated high
frequencies of hermaphrodites and phenolic chemotypes may be due to inbreeding.
Recent work however shows no evidence for greater homozygosity of hermaphrodites
(Tarayre and Thompson, 1997). Another possibility, currently under study, is that
females with a phenolic phenotype may have lower fertility andlor lower survival than
both hermaphrodites with a phenolic chemotype and females with a non-phenolic chemo-
type due to the greater resource cost of phenolic molecules.
70 John D. Thompson
Finally, the study of the spatial population structure has necessitated the develop-
ment of two different approaches. To understand the spatial dynamics of gynodioecy has
required a population genetics approach in which stochastic effects on gene frequency
play a key role. In contrast, the chemical polymorphism continues to provide a classic
example of how an ecological genetic approach (the study of selection versus drift and
variation across populations) can be used to elucidate the multiple selective factors that
may influence the dynamics of a genetic polymorphism. But in both cases, understand-
ing the dynamics of polymorphic variation has required recognition that plants occur
in patches, which form mosaics of local populations.
Each local population experiences the selective forces of the environment and the
regional processes of gene flow dynamics associated with the colonisation and extinction
of individual populations and occasional migration between established populations.
The dynamics of the two thyme polymorphisms is the result of an intricate balance
between the processes acting at these different levels of the spatial population structure.
Other Thymus species probably show similar patterns of variation as those discussed
here. Further study of these species will provide interesting comparative examples with
which to examine the general significance of the spatial population structure for the
evolution of genetic polymorphism in thyme.

ACKNOWLEDGEMENTS

I am particularly grateful to Isabelle Litrico who compiled Figure 2.2, and to Domenica
Manicacci, Yan Linhart, Anne-Gaelle Rolland, and Perrine Gauthier for their helpful
discussion of the manuscript.

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Varinard, 0. (1983) Contribution 2 l'ktude d u polymorphisme chimique et sexuel chez les
espi.ces vCg6tales, D.E.A., Universitk de Montpellier 2, Montpellier.
Vernet, P., Guillerm, J.L. and Gouyon, P.H. (1977a) Le polymorphisme chimique de Thymw
vulgaris L. (Labike) I. Repartition des formes chimiques en relation avec certains facteurs
6cologiques. Oecol. Plant., 12, 159-1 79.
Vernet, P., Guillerm, J.L. and Gouyon, P.H. (1977b) Le polymorphisme chimique de Thymus
vulgaris L. (Labike) 11. Carte 2 l'echelle 1125000 des formes chimiques dans la region de Saint-
Martin-de-Londres (Herault-France). Oecol. Plant., 12, 181-1 94.
Vernet, P., Gouyon, P.H. and Valdeyron, G . (1986) Genetic control of the oil content in T h y m u
valgaris L.: a case of polymorphism in a biosynthetic chain. Genetics, 69, 227-231.
Vokou, D. and Margaris, N.S. (1982) Volatile oils as allelopathic agents. In N. Margaris, A.
Koedam, and D. Vokou (eds), A7,omatic Plants : Basic and Applied Aspects. Martinus Nijhoff
publishers, The Hague, Boston, London, pp. 59-72.
Vokou, D. and Margaris, N.S. (1984) Effects of volatile oils from aromatic shrubs on soil micro-
organisms. Soil Biol. Biochem., 16, 509-5 13.
Vokou, D. and Margaris, N.S. (1986) Autoallelopathy of Thymus capitatus. Oecol. Plant., 7 , 157-163.
Williamson, G.B. (1990) Allelopathy, Koch's Postulates, and the Neck Riddle. In J.B. Grace
and T . Tilman (eds), Perspectives on Plant Competition. Academic press, Harcourt Brace Jovanovich,
Publishers, pp. 143-162.
3 Essential oil chemistry of the genus
Thymas - a global view
E lisabeth Stahl-Biskup

INTRODUCTION

The subject of plant chemistry has developed enormously in the last four decades and
this has been due to the increasingly successful identification of organic molecules in
minor quantities by means of sophisticated chemical techniques. It has also been due to
the awareness that secondary metabolites have a significant role in the complex interaction
occurring between plants and animals or plants and plants in their exposition to the
environment. Economic and medicinal interests as well as taxonomical studies, all three
in quest of new natural products, have always been the strongest stimulants for research in
plant chemistry.
Concerning the genus Thymus, we can state that its chemistry is fairly well known at
least concerning the two main classes of secondary products, the volatile essential oils on
the one hand and the polyphenols, especially the flavonoids, on the other hand. Both,
essential oils and flavonoids, are mainly responsible for the pharmacological activities
of Thymus plants (Simeon de Bouchberg etal., 1976, Van den Broucke, 1983).
Traditionally essential oils have been regarded as the relatively toxic waste products
of plant metabolic processes with no practical value to the plant. Nowadays it is thought
that they possess properties that assist the plant in repelling leaf-eating insects and in
preventing microbial attack. There is also evidence that terpenes leached from the
leaves contribute to the allelopathic effects on the ground inhibiting the germination
and growth of competitors. It has been suggested although not proven, that oil vapours
near the leaf surface may reduce water loss, and the oils in the flowers might release
odours attractive to pollinating agents.
In Lamiaceae, essential oils are widespread (Hegnauer, 1966; Richardson, 1992) and
many. species
- are used as aromatic herbs for flavouring foods. The essential oils them-
selves are products of great demand in the manufacture of perfumes and cosmetics, and
they are also used for medicinal purposes. This fact also holds good for the genus
Thynzw. Indeed all the Thymw species produce essential oils, and several representatives
are important herbs and spices used in all parts of the world. As will be shown the oils
of Thynzw species have been studied extensively.
In Lamiaceae, essential oils are stored in glandular peltate trichomes. They are situated
on the epidermal surface on both sides of the leaves and show a very typical anatomy
(Figure 3.1). Bruni and Modenesi (1983) intensively studied the trichomes and their
development in Thynzus vulgaris by conventional, fluorescent and electron scanning
microscopy. The glandular peltate trichomes are composed of one basal stalk cell, an
76 Elisabeth Stahl-Biskztp

Sub-cuticular
space
Secretory

Figure 3.1 Anatomy of the glandular peltate trichome of Thymus vulgarir L.

"endodermal" cell that prevents backflow of secreted substances through the apoplast, and
a gland head formed by 10-14 secretory cells whose prominent cytological characteristics
are a relatively large nucleus and a great number of small osmiophilic vacuoles. The
essential oils are produced in the secretory cells and are secreted into the sub-cuticular
space, where they are stored.
When the essential oil begins to penetrate into the sub-cuticular space, a separation
between the outermost and innermost layers of the secretory walls occurs. The outer-
most layer of the cell wall raises together with the cuticle and forms a framework
which the cuticle lies on. Furthermore, it was found that the mature peltate trichomes
possess a dehiscence mechanism whereby stored essential oils are released. It ends in
forming a crescent-shaped pore from which the sub-cuticular secreted material is
released, demonstrated in living field-collected leaves after a sunny day (Bruni and
Modenesi, 1983).
To date, the essential oils of 162 taxa of the genus Thymw have been chemically
investigated revealing about 360 different volatile components in total. Among these
the terpenes lead by almost 75 per cent, the monoterpenes being the most prominent
group (43 per cent). Sesquiterpenes cover 32 per cent of the volatiles, although there
are some ubiquitous sesquiterpenes present in most of the oils. Besides the terpenes
a small group of non-terpenoid aliphates (17 per cent) occur in many oils but in very
low concentrations. Simple benzene derivatives (6 per cent) and phenylpropanoids
(2 per cent) have been found only very sporadically.
After revision of about 270 papers dealing with the essential oil composition of
Thymw species it became obvious that they are not all of the same calibre. One has to
take into account that within the last three decades the analytical methods have developed
enormously. In the field of essential oils, Gas Chromatography (GC) was most concerned
being the most frequently applied analytical method. Especially the on-line coupling
with mass spectometry (GCIMS) nowadays allows effective oil analyses. Today identifi-
cation of 90-95 per cent of the essential oil constituents is the standard, while prior to
the 1960s only the main compounds could be identified.
 Essential oil chemistry ofthe genus Thymus 77
CONSTITUENTS OF THE ESSENTIAL OILS

Monoterpenes and sesquiterpenes

Most of the terpenoid volatiles detected in Thymus oils belong to the monoterpene group.
In the oils the monoterpenes usually make up more than 90 per cent. Sesquiterpenes are
always present, but with only few exceptions in minor percentages. About 270 terpenes
occur in Thymw oils, but their single presence is not significative when characterizing
the genus, thus, quantitative aspects also have to be considered. Only constituents with
concentrations above 10 per cent in at least one Thymus taxon will be mentioned here in
order to enhance clarity and manageability. Fifty-two terpenes are concerned, 34 of
them were selected as the most important volatiles within the genus Thymus. Their
skeletons are presented in Figure 3.2.
Further classification of the 52 individual terpenes can be made by evaluating the
number of Thymw taxa in whose oils they occur in concentrations beyond 10 per cent.
As a result, in Figure 3.3 the terpenes are arranged in order of their importance within
the genus Thynzus showing the 34 most significant constituents (y-axis). How many
Thymw taxa present the compound going beyond 10 per cent can be seen from the
x-axis. Thus, the diagram reflects with clarity the chemical character of the genus
Thymw. It once more shows the prominence of monoterpenes also among the most
important volatile compounds in Thymus.
The phenolic terpenes, thymol and carvacrol, rank highest in importance. They
occur in the oils of 77 (thymol) resp. 73 (carvacrol) different Thymw taxa in percentages
beyond 10 per cent. Analysing Table 3.2 (see below), which has served as a basis for
this diagram, one can gather that both phenols often amount to between 20 and
50 per cent of the oils. Their characteristic strong smell has always been closely associ-
ated with the genus Thymw, being T . vulgaris the most famous representative. Indeed,
the 162 Thymus taxa investigated can be classified into phenolic and non-phenolic taxa.
More than a half of the Thymus taxa (89 taxa=55 per cent) belong to the phenolic
group, while 73 (45 per cent) to the non-phenolic group. Among the phenolic taxa both
can be found: taxa with thymol plus carvacrol (46 taxa) and taxa with either thymol
(27 taxa) or carvacrol (16 taxa).
In the plant kingdom, outside the genus Thymw, thymol and carvacrol occur quite
restrictedly. Among the Lamiaceae some species of Corzdothynzus, Origanum, Satureja,
and Monarda are known to contain thymol and carvacrol as main components of their
essential oils. Aside from the Lamiaceae family it was only found in Trachyspermum cop-
ticum (Apiaceae). Therefore the genus Thymus is the most common source for the
monoterpenoid phenols as is the genus Mentha for menthol. The limited occurrence of
the phenols is one of the reasons why Thymus oils containing thymol or carvacrol have
always been of great interest. The search for phenol-containing species has always been
a great impetus for the chemical examination of the volatiles within the genus Thymus.
The high rank of the monoterpene hydrocarbonsp-cymene (56 taxa) and 7-terpinene
(38 taxa) can not be considered independently of the presence of thymol and carvacrol.
All four terpenes are closely connected by biogenetical processes. As will be shown later
p-cymene and y-terpinene are the precursors in the biochemical pathway of the phenols.
As a result they always occur simultaneously in the essential oils. Usually the hydroxy-
lated phenols are more abundant in the oils than the hydrocarbons. But this is not
obligatory as in few oils also the opposite is realised, with one of the hydrocarbons,
 0 C'. pH
Acyclic Monoterpenes

?CH3

f TH7
CH2OH
Myrcene ~ ~ ~ ~Geraniol
~ ~ . Geranyl
g acetate
~ l

-
~ C H QH0
O

Linalool Linalyl acetate Geranial Neral

Citral

9$
Monocyclic Monoterpenes

$OH ~ o c o c H ~ O H $ QoH

T h ~ m o l Thymy1 acetate Carvacrol p-Cymene y-Terpinene 1,8-Cineole a-Terpineol

Bicyclic Monoterpenes

&OH &.$..@&+ HQ

B O r n ~ acetate
i Camphor Camphene cu-Pinene tmns-Sabinene hydrate

Sesquiterpenes

trans-Nerolidol
,
P-Caryophyllene Germacrene D Germacra-1(10),5-dien-4-01 Germacra-1(10),4-dien-6-01

Hedycaryol
q4
Fzgz~re 3.2 Skeletons of the most important volatlles within the genus Thynzus.
 Essential oil chenzistry ofthe genus Thymus 79

carvacrol

borneol
1,8-cineole
geraniol

alpha-terpineol

-
- camphor
linalyl acetate

-
-
citral
myrcene
terpinen-4-01

-
n--sabinene hydrate
alpha-pinene
camphene
nerolidol
llmonene
germacrene D
MP-bisabolene
Iphellandrene
Igermacren-4-01
bornyl acetate
Idihydrocarvon
Itr-tr-iarnesol
Ihedycaryol
Imyrcenol-8
IT-cad~nol
Igermacrad~en-6-01
Isab~nene
I

F i g z ~ ~3.3
e Components of Thymu essential oils in the order of their importance for the genus Thynzw.

p-cymene or y-terpinene, being the main component of the oil and going clearly
beyond the phenols. It seems plausible that such oils are also treated as "phenolic" oils.
Linalool ranks third in importance for the genus Thymus. In the oils of 56 taxa it is
found in percentages above 10 per cent. Its fine sweet smell is very opposed to that of
the phenols and gives the plants quite a different character. The same is true for geraniol
which occurs in 33 taxa. Linalool is widely distributed within essential oils in general
and the genus Thymus has never been an important source of linalool. In this respect
other Lamiaceae, namely lavender (Lavandula angustifolia) or clary sage (Salvia sclarea),
and among the Apiaceae coriander (Coriandrumsativam), have always been more important.
As will be shown later, within Thymw several taxa contain both, phenolic plants and
plants containing linalool.
With borneol on the sixth place, a bicyclic monoterpene skeleton has got great
importance. In 37 species it could be detected in concentrations above 10 per cent mostly
accompanied by structurally-related monoterpenes such as camphor and camphene.
1,s-Cineole ranks seventh in frequency in Thymw essential oils and is represented by
36 taxa. In the essential oils it often occurs together with camphor and borneol, being
responsible for the relatively high rank of camphor, which is also abundant in borneol-type
oils. 1,8-cineole is known to be widely distributed in the Myrtaceae family, especially
in the essential oils of Eucalyptus species, but is also known to be the main component
of oil from Rosmarinw officinalis (Lamiaceae). From geraniol on, the importance of
individual terpenes declines continuously. Nevertheless, some widely distributed
monoterpenes can be met, such as aiterpinyl acetate (26 taxa), a-terpineol(22 taxa), gera-
nyl acetate (20 taxa), camphor (18 taxa), citral (geranial+ neral, 12 taxa), linalyl acetate
(12 taxa), myrcene (1 1 taxa), and terpinen-4-ol(l1 taxa).
As mentioned above, within the genus Thymw sesquiterpenes are not very important.
The most frequently represented is /I-caryophyllene, more or less ubiquitous in the
essential oil kingdom. Nevertheless, it was detected in concentrations above 10 per
cent in 20 Thymus taxa but never formed the main component of the oils and therefore
hardly gives a special character to the oils. The same stands for germacrene D, ubiquitous
in essential oils but hardly reaching more than 10 per cent in the oils. More extraordinary
is the presence of the oxygenated germacranes, namely germacra-l(lO), 5-dien-4-01,
germacra-l(l0),4-dien-6-01 and hedycaryol. The latter two are known to be thermo-
labile and to decompose during GC, forming elemol and shyobunol respectively, docu-
mented by broad peaks in the gas chromatogram (Stahl, 198413). Together with two
other sesquiterpenes, namely T-cadinol and nerolidol, they are widely distributed within
the Thymus species of northern Europe and therefore deserve to be mentioned.
Eighteen terpenes though present in concentrations above 10 per cent are not included
in the diagram because they reach the 10 per cent limit only within one taxon. Compared
with the high number of taxa investigated (162) their occurrence must be classified
as sporadic. Nevertheless, they are listed here: a-cadinol, carveol, carvone, cinnamol,
citronellol, elemol, fenchone, geranyl butyrate, germacrene B, intermedeol, isoborneol,
isoeugenol, cis-myrcen-8-yl acetate, neryl acetate, spathulenol, a-terpinene, thymyl methyl
ether, and thymyl acetate. A few of them give doubt of a correct identification.

Non-terpenoid aliphates
Non-terpenoid aliphates are present in many Thy~zwoils but only in minor percentages.
Compounds with a chain length of 8 carbon atoms are the most frequent ones, e.g.
octanol-3, octen-1-01-3, octanone-3, octyl-3 acetate, octen-1-yl-3 acetate. The correspond-
ing hexane derivatives rank second in frequency followed by nonane derivatives. In
addition, these branched chains are also common, e.g. 6-methyl-5-heptanol, 5-methyl-
3-heptanone, isoamyl acetate, methyl isovalerate, etc. However more than 62 different
non-terpenoid aliphates could be detected in the oils, thus representing 17.2 per cent of
the oil constituents within Thymus.

Non-terpenoid aromatics a n d phenylpropane derivatives

This group comprises C6C1-, C6C2-, and C6C3-derivatives, the latter better known as
phenylpropanoids. Together they represent about 7.8 per cent of the oil constituents of
Thymus. Among these compounds isoeugenol and cinnamol are the most prominent
compounds, because they occur in at least one Thynzw species in concentrations above
10 per cent and therefore they deserve to be mentioned here. All the others can more or
less be found only in traces and sometimes their identification is doubtful.
 Essential oil chemistry ofthe genw Thymus 81

Enantiomeric composition of essential oil compounds

Only few essential oils of the genus Thymw have been the object of enantioselective
analyses. This is due to the fact that thymol and carvacrol, the most interesting compounds
in Thymus oils, are both achiral terpenes because of their aromatic ring system. The same
is true for 1,s-cineole with its symmetrical skeleton. Even the enantiomeric puritiy of
linalool showing two enantiomers, R-(+)- and S-(-)-linalool, has never been studied in
Thymus oils. Only two publications can be found focusing on some minor terpenes in
oils of the phenolic group (Kreis etal., 1990, 1991). Because of a very limited number
of samples these studies cannot claim to reflect the true situation within the Thymus
oils. Nevertheless, they can be considered as a first attempt to use enantioselective analysis
as an analytical tool for quality control of thyme oils as it is common for the authenticity
control e.g. of lavender oils (Kreis etal., 1993) and Neroli oils (Juchelka etal., 1996).
In the course of a screening of several medicinal essential oils, the oils of thyme
(T. vulgaris) and wild thyme (T. serpylhm) were investigated concerning the enantio-
meric proportions of a-pinene (1s- and 1R-), P-pinene (IS- and 1R-) and limonene
(4S- and 4R-) (Kreis etal., 1990). With percentages between 1 and 3 all three terpenes
are minor constituents of these oils. Applying enantioselective GC on P-cyclodextrane
phases P-pinene was shown to be the enantiomerically most pure compound with 4 per
cent R- and 96 per cent S-P-pinene in thyme oil and 7 per cent R- and 93 per cent
S-P-pinene in wild-thyme oil. The enantiomeric proportion of a-pinene in thyme oil was
8 9 per cent S- and 11 per cent R-, in wild-thyme oil 86 per cent S- and 14 per cent R-.
For limonene the proportion 70 per cent S- and 30 per cent R- was found in thyme oil
and 73 per cent S- and 27 per cent R- in wild-thyme oil.
In another publication the authors focused on the borneol and isoborneol contents in
the essential oils of T. vulgaris, T. serpyllum, and T. zygis (Kreis etal., 1991). The borneol
content in these oils amounts to a maximum of 3 per cent, that of isoborneol clearly
below that limit. Borneo1 as well as isoborneol show two enantiomers each, (-)-borne01
(IS, 2R, 4S-), (+)-borne01 ( l R , 2S, 4R-) and (-)-isoborneol ( l R , 2R, 4R-), (+)-isoborneol
(IS, 2S, 4s-). For the enantioselective analysis a combination of thin-layer chromato-
graphy (TLC) with GC on permethylated P-cyclodextrin as chiral stationary phase
was applied, the (-)-enantiomers eluting before the (+)-isomers. Analysing lab-distilled
oils from dried herbs, it was found that the proportion of (-)-borne01 to (+)-borne01
was quite homogeneous with 98.1-99.6 :0.4-1.5 in four T. vulgaris oils, and >99.9 : <0.1
in both, T. zygh and T. serpyllum oils. The enantiomeric proportion of isoborneol
was investigated only in 7 samples of commercial thyme oils (T. vulgaris) showing
extremely varied results with 21.2-57.0 per cent (-)-isoborneol and 43.0-78.8 per cent
(+)-isoborneol.

BIOSYNTHESIS OF THE AROMATIC TERPENES

Volatile terpenoids in plants are usually of aliphatic character. Only few exceptions
exist, e.g. p-cymene, thymol, carvacrol, p-cymen-8-01, cuminalcohol, calamenene, and
xanthorrhizol. Therefore the processes leading to the aromatization of the cyclohexane
ring have always been of great interest. T. vulgaris served as the main object for the
elucidation of the biogenetic pathway of the aromatic monoterpenes due to the fact that
the volatile oil of thyme consists mainly of thymol, carvacrol, and p-cymene. Here
special attention will be dedicated to the biosynthesis of these terpene phenols, whereas
the biosynthesis of monoterpenes and sesquiterpenes in general will only be touched.
Terpenes contain a sequence of two or more isoprenoid units (C5H8) joined either
head to tail (more common) or head to head (less common) followed by secondary
chemical transformations. The early steps in terpenoid biosynthesis are the reactions
resulting in the isoprenoid units, namely isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP). Nowadays two different pathways are known (Figure 3.4). The
first defined and today called the "classical" pathway (Figure 3.4, left column) was the
acetateimevalonate pathway (Little and Croteau, 1999) in which three molecules of
acetyl-Coenzyme A (acetyl-CoA) are fused by the enzymes acetyl-CoA acyltransferase
and hydroxymethylglutaryl-CoA (HMG-CoA) synthase to produce the C6 compound
HMG-CoA. A reduction of HMG-CoA forms mevalonic acid which is then converted
to the C5 compound IPP, the central precursor of the terpenoid synthesis.
As a result of detailed feeding experiments it became obvious that mevalonic acid can
not be the only intermediate in the terpenoid biosynthesis, but that IPP was derived by
assembly of glycolytic intermediates. As a result of this finding the pyruvatelglyceral-
dehyde-+phosphate pathway has been discovered (Figure 3.4, right column). The initial
step of this pathway has been formulated as involving a transketolase reaction of glycer-
aldehyde-3-phosphate with carbons 2 and 3 of pyruvate to yield the C5 intermediate
1-deoxy-d-xylulose followed by a series of reduction and dehydration steps and a phos-
phorylation affording IPP or DMAPP as end products (Rohmer etal., 1996). The classical
acetateimevalonate pathway seems to predominate in the cytosol leading to sesquiter-
penes and triterpenes, whereas the pyruvate/glyceraldehyde-3-phosphatepathway (also
called the deoxyxylulose pathway) is realized in the plastids to form monoterpenes,
diterpenes, and tetraterpenes. Consequently, as could be proved the isoprenoic units of
thymol in T. vulgaris are formed via the latter pathway (Eisenreich etal., 1997). Label-
ling patterns of some monoterpenes and sesquiterpenes were found to bear two isoprene
units derived from both pathways (Adam and Zapp, 1998).
The coupling of two isoprene units, IPP and DMAPP, yields a C10 molecule,
namely geranyl pyrophosphate (GPP). It serves as a precursor to the monoterpenes.
A head-to-tail coupling of a third isoprene unit provides farnesyl pyrophosphate (FPP),
the precursor of the sesquiterpenes with 15 carbon atoms. Over twenty monoterpene
synthases, mostly cyclases, convert the acyclic precursors (geranyl, neryl, linalyl pyro-
phosphates) to various cyclohexanoid monoterpenes (Charlwood and Banthorpe, 1978;
Croteau, 1987).
Focusing on the phenolic monoterpenes, it was postulated in 1964 that y-terpinene
was the starting product for the biosynthesis of the aromatic monoterpenes, and
p-cymene was considered to be formed as the first aromatic product via a non-enzymatic
aromatization of 7-terpinene (Granger etal., 1964). In principle this path was affirmed
several years later and, indeed, p-cymene could be attributed the role as a key inter-
mediate, but the non-enzymatic process was refuted and proved to be strongly enzymatic
(Poulose and Croteau, 1978). Young, rapidly expanding thyme leaves (T. vulgaris)
were utilized for these latter biosynthetic experiments. The time course of incorporation
of 14c0,into the volatile terpenoids of thyme cuttings suggested a biosynthetic
sequence by which y-tetpinene gave rise to p-cymene. Further incorporation experiments
with exogeneous - . / - ( ~ - ' ~ ) t e r ~ i naenn de p - ( ~ - ' ~ ) c ~ m egave
n e strong evidence that thymol
is biosynthesized by hydroxylation ofp-cymene. Thus, p-cymene is the central precursor
of the oxygenated compounds (Figure 3.5).
 Essential oil chenzzstry of the genus Thymus 83

Pyruvate Glyceraldehyde-3-P

HOO
SCoA

Mevalonic acid

\
several steps

A CH20PP
-
-
CH20PP

IPP DMAPP

I f

p o p : IPP OPP

fi OPP

Figure 3.4 Terpenoid biosynthesis. Left column: classical pathway (acetate/mevalonate pathway);
right column: the pyruvate/glyceraldehyde-3-phosphatepathway.

In the meantime the isolation of y-terpinene synthase, a soluble enzyme which catalyses
the cyclisation from neryl and geranyl pyrophosphate to y-terpinene, was successful
(Alonso and Croteau, 1991). It could be achieved with 21-day-old plants which had
been subjected to an epidermal abrasion technique which selectively extract the contents
 Carvacrol
Geranyl pyrophosphate

y-Terpinene

A
Neryl pyrophosphate
AThymol

Fzgure 3.5 Biosynrhesis of thymol and carvacrol

of the epidermal oil glands (Gershenzon etal., 1987). The phenolic and lipophilic mater-
ials present in the crude enzyme solution were removed by adsorption before the enzyme
was purified by isoelectric focusing and dye-ligand anion-exchange. As a result of the
production test with the y-terpinene cyclase reacting with geranyl pyrophosphate it
was observed that besides y-terpinene (the major product set at 100 parts) a plot of side
products were generated: a-thujene (16 parts), myrcene (6 parts), a-terpinene (7 parts),
limonene (4 parts), linalool (5 parts), terpinene-4-01 (3 parts), a-terpineol (5 parts), and
geraniol (8 parts). These results suggest that all of these monoterpenes are synthesised
as co-products by the y-terpinene synthase in vitro (Alonso and Croteau, 1992).
In Lamiaceae, the localisation of the biosynthesis and accumulation of monoter-
penoids in the peltate glandular trichomes have never been doubted although they were
experimentally proved by Croteau only in 1977 investigating Majorana (Croteau,
1777). He reported that the excised Majorana leaf epidermis with glandular trichomes
incorporated the radioactivity of sucrose sucrose
into monoterpenes. However, it was
not clear whether the biosynthesis or accumulation of monoterpenes was restricted to
peltate glandular trichomes alone or occurred in both peltate glandular trichomes and
capitate glandular trichomes. This question was studied with T. vulgaris being the
experimental object (Yamaura etal., 1792). Quantitative analyses of the essential oils in
intact glandular trichomes isolated from thyme cotyledons with the use of adhesive
tape and a glass capillary tube showed that the content of thymol per cotyledon was
approximately equal to the total sum of thymol in peltate and capitate glandular tri-
chomes. The radioactivity of (u-'*c)-sucrose administered to cotyledonal segments was
incorporated into y-terpinene and thymol most actively by the peripheral part abun-
dant in peltate glandular trichomes. This enzymatic reaction failed when peltate glan-
dular trichomes were removed from cotyledons, indicating that the biosynthesis and
accumulation of monoterpenes in thyme seedlings take place primarily in peltate glan-
dular trichomes and only to a minor extent in the capitate glandular trichomes
(Yamaura etal., 1992). In previous experiments the light-dependent formation of
peltate glandular trichomes and monoterpenes in thyme seedlings had already been
 Essential oil chemistry oftbe genus Thymus 85
demonstrated (Yamaura etal.,1989) as well as the participation of phytochrome in the
photoregulation of monoterpene production (Tanaka etal., 1989; Yamaura etal., 1991).

SUMMARY TABLE OF THE PRINCIPAL OIL COMPOUNDS

OF ALL THYMUS SPECIES STUDIED

The presentation of information found in all the publications on Thymw essential oils
adequately requires a special attention due to the high variability in techniques, sources,
etc. The time prior to 1960 is covered by the publication of Gildemeister and Hoffmann
(1961). From the analytical point of view, the publication of Gildemeister and Hoffmann
represents a certain borderline because since that time analytical techniques, especially
GC and later GCIMS, have developed considerably. With respect to these earlier publica-
tions, a list of all Thymw species described only in the publication of Gildemeister and
Hoffmann (1961) is given in Table 3.1. All the results from chemical research work on
Thymus species from 1960 to 2000 are summarised in Table 3.2. This Table 3.2 is
intended to help the interested reader to consult original publications for further study.
Table 3.2 is an expanded and updated version of Stahl-Biskup's 1991 table.
It must be stressed that questioning chemical data were adopted without filtering
meaning, neither published nor checking although in some cases the correctness of the
results is in doubt. Not all the authors present their results with sufficient accuracy. This
may also concern the correct assignment of the plant material investigated. Never-
theless, Table 3.2 will give a very valuable basis for reflections on the chemical nature of
volatiles in Thymus. In Table 3.2 for each species analysed the oil description is given
presenting the five strongest constituents of the essential oils together with their
percentages in the oils if beyond 10 per cent. Those whose concentrations lay below
10 per cent are listed in decreasing order.
The findings are arranged with respect to geographical aspects. Political names of
the countries were chosen because in this way the plant source seemed to be associated
sufficiently correctly. In the case of "Caucasia", the geographical term was preferred
since the small states in the Caucasian region are hardly known and therefore do not
allow correct location. Previously they were part of the former Soviet Union. The order
of the countries follows two lines: it begins in the north with Greenland and Iceland
going via Great Britain and Scandinavia to the east to Siberia. The second line begins with
Morocco in the western Mediterranean region and goes via the Iberian Peninsula eastward
to the Balkan Peninsula touching France, Austria, Germany, and Italy. Turkey and the

Table 3.1 Thynzu species exclusively treated in the Gildemeister and Hoffmann (1961)

T. brachyphyllzls Opiz
T . rephalotus L.
T . czrnicinus Blum.
T. clivorzlnz Lyka f. borosianus
T . eltonrcus Klok. et Schost.
T. odoratissinzzls Bieb.
T. serpyllzl7n L. ssp. carnioliczl~
T . squarroszls Fisch. et Mey.
Tabb 1.2Summary table of principal results of oils analysed from all Thynzw species studied

Thynzm species Essential oil conzposition *: (Main conzponents only)

Greenland (East Coast)

T. praecox Opiz ssp. in all the 4 types, linalyl acetate is the main componenc (61.4-73.1%),
a~ectilw(E. Durand) sesqulterpene alcohols are type characterislng: (1) linalyl acetate, hedycaryol
Jalas (Stahl, 1984a) 12.7%, nerolidol, ,O-caryophyllene, oct-1-en-3-yl acetate, (2) linalyl acetate,
nerolidol 11.4%, P-caryophyllene 10.6%, oct-1-en-3-yl acetate, (3) linalyl
acetate, 0-caryophyllene, germacrene D, ,O-sesquiphellandrene, and (4) linalyl
acetate, hedycaryol, P-caryophyllene, oct-1-en-3-yl acetate
Iceland
T. plzlecox Opiz ssp. types 1 to 7 contain linalyl acetate as main component (about 70%),
arcticz~s(E. Durand) sesquiterpene alcohols are type characterizing, type 8: no linalyl acetate.
Jalas (Stahl, 1984b) (1) linalyl acetate, hedycaryol, nerolidol, T-cadinol, (2) linalyl acecate,
hedycaryol, nerolidol, (3) linalyl acetate, no sesquiterpene alcohols,
(4)linalyl acetate, hedycaryol, T-cadinol, (5) linalyl acetate, hedycaryol,
(6) linalyl acetate, T-cadinol, (7) linalyl acetate, nerolldol, and
(8) hedycaryol45%, P-caryophyllene, germacrene D, 0-bisabolene
Great Britain
(Scotland, Ireland, south
sf England)
T. praecox Opiz ssp. polymorphous, type characterizing compounds: hedycaryol40.2%, linalool
arcticz~s(E. Durand) 25.5%/1inalyl acetate 25.2%, germacra-l(l0), 4-dien-6-01 35.4%, tr-nerolidol
Jalas (Bischof-Deichnik, 36.2%, T-cadinol 24.5%/hedycaryol 22.1%, P-caryophyllene 31.5%, linalool
1987; Schmidt, 1998) 61.2%, a-cadinol 23.6%/hedycaryol 19.3%, germacra-1(10),5-dien-4-01
32.7%, tr-tr-farnesol 23.6%, tr-sabinene hydrate 2 1.5%/germacra-l(l0),
4-dien-6-01 19.996, a-pinene 19.2%, geranyl acetate 27.7%, y-terpinene
35.1 %, a-terplneol 5 1.7%, a-terpinyl acetate 36.O%/a-terpineol20%,
cis-myrcenyl-8-acetate 29.6%
T. pz~legzoihsL. (1) thymol 38.3%, 7-terpinene 12.2%, p-cymene, 3-octanone, 0-bisabolene,
(Schmidt, 1998) (2) linalool 68.996, thyrnol, geraniol, P-bisabolene, thymyl methyl ether,
(3) geraniol 36.496, neral, 0-bisabolene, gerrnacra-1(10),5-dien-4-01,
germacrene D, and (4) carvacrol 28.196, y-terpinene 2 1.4%, p-cymene 10.8%,
linalool, germacra-1(10),4-dien-6-01
Norway (West Coast)
T. praecox Opiz ssp. in all types linalyl acetate is the main componenc (about 70%), sesquiterpene
arcticus (E. Durand) Jalas alcohols are type characterizing: (1) linalyl acetate, hedycaryol, nerolidol,
(Stahl-Biskup, 1986a) T-cadinol, (2) linalyl acetate, hedycaryol, nerolidol, (3) linalyl acetate, no
sesquiterpene alcohols, (4) linalyl acetate, hedycaryol, T-cadinol, (5) linalyl
acetate, hedycaryol, and (6) linalyl acetate, T-cadinol
T. pulegzoides L. (1) carvacrol 35.296, y-terpinene 24.8%,p-cymene 10.296, P-caryophyllene,
(Stahl-Biskup, 1986b) pbisabolene, and (2) thymol 37.2%, yterpinene 23.2%,p-cymene, pcaryophyl-
lene, thymyl methyl ether
Finland
T. serpyllum L. ssp. (1) monoterpene hydrocarbons 33%, 1,8-cineole 12.5-1 5.0%,
serpylhnz (Stahl-Biskup germacra-1(10),5-dien-4-01 3-12%, germacrene D 10.0-1 2.0%,
and Laakso, 1990) germacra-l(l0),4-dien-6-01,(2) monoterpene hydrocarbons 30%,
1,s-cineole 26%, P-caryophyllene, germacrene D, hedycaryol, and (3)
monoterpene hydrocarbons 27%, 1,8-cineole 19%, germacrene D,
P-caryophyllene, camphor
T. serpylluni L. ssp. (1) linalool 2 1 . 9 4 3 . 8 % , linalyl acetate 8.9-17.6%, P-caryophyllene,
tanaensis (Hyl.) Jalas 1,8-cineole, camphor, and (2) 1,s-cineole 17.2-27.6%, myrcene 15.4-22.4%,
(Ivars, 1964; Von P-caryophyllene 6.8-19.1%, camphor, linalool
Schantz and Ivars, 1964)
(Stahl-Biskup and (1) linalool 52.2%, monoterpene hydrocarbons 13%, germacrene D,
Laakso, 1990) germacra-1(10),4-dien-6-01, (2) linalyl acetate 58.3%, monoterpene
hydrocarbons 15%, gerrnacrene D , germacra-1(10),4-d1en-6-ol,and
(3) monoterpene hydrocarbons 33%, 1,8-cineole 12.5-15.0%,
germacra-1(10),5-dien-4-013-1 2%, germacrene D 10.0-12.0%,
germacra- 1(10),4-dien-6-01
Lithuania
T. pulegioides L. (1) geraniol 16.3-29.2%, geranial 9.7-16.1%, linalool 0.4-l3.7%,
(Mockut6 and P-caryophyllene, neral, (2) carvacrol 16.0-22.2%, /3caryophyllene
BernotienC, 1998, 11.4-15.9%, P-bisabolene 11.1-12.2%, y-terpinene (5.9-14.5), and
1999,2001) (3) a-terpinyl acetate 49.5-70.4%, ,8-caryophyllene 6.2-1 1.5%, geranlol,
P-bisabolene, a-terplneol
T. serpyllz~77zL. s.1. 1,s-cineole 16.3-19.0%, P-caryophyllene 9.6-1 1.3%, myrcene 9.7-lo.?%,
(Loiiene et dl., 1998) germacrene D, camphor
W h i t e Russia
T. serpyllum L. (Popov y-terpinene 21.4%,p-cymene 19.0%, thymol, a-terpineol, carvacrol
and Odynets, 1977)
Kazakhstan
T. rnarschallzanus Willd. p-cymene 22.4%, thymol 20.0%, y-terpinene 19.3%, P-bisabolene, thymy1
(Dembitski~etal., 1985) methyl ether
Siberia
T. krylovii Byczenn. 7-terpinene 26.2%,p-cymene 17.5%, myrcene, limonene, a-terpineol
(Tikhonov etal., 1988)
Morocco
T. algerzensis Boiss. (1) thymol 14.4-65.1%, and (2) carvacrol 22.8-70.3%, both types additionally:
(Benjilali etal., 1987a,b) p-cymene 0-36%, 7-terpinene 0-16%, linalool0.7-1 I % , borneol 1-15.2%
T. broussonettii Boiss. (1) carvacrol 53-83.2%, (2) borneol 2 4 4 2 . 9 % , p-cyrnene 19-24.3%, and
(Benjilali etal., 198713) (3) thymol 18.2-58.5%, additional intermediates
(Tantaoui-Elaraki etal., carvacrol 53.3 %, p-cyrnene 13.5 %, a-plnene, a-terpinene
1993)
T. rilzatus (Desf.) Bench. polymorphous, most important compounds: thymol 0.3-29.3%, carvacrol
(Benjilali etal., 1987b) 0.4-21.7%, a-terpinyl acetate 16.4142.9%, geranyl butyrate 14.6-26.7%,
geranyl acetate 21.7%, camphor 0.4-28.4%, borneol 0.1-31.6%
T. hirtu Willd. (I) carvacrol91.6%, and (2) thymol 19.2%, geraniol, camphor, caryophyllene
(Benjilali etal., 1987b) epoxide, carvacryl acetate
T. maroccanus Ball carvacrol 74.015 5.596, thymol 0.4118.4%, p-cymene 10.0/5.6%, linalool,
(Richard etal., 1985) y-terpinene
T. pallidus C O S S Oex~ (1) thymo120.6, borneol l2.7%,p-cymene 23.6%, -/-terpinene 14.3%, carvacrol,
Blatt (Richard etal., (2) carvacrol24.2%, borneol 17.496, thymol 17.7%, -/-terpinene 1l.l%,p-cyrnene
1985; Benjilali etal., 10.3%, (3) tr-dihydrocarvone 39.9-61.1%, cis-dihydrocarvone 6.2-26%, and
1987a) (4) camphor 54.8%, camphene 13.8%, borneol 11.0%, a-pinene, linalool
T. rzutarzinz Humbert et carvacrol 24.5%, p-cymene I?.?%, -/-terpinene 17.6%, borneol, thymol
Maire (Velasco
Negueruela et al., 1991a)
(Iglesias et al., 1991) carvacrol 22.3%, p-cymene 17.5%, 7-terpinene 10.3%, borneol, P-bisabolene
T. satureioides Cosson (1) borneol 26-77.6%, phenols (thymol+ carvacrol) 8.7-21.9%, a-terpineol
(Benjilali etal., 1987; 5.8-21%, camphene 0.1-1 1.2%, bornyl acetate, and (2) phenols
Richard etal., 1985) (thymol+ carvacrol) 34.7-50%, borneol 13.0-19.0%
(Tantaoui-Elaraki etal., borneol 31.2 %, camphene 27.4%, a-pinene 17.5%, linaloo1,p-cyrnene
1993)
Table 3.2 (Continued)

Thymus species Essential oil composition * (Main components only)

T. zygis L. (Richard (1) thymol 30.7%,p-cymene. 23.3%, ,!-caryophyllene epoxide, carvacrol,

etal., 1985) thymyl methyl ether, and (2) camacrol42.9%,p-cymene 28.5%, y-terpinene,
linalool, borneol
(Tantaoui-Elaraki etal., p-cymene 50.6%, camacrol, borneol, thymol, linalool
1993)
Spain
T. albzcans Hoffmanns. (1) linalool 51.1%, 1,s-cineole 32.9%, a-terpineol, ,!-pinene, a-pinene, and
et Link (Morales, 1986) (2) 1,s-cineole 70.5%, a-terpineol, ,!-pinene, a-pinene, alloaromadendrene
T. antonrnae Rouy et 1,8-cineole 38.3%, camphor 17.5%, borneol 11.0%, ,!-caryophyllene
Coincy (Velasco
Negueruela and Perez
Alonso, 1986)
(Vila etal., 1991a; 1,8-cineole 19.2124.4%, camphor 15.6114.9%, borneol, camphene, myrcene
Caiiigueral et dl., 1994)
T. x arunaknw Willk. 1,8-cineole 46.4%, linalool 11.7%, limonene, tr-sabinene hydrate, p-cymene
(Soriano Cano etal., 1997)
T. baeticus Boiss. ex (1) a-pinene 28.6%,p-cymene 13.9%, 7-terpinene 12.9%, terpinen-4-01,
Lacaita (Morales, 1986) linalool, (2) terpinen-4-01 24.1%, a-terpineol 23.5%,p-cymene, a-pinene,
borneol, and (3) llnalool 35.4%, borneol, a-terpineol, p-cymene, a-pinene
(Adzet etal., 1988) 1,8-cineole 14.4%, a-pinene 10.0%,p-cymene, terpinen-4-01, borneol
(Garcia Vallejo etal., polymorphous; 1,s-cineole 0.5-15.1%, geraniol 0.2-14.9%,
19924 terpinen-4-01 2.4-12.6%, borneol 0.9-10.0%,p-cymene, a-pinene
(Cruz etal., 1993; Cabo female: 1,8-cineole 20.9110.3%, terplnen-4-01 11.2122.8%, borneol 9.8112.7%,
etal., 1990, 1992) a-terp~neol+a-terpinyl acetate, linalool+ linalyl acetate, hermaphrodite:
1,8-cineole 13.8-21.4%, geraniol 20.7/7.3%, borneol 3.511 1.2%,
terpinen-4-01 8.1/10.3%, citral 10.8-8.9%
(Soriano Cano etal., 1997) tr-sabinene hydrate 18.2%, borneol 13.7%, a-pinene, verbenone,p-cymene
(SBez, 1999) polymorphous, type characterising components: terpinen-4-01, tr-sabinene
hydrate,p-cymene, I ,8-cineole, borneol, linalool, geranyl acetate, linalyl
acetate, geranial
T. bwgiae Rivas-Martinez, carvacrol 59.7%, camphor, P-caryophyllene, germacrene D, germacrene B
Molina et Navarro
(Blizquez etal., 1990)
T. bractedtzds Lange ex (1) linalool 74.3%, borneol, camphor, and (2) Linalool 17.2%, carvacrol
Cutanda (Morales, 1986) 16.9%, borneol 12.8%, a-terpineol, terpinen-4-01
T. caespititizs Brot . a-terpineol 68%, borneol 11.0%, p-cymene, linalool
(Seoane et dl. , 1972)
(Morales, 1986) a-terpineol 26.2%,p-cymene 24.5%, camphene, a-pinene, rhyrnol
T. capitatu Hoffmanns. carvacrol 61.0%,p-cymene, -/-terpinene, P-caryophyllene, myrcene
et Link, today: Thymbra
capitata (L.) Cav. (Mateo
etal., 1976; Morales, 1986;
Velasco Negueruela and
Perez Alonso, 1986)
T. carnosus Boiss. borneol 41.2143.7%, camphene 10.8110.7%, bornyl acetate, terpinen-4-01,
(Marhuenda and Alarc6n camphor
de la Lastra, 1987;
Marhuenda etal.,
1987, 1988)
T. x enicensic Blanca p-cymene 36.4%, thyrnol29.9%, y-terpinene, borneol, a-pinene
(Siez, 1995a,b)
T. ericoides Rouy p-cymene 51.1%, 7-terpinene 10.2%, a-pinene, borneol, carvacrol
(Mateo etal., 1978)
T.fontqueri Ualas) Molero unknown 14.8%, myrcene 14.4%, 0-citral 13.4%, 1,8-cineole 11.5%
et Rovira (Molero and
Rovira, 1983)
T.funkii Coss (Mateoetal., 1,s-cineole 48.0%, camphor 11.2%, camphene, a-pinene, P-pinene
1978; Morales, 1986)
(Morales, 1986) 1,8-cineole 58.0148.0122.1%, camphor 6.911 1.2118.8%, borneol
1.912.7111.4%, myrcene, a-terpineol
(Velasco Negueruela 1,8-cineole 52.0120.0%, camphor 6.511 1.0%, borneol, camphene, myrcene
and Perez Alonso, 1986)
(Adzet etal., 1988) 1,s-cineole 47.6%, camphor 10.0%, P-pinene, camphene, a-pinene
(Vila etal., 1995) 1,8-cineole 28.0127.5%, camphor 17.7114.2%, borneol, camphene, P-pinene
T. glandulosus Lag. ex p-cymene 58.0%, borneol, a-pinene, camphene, y-terpinene
H . del Villar (Adzet
etal., 198913)
T. godayanus (BlPzquez a-terpinyl acetate 23.616.1%, bornyl acetate 3.8/18.2%, carvacrol4.4117.3%,
and Zafra-Polo, 1989) geranyl acetate 1.011 1.0%, 1,8-cineole
T. grandtensis Boiss. myrcene 18.6%, P-caryophyllene 14.0%, camphor 10.6%, borneol, lirnonene
(Cabo etal., 1986a, b)
T. hyemdlz~Lange polymorphous, type characterizing main components: (1)p-cymenelcarvacrol,
(Adzet etal., 1976) (2)p-cymenelthymol, (3) Carvacrol, and (4) Borneol/camphorll,8-cineole
(Mateo etal., 1978) p-cymene 38.0%, thymol 26.3%, y-terpinene, linalool, myrcene
(Morales, 1986) p-cymene 51.1%, yterpinene 10.2%, a-pinene, borneol, carvacrol
(Caboetal., 1980,1986~) (1)p-cyrnene 52.7%, 1,8-cineole 15.5%, linalool, a-pinene, thymol, and
(2) myrcene 16.9131.5%, 1,8-cineole 17.1113.6%, camphor 12.011.2%,
terpinen-4-01, 0.1116.8%
(Cabo etal., 1987) myrcene 3 I.?%, 1,8-cineole 17.1%, camphor 12.5%, camphene, a-pinene, limonene
(Jimenez Martin et dl., (1) p-cyrnene 3 1.5%, borneol 17.8%, carvacrol, l i n a l ~ o l l l i n a lacetate,
~l
1989,1992) camphene, (2)p-cymene 43.8-49.2%, thymol 5.4-13.9%, carvacrol
1.4-13.3%, y-terpinene, borneol, and (3) thymol 52.3%, p-cymene 13.9%,
carvacrol, borneol, linaloolllinalyl acetate
(1) thymol 36.7%, y-terpinene 27.5%,p-cymene 17.5%, (2) carvacrol 39.9%,
p-cymene 22.4%, y-terpinene 10.6%, a-pinene, verbenone, (3) linalyl
acetate 53.0%, linalool 20.2%, a-terpineol, (4) linalool 34.4%,p-cyrnene
16.4%, carvacrol 15.1%, a-pinene, borneol, (5)p-cymene 35.8%, carvacrol
24.7%, borneol, -/-terpinene, (6)p-cymene 34.3%, mi-terpinene 12.4%, borneol
12.4%, a-pinene 10.9%, thymol, and (7) p-cymene 45.3%, borneol 16.9%,
camphene 11.1%, y-terpinene
T. LyemliJ Lange x p-cymene 44.7%, -i-terpinene 12.3%, carvacrol 11.2%, borneol 10.4%,
T. vulgaris L. (Siez, 1995a) camphene
T. x indalicus Blanca 1,8-cineole 25.7%,p-cymene 22.796, y-terpinene 12.3%, thymol, borneol
(Siez, 1995a)
T. bcaitae Pau (Garcia 1,s-cineole 59.2-68.8%, P-pinene, limonene, sabinene, myrcene
Martin and Garcia
Vallejo, 1984)
(Velasco Negueruela and 1,8-cineole 61.3%, lirnonene, P-pinene, sabinene, nerolidol
PCrez-Alonso, l985a)
(Morales, 1986) 1,8-cineole 32.9%, rnyrcene, a-pinene, P-caryophyllene, carvacrol
Table 3.2 (Continued)

Thylnw species Essential oil co~izposition* (Main ronzponents only)

T . leptophylhs ssp. 1,8-cineole 21.6%, linalool, thymol, p-cymene, myrcene

leptophyllus Lange
(Mateo etal., 1978;
Morales, 1986)
(Zafra-Polo etal., 1988a; linalyl acetate 68.5%, linalool 17.0%, 1,8-cineole, a-terpineol
Blizquez etal., 1989)
T. longijilorus Boiss. 1,8-cineole 58.0%, camphor, a-pinene, camphene, ,!?-pinene
(Mateo etal., 1978)
(Morales, 1986) (1) 1,8-cineole 24.8%, borneol 10.6%, myrcene, camphor, camphene, and
(2) terpinen-4-01 26.2%, 7-terpinene, myrcene, a-pinene, limonene
(Velasco Negueruela and camphor 34.696, 1,8-cineole 22.6%, borneol li.O%, terpinen-4-01,
Perez-Alonso, 1986) a-terpineol
(Cruz et dl., 1988) 1,8-cineole 40.6%, camphor, camphene, a-pinene, borneol
T. loscosii Willk. (Molero citral 15.0%, unknown 12.8%, camphene, camphor
and Rovira, 1983)
(Morales, 1986) 1,s-cineole 39.8150.4%, camphor 5.5114.0%, a-terpineol, borneo1,p-cymene
T . mastigophorus Lacaita P-caryophyllene 14.2%, myrcene 13.8%, camphene, linalool, or-pinene
(Morales, 1986)
(Velasco Negueruela and myrcene 25.0%, 1,8-cineole 19.2%,camphene 18.5%, 0-caryophyllene,
Perez-Alonso, 1986) a-pinene
(Garcia Vallejo and P-caryophyllene 7.9-27.0%, myrcene 0.1-23.1%, germacrene D 4.8-14.0%,
Garcia Martin, 1986) elemol 0.7-10.9%
T. mastichina L. (Garcia- linalool 82.596, limonene + 1,s-cineole, camphor, p-cymene
Martin etal., 1974)
(Adzet 1977a, b) (1) linalool 74%, camphor, borneol, 1,s-cineole (Portugal), (2) 1,8-cineole
60-75%, linalool 8.5-20.5%, borneol, camphor (Spain), and (3) linalool43%,
1,s-cineole 41%, borneol, camphor (Spain)
(Garcia Vallejo et dl., (1) 1,s-cineole 81.3%, (2) linalool 82.9%, and (3) 1,8-cineole 47.7%, linalool
1984) 29.0%
(Mateo etal., 1978; 1,8-cineole 60.1%, linalool 14.5%, a-pinene, P-pinene, a-terpineol
Morales, 1986)
(Velasco Negueruela and 1,8-cineole 74.8%, borneol+ bornyl acetate, linalool, a-phellandrene
Perez-Alonso, 1986)
(Velasco Negueruela (1) linalool, (2) 1,8-cineole 7 1%, and (3) l,8-cineole/linalool
et al., 1992)
(Soriano Cano etal., 1997) 1,8-cineole 42.6%, linalool 32.8%, ,L-pinene, sabinene, a-terpineol
T. ~izastichinaL. ssp. 1,s-cineole 66.596, linalool, ,L-pinene, a-pinene, a-terpineol
lnastichina (Morales,
1986)
T. melizbranaceus Bo~ss. 1,8-cineole 35.9162.4/55.1%, camphor 17.812.8/17.8%, camphene, myrcene,
incl. T. l~zurciczl~
Porta P-pinene
(Mateo etal., 1978;
Morales, 1986)
(Velasco Negueruela and camphor 29.1%, 1,8-cineole 27.4%, borneol 10.4%, a-terpinene
PCrez-Alonso, 1986)
(Zarzuelo et dl., 1987) 1,8-cineole 28.9%, camphor 16.7%, borneol, camphene, carvacrol
(Vila etal., 1987) 1,s-cineole 41.2%, camphor 13.7%, camphene, a-pinene, P-pinene
T. nzeuzbranaceus Boiss. x 1,8-cineole 32.9%, camphor 15.5%, camphene, a-pinene, borneol
T. lnoroderi Pau ex R.
Morales (Vila etal., 1987)
T. x nzonrealensis Pau ex thymol41.144.2%, y-terpinene 13.9-15.4%,p-cymene 10.1-15.196,
R. Morales (Soriano 1,8-cineole, carvacrol
Cano et al., 1992)
T. x nzo~zrealensisPau ex geranyl acetate 24.396, camphene 18.896, camphor 14.5%, myrcene, a-pinene
R. Morales nothosubsp.
garcza-vallejoz
Sinchez-G6mez et
Alcaraz (Siez, 1995b)
T. nzoroderi Pau ex 1,8-cineole 24.5%, camphor 22.8%, camphene 10.696, a-pinene, borneol
Martinez (Adzet et al.,
1989a)
(Caiiigueral et al., 1994) 1,%cineole 28.1118.5%, camphor 19.8120.3%, camphene, borneol, a-pinene,
additional sesquiterpene alcohols are mentioned as type characterizing
components
T. orospeddnzlr H. del p-cymene 22.596, y-terpinene 22.4%, carvacrol 15.6%, thymol, linaiool
Villar (Crespo etal., 1986)
(Velasco Negueruela and 1,s-cineole 25.2%, camphor 18.1%, borneol 12.3%, camphene, 7-terpinene
Perez-Alonso, 198513)
T. ptperella L. (Adzet (1)p-cymene 26.3/>0%,carvacrol 38.6121.8%, -/-terpinene 14.512.1%,
and Passet, 1976) linalool, thymol, and (2) thymol 30.448.5%,p-cymene 21.2-31%,
y-terpinene, terpinen-4-01, linalool
(Morales, 1986) p-cymene 47.0%, carvacrol 11.5%, -(-terpinene, 0-caryophyllene, borneol
(Blanquer et al., 1998; (1)p-cymene 43.3%, carvacrol 15.8%, y-terpinene 14.0%, limonene,
Boira and Blanquer, ,R-caryophyllene,(2)p-cymene 44.8%, thymol 23.0%, -/-terpinene, borneol,
1998) and (3) p-cymene 52.1%, carvacrol 18.1%, y-terpinene, borneol
T. serpylloides Bory ssp. carvacrol 41.2%, p-cymene 24.9%, y-terpinene 15.596, borneol, myrcene
gadorensis (Pau) Jalas
(Morales, 1986)
(Crespo et a/. , 1988) camacrol 14.496, 7-terpinene 25.6%,p-cymene 19.396, u-phellandrene, linalool
(Arrebola etal., 1995) carvacrol 73.5150.1124.5138.596, yterpinene 0.7311 1.31/26.7112.1%,
p-cymene 0.94116.1112.819.3%, thymol, borneol
(Arrebola et al., 1997) carvacrol 70,696, p-cymene, 7-terpinene, a-pinene, thymol
(Siez, 2001) polymorphous (1) geraniol 80.0%, isobornyl acetate, borneol, citronellol,
(2)p-cymene 30.9%, carvacrol 20.1%, thymol 18.596, tr-ocimene 10.396,
7-terpinene, (3) linalool 27.496, y-terpioene 11.7%, thymol 11.6%, p-cymene
11.3%, a-terpinene, (4) linalyl acetate 39.4%, linalool 24.5%, camphor,
camphene, a-terpineol, (5) myrcene 17.1%, p-cymene 15.1%, 1,8-cineole
10.5%, terpinen-4-01 10.896, and (6) myrcene 30.4%, a-terpineol 27.0%,
terpinen-4-01 1 I.?%, 1,8-cineole 10.1%
T. serpyllozdes Bory ssp. carvacrol 45.0-62.5%, y-terpinene 8.8-19.9%,p-cymene 8.3-16.0%,
serpylloides (Arrebola thymol, terpinen-4-01, a-terpinene
et al., 1994)
T. villosus L. ssp. camphor 30.296, borneol 15.596, camphene, a-pinene, terpinen-4-01
lusitani~~us(Boiss.)
Coutinho (Morales, 1986)
(Perez Alonso and camphor 37.0125.7/14.2%, borneol 15.618.814.4%, linalool
Velasco Negueruela 1.4112.8%,18.2%, 1,8-cineole 1.9114.7113.8%, camphene
1984; Velasco
Negueruela etal., 1992)
Tuble 3.2 (Continued)
--- - -

Tbymw speczes Essentzal ozl composttzon * (Muzn components only)

T. vulguris L. (only of 1,s-cineole 3 5 . 7 4 4 . 4 % , camphor 11.6-16.3%, camphene 8.1-10.996,

wild origin) (Garcia- linalool, borneol
Martin etul., 1974)
(Elena-Rossell6, 1976) +
(1) linalool linalyl acetate 90-98%, (2) a-terpineol + terpinyl acetate
90-96%, and (3) thymol+p-cymene 50-65s
(Adzet etul., 1977b) (1) 1,%cineole, (2) thymol, (3) carvacrol, (4) tr-sabinene hydrate1
terpinen-4-01, (5) linalool, and (6) a-terpineol

(Mateo etal., 1978; 1,s-cineole 33.096, camphor 14.596, camphene 11.4%,p-cymene, -/-terpinene
Morales, 1986)
(Garcia Vallejo et dl., 1,s-cineole 6.0-38.996, camphor 3.5-18.0%, camphene 2.9-13.2%,
199%) 7-terpinene 0-12.4%, myrcene 1.2-10.6%
T. vz~lgurisssp. aestivu 1,8-cineole 23.296, thymol 17.2%, camphor 12.8%,p-cymene, borneol
(Reuter ex Willk.) H.
Bol6s et 0. Bolbs, syn.
T. aestivzs Reut. ex Willk.
(Mateo etul., 1978)
(Adzer etal., 1988) linalool 62.896, geraniol, 1,s-cineole, borneol, camphor
(Morales, 1986; Elena- (1) 1,s-cineole 22.5155.1%, a-pinene 13,096, 0-pinene 12.6%, myrcene,
Rossell6, 1976; Adzet y-terpinene, (2) linalool 62.896, geraniol, 1,8-cineole, borneol, camphor,
etul., 1988; Blizquez (3) a-terpineol, and (4) 1,s-cineole 22.296, geranyl acetate 20.0%, geraniol
and Zafra-Polo, 1990) 17.4%, linalool, borneol
T. vulguris L. ssp. 1,s-cineole 23.296, thymol 17.2%, camphor 12.8%,p-cymene, borneol
vulgarir (Morales, 1986)
T. webbianus Rouy germacrene B 18.896, terpinen-4-01, P-caryophyllene, 1,s-cineole, borneol
(Zafra-Polo etal., 1988b)
T. willkommii Ronn polymorphous, main types: (1) linalool 30-57%, (2) a-terpinyl acetate
(Adzet etul., 1991) 36-69%, linalool 0.4-14%, (3) tr-sabinene hydrate, terpinen-4-01,
myrcen-8-01, and (4) linalool, terplnyl acetate, 1,8-cineole
T. zygis L. (Adzet et dl., (1) thymol, and (2) linalool
1977b)
(Mareo etul., 1978) (1) thymol 20.9-61.196,~-cymene 9.1-18.096, 1,s-cineole 0.2-14.4%,
camphor 0-11.3%, y-terpinene, and (2)p-cymene 22.496, carvacrol20.6%,
y-terpinene 13.0%, thymol 11.796, borneol
(Cabo et dl., 198 1) (1)p-cymene 30.396, carvacrol 22.2%, borneol, 1,s-cineole, thymol, and
(2) thymol 36.0%,p-cymene 19.8%, 1,s-cineole 15.3%, borneol, carvacrol
(Garcia Martin and (1) thymol 49.896, p-cymene 18.9%, y-cerpinene, linalool, a-pinene,
Garcia Vallejo, 1983) (2) carvacrol 43.9%,p-cymene 20.8%, y-terpinene 11.996, linalool, a-pinene,
(3) linalool 79.0%, linalyl acetate, camacrol, myrcene, (4) geranyl acetate
68.696, geraniol 1 6 . l % , linalool, (5) a-terpinyl acetate 70.396,
+
a-terpineol borneol 13.396, limonene, and (6) myrcenol 28.696,
terpinen-4-01 10.096, myrcene, tr-thujanol, y-terpinene,
(Garcia Martin and thymol 21.3-38.196,~-cymene 25.3-35.896, y-terpinene 6.5-1 1.696,
Garcia Vallejo, 1984) linalool, a-pinene, carvacrol
(Jimenez et dl., 1993) thymol 74.096,p-cymene 10.396, carvacrol, terpinen-4-01
T. zygygii L. ssp. grucilis (1) thymol 37.2%161.1%,p-cymene 20.6118.0%, terpinen-4-01 11.310.8%,
(Boiss.) Morales y-terpinene, linalool, and (2) carvacrol20.6%,p-cymene 22.4%, y-terpinene
(Morales, 1986) 13.096, thymol 11.7%, borneol
(Sinchez G6mez et dl., linalool 33.3%, myrcene, terpinen-4-01, yterpinene, tr-sabinene hydrate
1995)
(Siez, 1995b) (1) tthymol 7 1.8%, linalool, carvacrol, p-cyrnene, (2) linalool 28.6%,
a-terpineol 17.0%,p-cymene 13.4%, a-pinene, myrcene, (3) linalool 91.496,
and (4) thymol 25.5%, carvacrol 22.8%,p-cymene 18.8%, -/-terpinene,
geranyl acetate
T. zygis L. ssp sylvestris thymol 37.4-53.2%,p-cymene 10.1-17.3%, linalool 1.9-11.4%, borneol
(Hoffmanns. ec Link) 1.0-10.2%, y-terpinene
Brot. ex Coutinho
(Velasco Negueruela and
Perez Alonso, 1984)
(Morales, 1986) thymol 20.946.8%,p-cymene 9.1-15.2%, 1,8-cineole 0.5-14.4%,
camphor 5.6-1 1.3%, y-terpinene
(Siez, 1995b) (1) 1,s-cineole 34. 5%, limonene 19.0%, thymol, linalool, p-cymene,
(2) thymol 34.296,~-cymene27.6%, y-terpinene 11.0%, linalool, a-pinene,
(3) linalool 73.0%, 1,8-cineole 16.1%, borneol, and (4)p-cymene 28.2%,
thymol 24.4%, carvacrol 18.2%, y-terpinene 10.9%, linalool
T. zygis L. ssp. zygis linalool 32.8%,p-cymene 17.9%, thymol 15.1%, camphor, carvacrol
(Garcia Martin et dl.,
1974)
(Morales, 1986) (1) terpinyl acetate 73.1/65.4%, a-terpineol, carvacrol, linalyl acetate,
thymol, (2) terpinyl acetate 37.8%, linalool 37.5%, thymol, a-terpineol,
(3) linalool 57.1%, borneol, carnphene, terpinen-4-01, and (4) carvacrol 54.5%,
thymol 13.5%,p-cymene 12.3%, y-terpinene, 0-caryophyllene
T. x zygophorus R. Morales linalool + linalyl acetate 42.6%, 1,s-cineole, a-terpineol, camphene, a-pinene
(GarciaMartin and Garcia
Vallejo, 1984)
Portugal
T. ulbicans Hoffmanns. 1,s-cineole 50-65%, borneol, a-pinene, P-pinene, terpineol
et Link (Miguel etul.,
1999)
(Salgueiro etul., 1 9 9 7 ~ ) (1) 1,s-cineole 42.6%, a-terpineol, borneol, linalool, ppinene, (2) linalool
44.5%, borneol, a-terplneol, 1,8-cineole, camphene, and (3) Linaiool40.8%,
1,8-cineole 25.8%, a-terpineol, borneol, 0-caryophyllene
T. cuespititzw Brot. (1) a-terpineol 30.6-40.5%, p-cymene, T-cadinol, y-terpinene, y-cadinene,
(Salgueiro et al., 199713) and (2) carvacrol 36.3%, thymol 16.1%, carvacryl acetate,p-cymene,
a-terpineol-type 2: on the Azores only
(Pereira et dl., 1999) (1) sabinene 66.1!74.3%, (2) thymol 30.2!39.0%, sabinene 26.8/9.7%,
carvacrol, y-terpinene, (3) a-terpineol 34.3-55.9%, thymol 1.6-12.2%,
y-terpinene, and (4) Carvacrol 36.3143.4, thymol 20.9112.0%, a-terpineol
T. cavzphoratus +
1,8-cineole 19.9%, borneol a-terpineol+ bornyl acetate 15.5%,
Hoffmanns. et Link terpinen-4-01 10.2%, camphene 10.0%
(Velasco Negueruela and
P6rez-Alonso, 1987)
(Adzet et al., 1988) terpinen-4-01 29.3%, 7-terpinene 12.2%,p-cymene, a-terpinene,
borneol
(Salgueiro, 1992) (1) 1,8-cineole, (2) linaloolllinalyl acetate, (3) campheneiborneol, (4)
camphenell ,8-cineolelborneol, (5) a-pineneilinalool, and (6) a-pinene!
1,s-cineole; mean percentages: borneol 1.2-35.0%, 1,s-cineole 0.6-35.5%,
linalool 1.1-26.1%, camphene 0.2-13.5%, linalyl acetate 0.3-13.2%,
a-pinene 0.9-10.5%
(Salgueiro et dl., 1997a) (1) linalool 16.7%, linalyl acetate, T-cadinol, geranyl acetate, intermedeol,
(2) borneol 29.5%, camphene 11.4%, camphor 10.7%, a-pinene, I~nalool,
(3) 1,8-cineole 32.8%, T-cadinol, a-pinene, linalool, y-cadinene, and
(4) 1,8-cineole 22.9%, borneol 18.2%, a-pinene, camphene, camphor
Table 3.2 (Continued)

Thynzus species Essential oil conzposztion * (Main components only)

T. capztatus Hoffmanns. carvacrol 69%, y-terpinene, p-cymene, a-pinene, myrcene

et Link, today: Thynzbra
capitata (L.) Cav.
(Proenp da Cunha and
Roque, 1986)
T. capitellatf~s
Hoffmanns. linalool 31.6%, linalyl acetate, a-terpineol, 1,8-cineole, borneol
et Link (Velasco
Negueruela etal., 1991b)
(Salgueiro, 1992) (1) 1,s-cineole, (2) Camphenel1,X-cineoleiborneol,and (3) linaloolilinalyl
acetate; mean percentages: 1,8-cineole 25.1-59.1 %, borneol 0.5-2 1.0%,
linalool 0.8-13.5%, camphene 0.6-10.9%
(Figueiredo et dl., 1993) 1,8-cineole 50.4155.5%, borneol, a-pinene, sabinene, linalool
T. carnosw Boiss. (1) borneol 25.2%, czs-sabinene hydrate 12.7%, terpinen-4-01 10.196,
(Salgueiro etal., 1995) tr-sabinene hydrate, camphene, (2) borneol 30.0%, camphene 10.896, bornyl
acetate, terpinen-4-01, czs-sabinene hydrate, and (3) linalool 26.9%, borneol
17.5%, tr-sabinene hydrate, tr-sabinene hydrate, terpinen-4-01
T. lotocephalus 1,S-cineole 10.3124.1%*, linalyl acetate 22.8/5.5%, linalool I l.il6.0%,
G. L6pez et R. Morales a-pinene, a-terpineol; * flowersileaves
(Figueiredo et al., 1993)
(Salgueiro et al., 2000b) (1) linalool 24.696, P-caryophyllene 10.1%, camphor, intermedeol,
viridiflorol, (2) 1,s-cineole 18.4%, camphor, a-pinene, viridiflorol, borneol,
(3) linalool 13.9%, 1,s-cineole 11.7%, a-pinene, camphor, viridiflorol,
(4) linalyl acetate 16.1%, linalool 11.5%, caryophyllene oxide 10.6%,
1,8-c~neole,camphor, and (5) geranyl acetate 20.3%, intermedeol 10.9%,
camphor, caryophyllene oxide, viridiflorol
T. nzastichzna (L.) L. ssp. 1,8-cineole 38.4%, borneol 15.3%, camphene, a-terpineol, /I-pinene
donyanae R. Morales
(Salgueiro etal., 1 9 9 7 ~ )
T. nzastichina (L.) L. ssp. (1) 1,8-cineole 60%, a-terpineol, 0-pinene, a-pinene, sabinene, (2) linalool
~7zastic~hi1za
(Salgueiro 58.5%, 1,8-cineole, camphor camphene, borneol, and (3) linalool 38.0%,
eta/., 1 9 9 7 ~ ) 1,8-cineole 2 1.996, p-pinene, a-terpineol, a-pinene
(Miguel et al., 1999) 1,8-cineole 40-50%, camphor, camphene
T.x nzourae (Salgueiro 1,s-cineole 23.5%, borneol 14.7%, camphor, intermedeol, Pcaryophyllene
etal., 2000 b)
T. pzdegioides L. thyrnol 40.0%,p-cymene 12.5%, Y-terpinene 12.2%, octan-3-one, carvacrol
(Salgueiro et dl., 1993)
T. x viciosoi (Pau) Morales carvacrol 30.0%,p-cymene 18.0%, thymol 17.3%, y-terpinene, linalool
(Salgueiro etal., 1993)
T. villosns L. ssp. (1) linalool41.0%, terpinen-4-01 16.4%, tr-sabinene hydrate 11.2%,
luritanicz~s(Boiss.) (2) linalool 3 1.596, 1,s-cineole 22.3%, (3) linalool69.7%, and
Coutinho (Salgue~ro (4) geranyl acetate 39.2%, geraniol 24.2%, 1,8-cineole 19.5%
et al., 2 0 0 0 4
T. villosf~sL. ssp. uillosus (1)p-cymene/borneol, and (2)p-cymenelcamphor; mean percentages:
(Salgueiro, 1992) p-cymene 22.5-39.S%, borneol 10.1-22.5%, camphor 2.5-13.9%,
linalool, 7-terplnene
(Salgueiro etal., 1997d; (1)p-cymene 24.0%, camphor 11.0%, linalool 7-terpinene, borneol,
Salguelro and P r o e n p (2)p-cymene 2 3 . 2 % ~borneol
~ 19.696, camphor, I~nalool,a-pinene, (3) linalool
da Cunha, 1992) 20.9%, geraniol 12.8%, geranyl acetate 10.2%, camphor, a-pinene, and
(4) a-terpineol 16.1%, camphor 13.2%, myrcene 10.6%, a-pinene, linalool
T. zygis L. ssp. syluestrzs thyrnol 14.8%,p-cymene 15.5%, geraniol 14.5%, geranyl acetate 12.0%,
(Hoffmanns. et Link) borneol + a-terpineol
Brot. ex Coutinho (Roque
and Salgueiro, 1987)
(Salgueiro and Proenga (1) linalool 87.096, thymol, P-caryophyllene, linalyl acetate, (2) thymol
da Cunha, 1989) 49.2%,p-cyrnene 19.4%, y-terpinene, borneol+ a-terpineol, camphor,
(3) geraniol 52.5%, geranyl acetate 38.0%, borneol + a-terpineol, (4) linalool
+
42.1%,1,s-cineole 32.5%, borneol a-terpineol, P-pinene, thymol and
(5) 1,s-cineole 29.2%, thymol 25.6%,p-cymene 10.3%, y-terpinene,
+
borneol a-terpineol
(Proenga da Cunha and (1) linalool 68.0-92.196, (2) thymol 25.0-62.1%, p-cymene 11.2-49.9%,
Salgueiro, 1991) 7-terpinene 3.5-28.5%, (3) carvacrol 32.5-65.2%, p-cymene 12.5-31.0%,
7-terpinene 3.9-15.5%, (4) geraniol 21.1-67.5%, geranyl acetate
13.1-59.1%, (5) 1,8-cineole 2 5 . 0 4 2 . 5 % , linalool 2 4 . 5 4 9 . 1 % ,
(6) 1,s-cineole 19.0-39.5%, thymol 18.5-37.1%, p-cymene 9.5-16.0%,
(7) a-terpineol+ a-terpinyl acetate 60.8-7 1.1%, (8) linalool 20.2-58.5%,
thymol 16.0-33.5%,p-cymene 10.1-18.5%, and (9) 1,8-cineole 15.0-19.5%,
linalool 28.2-39.9%, thymol 12.5-21.5%
T. zygis L. ssp. zygls geranyl acetate 0.1-59.9%, thymol 0.4-38.5%, carvacrol 0.5-28.6%,
(Salgueiro et al., 1992) p-cymene 0.5-29.0%, y-terpinene 0.5-19.5%

(Salgueiro etal., 1993) carvacrol 42.0%,p-cymene 19.9%, y-terpinene 15.0%, linalool;

0-caryophyllene
South of France
T. nitens Lamotre (1) geraniol 90%, (2) a-ter~ineol90%, (3) thymol 50%,p-cymene 30%,
(Granger et al., 1973) (4) carvacrol 1540%,p-cymene 50-70%, and (5)p-cymene 80%
(Adzet et al., 197713) ( I ) phenol type, (2) geraniol type, and (3) a-terpineol type; percentages not
given
T. serpyllz~7zpfaecox geranyl acetate 25.0%, p-caryophyllene 13.8%, geraniol 11.8%, nerolidol,
(Opiz) Wollrn. (Vernin T-cadinol
etal., 1994)
7. uz~lgdrisL. (only of (1) geraniol +geranyl acetate up to 9396, (2) llnalool+linalyl acetate 95%,
wild origin) (Granger (3) a-terplneol+ a-terpinyl acetate 90-96%, (4) trans-sabinene hydrate up to
and Passet, 1971, 1973) 56%, terpinen-4-ol + terplnen-4-yl acetate up to 43%, cic-myrcen-8-01
10-20%, (5) carvacrol up to 85%, and (6) thymol up to 65%
Sardinia
T. L-apitati~s
Hoffmanns. carvacrol 73.4%, p-cyrnene 10.5%, 7-terpinene, borneol, ,O-caryophyllene
et Link, today: Thj177zh~a
capitata (L.) Cav. (Arras
and Grella, 1992)
7. herha-barona Lo~sel (1) carvacrol43%, and (2) thymol 45%
(Falchi, 1967)
Corsica
T. herha-ba~07zaLoisel (1) carvacrol 59%; p-cymene, 7-terpinene, (2) carvacrol 44%, p-cymene 13%,
(Granger and Passet, y-terpinene, (3) carvone 75%, dihydrocarvone l o % , limonene, (4) carvone
1974) 85%, limonene, and (5) carvacrol 50%, dihydrocarvone 20%
(Corticchiato et al., (1) carvone 74.6%, limonene, carvacrol, a-terpineol, cis-dihydrocarvone,
1995, 1998) (2) a-terpinyl acetate 55.9%, a-rerpineol 15.7%, linalyl acetate, geranial,
linalool, (3) carvacrol 59.4%, p-cymene, 2-heptanone, y-terpinene, borneol,
(4) thymol47.9%, carvacrol, p-cymene, y-terpinene, 2-heptanone, (5) linalool
31.8%, linalyl acetate 16.196, a-terpineol, geranial, geraniol, (6) cis-dihydrocarvone
77.1%, tr-dihydrocarvone, borneol, 1,s-cineole, a-terpineol, and (7) geraniol
60.6%, geranyl acetate 19.I % , terpinen-4-01, p-caryophyllene, a-terpineol
Table 3.2 (Continued)

Thy?izwspecies Essential ozl compositzon * (Main co77zponents only)

Romania
T. bali.anw Borb. a-terpineol 21.1%, linalool 14.1%, linalyl acetate 10.3%, a-terplnyl acetate,
(Kisgyorgy etal., 1983) neryl acetate
T. colnosus Heuff. neryl acetate 24.4%, carvacrol 12.5%, thymol 10.5%, camphene
(Kisgyorgy etal., 1983)
T. dacicz~sBorb. carvacrol 30.0%, thyrnol 16.8%, nerol, a-terpineol, linalyl acetate
(Kisgyorgy et dl., 1983)
T. glabrescens Willd. unknown compound 2 I.)%, geraniol+ geranyl acetate 15.596, linalool, nerol,
(Kisgyorgy etal., 1983) neryl acetate
T. pulegiozdes L. carvacrol 33.6%, thymol 31.2%,p-cymene 12.9%, neryl acetate, nerol
(Kisgyorgy etal., 1983)
Croatia
T. capitatz1~Hoffmanns. carvacrol 75.9182.6%, 1,s-cineole, limonene, bornyl acetate, linalyl acetate
et Link, today: Thyrnbra
capttata (L.) Cav.
(KuStrak etal., 1990)
T. glabrescens Willd. (1) 1,8-cineole 29.496, myrcene, camphene, a-pinene, P-pinene, and
(KuStrak etal., 1990) (2) thymyl acetate 14.396, carvacrol 10.796,p-cymene 10.0%, thymol,
bornyl acetate
T. longicaulzs C. Presl thymol40.1%,p-cymene 26.396, carvacrol, a-terpineol, -/-terpinene
(KuStrak etal., 1990)
T. pulegiozdes L. geraniol 38.3%14.7%, linalool 28.2128.6%, P-caryophyllene, thyrnol, geranyl
(Mastelic etal., 1992) acetate
(KuStrak etal., 1990) (1) carvacrol 29,896, p-cymene 15.1%, y-terpinene 11.8%, 0-caryophyllene
11.5%, a-pinene, (2) thymol 21.7%, carvacro1,p-cymene, thymyl acetate,
borneol, and (3) linalool49.4%, carvacrol 13.296,~-cymene,thymol,
P-caryophyllene
Bosnia-Herzegowina
T. glabrescens Willd a-terpinyl acetate 32.0%, terpinen-4-01, thymol, myrcene, a-plnene
(Karuza-Stojakovic
etal., 1989)
T. jazkae Celak. p-cymene 30.3115.5%, carvacrol 21.216.596, geraniol 2.3126.3%,
(Karuza-Stojakovic geranyl acetate 0.5/12.0%, a-terpinyl acetate
etal., 1989)
T. maly Ronn. in Hayek terpinyl acetate 4.811 3.8%, a - p ~ n e n e4.311 3.596, geraniol, camphene, myrcene
(Karuza-Stojakovic
etal., 1989)
T. ~7~arschallia~zus
Willd. thymol 13.7%,p-cymene, carvacrol, terpinen-4-01, a-plnene
(Karma-Stojakovic
etal., 1989)
T. pannonicz~sAll. (syn. a-terpinyl acetate 31.396, terpinen-4-01, carvacrol, thymol, geranyl acetate
T. kosteleckya~zwOpiz)
(Karuza-Stojakovic
et al., 1989)
T. pulegioides L. (Karuza- geraniol 29.817.1%, linalool 20.015.4%, thymol 6.3114.2%, carvacrol
Scojakovic etal., 1989) 4.311 1.1%, a-terpinyl acetate 8.1/16.7%
T. styzatu Vahl. (Karuza- terpinen-4-01 23.311 1.0%, a-terpinyl acetate 8.1/11.2%, linalool 6.6110.696,
Stojakovic etal., 1989) myrcene, limonene
Austria
T. praecox Opiz ssp. very polymorphous, type characterizing components: thymol, geranioligeranyl
polytrichus (Kerner ex acetate, tr-sabinene hydrateiterpinen-4-01, a-terpineol, linalool, linalool/linalyl
Borbas) Ronn. emend. acetate, tr-nerolidol, hedycaryol, T-cadinol, germacra-1(10),5-dien-4-01,
Jalas (Bischof-Deichnik germacra-l(l0),4-dien-6-01, borneol.
etal., 2000)

Hungary
T. serpyllum L. carvacrol 39.5145.9%, thymol,p-cymene, linalool, nerol
(Oszagyin et dl., 1996)

Slovakia
T. ulpestrzs Tausch (1) thymol 41.0%, P-caryophyllene 10.5%,p-cymene, 7-terpinene, carvacrol,
(Mirtonfi, 1992a) and (2) carvacrol47.0%, P-caryophyllene, y-terpinene,p-cymene, thymol
T. kosteleckyanus Opiz, ( I ) a-terpinyl acetate 74.9-84.5%, limonene 3.3-13.0%, a-terpineol,
syn. T. pannonicus All. (2) thymol41 .O-50.5%, p-cymene 16.7-25.5%, y-terpinene 7.2-14.8%,
(Mechtler etal., 1994a) geraniol, (3) linalool 70.0-72.0%, ( 4 )thymol 12.343.l%,p-cymene
6.5-36.7%, geraniol 1.3-29.6%, y-terpinene 1.6-27.3%, thymyl methyl
ether, and (5)p-cymene 32.6-66.2%, thymol 11.7-29.9%, thymyl methyl
ether 6.7-lo.?%, y-terpinene, geraniol
T. pruecox Opiz carvacrol 23-52%, p-cymene 15-19%, P-caryophyllene 16-2 1%
(Mechtler et al., 1994b)
T. pulrherrimu Schur percentages not given, main components: P-caryophyllene, P-bisabolene
(Mechtler etal., 1994b)
T. pulegioides L. (1) thymol 17.9-49.6%, ,L-caryophyllene 14.8-29.6%, y-rerpinene
(Mirtonfi et dl., 1994) 7.5-19.2%, carvacrol 0.7-12.9%, citral 1.3-11.3%, (2) carvacrol
24.0-58.1%, b-caryophyllene 11.0-34.9%, y-terpinene 1.9-25.6%,
p-cymene 0-17.796, citral0-10.2%, (3) linalool 33.4-92.3%, P-caryophyllene
0-32.096, citral 0-23.8%, geraniol 0-15.8%, carvacrol 0-7.9%, (4) citral
24.7-65.5%, geraniol 14.3-57.0%, P-caryophyllene 10.2-30.4%, linalool
1.1-22.9%, carvacrol, and (5) fenchone 1 8 . 5 4 6 . 3 % , P-caryophyllene
9.7-18.5%, citral, y-terpinene, carvacrol
(Mechtler etal., 1994b) p-cymene 18-27%, thymol 18-2596, geraniol 15-19%, P-caryophyllene 8-12%
T. pulegzoides L. ssp. ( I ) thymol 20.8%, P-caryophyllene 15.2%, -/-terpinene 14.9%, carvacrol
chumuedrys (Fries) 10.896,~-cymene,(2) carvacrol 32.9%, y-terpinene 17.2%, P-caryophyllene
Gusul. (Mirtonfi, 16.696, citra1,p-cymene, (3) linalool 54.8%, citral, P-caryophyllene,
1992b) geraniol, (4) citral 29.1%, geraniol 22.4%, P-caryophyllene 14.6%, linalool,
carvacrol, and (5) fenchone 33.9%, P-caryophyllene 10.4%, citral,
y-terpinene, carvacrol

Ukraine
T. borystenicum (Sur (1) thymol 33.6%, borneol+a-terpineol 24%, limonene 18.3%, unknown
et dl., 1988) 14.5%,p-cymene, and (2) borneol + a-terpineol45.9%, limonene, 1,8-cineole,
linalool, thymol
T. dimorphzls Klok. et carvacrol 13.1%, y-terpinene 12.7%, 1,s-cineole,p-cymene, P-myrcene
Shost. (Prikhod'ko etal.,
1999)
T. murschallidnusWilld. (1) thymol 29.0-59.3%, y-terpinene,p-cymene, carvacrol, (2) carvacrol
(Sur et dl., 1988) 39.1-61.4%, thymol, -/-terpinene,p-cymene, (3) thymol 26.4/19.7%,
carvacrol 28.1117.1%, y-terpinene,p-cymene, (4) geraniol 33.7-70.2%,
geranyl acetate 5.4-1 1.6%, geranial 1.7-10.1%, and (5) carveol 65.8%,
borneol, a-terpineol
T. serpyllum L. (Sur etul., (1) thymol 50.0/35.1%, y-terpinene 12.7/18.0%,p-cymene 8.6114.1%, and
1988) (2) ca~acrol48.4155.2%,-/-terpinene 10.1/27.1%,p-cymene 8.017.1%
Table 3.2 (Continued)

Thy?izusspecies Essential oil composition * (Main components only)

Romania
T. balcanus Borb. a-terpineol 21.1%, linalool 14.1%, linalyl acetate 10.396, a-terpinyl acetate,
(Kisgyorgy etal., 1983) neryl acetate
T. co?~zo~us
Heuff. neryl acetate 24.496, carvacrol 12.5%, thymol 10.5%, camphene
(Kisgyorgy et al., 1983)
T. dacicus Borb. carvacrol 30.0%, thymol 16.896, nerol, a-terpineol, linalyl acetate
(Kisgyorgy etal., 1983)
T. glabrescens Willd. unknown compound 2 1.3%, geraniol+ geranyl acetate 15.5%, linalool, nerol,
(Kisgyorgy etnl., 1983) neryl acetate
T. pz~legioidesL. carvacrol 31.696, thymol 31.296,p-cymene 12.996, neryl acetate, nerol
(Kisgyorgy et dl., 1983)
Croatia
T. capitatus Hoffmanns. carvacrol 75.9182.696, 1,8-cineole, llmonene, bornyl acetate, linalyl acetate
et Link, today: Thynzbra
~apitata(L.) Cav.
(KuStrak etal., 1990)
T. glabrescens Willd. (1) 1,8-cineole 29.496, myrcene, camphene, a-pinene, P-pinene, and
(KuStrak et al., 1990) (2) thymyl acetate 14.396, carvacrol 10.7%,p-cymene 10.0%, thymol,
bornyl acetate
T. longicaulis C. Presl thymol40.196, p-cymene 26.396, carvacrol, a-terpineol, -/-terpinene
(KuStrak et al., 1990)
T. pulegioides L. geraniol 38.3%!4.7%, linalool 28.2!28.6%, ,!-caryophyllene, thymol, geranyl
(Mastelic etal., 1992) acetate
(KuStrak et al., 1990) (1) carvacrol 29.8%, p-cymene 15.196, -{-terpinene 11.896, /%caryophyllene
11.596, a-pinene, (2) thymol 21.796, carvacro1,p-cymene, thymyl acetate,
borneol, and (3) linalool49.4%, carvacrol 13.296,p-cyrnene, thymol,
,!-caryophyllene
Bosnia-Herzegowina
1.
glabresrens Willd. a-terpinyl acetate 32.0%, terpinen-4-01, thymol, myrcene, a-pinene
(Karuza-Stojakovic
et al., 1989)
T. lankae Celak. p-cymene 30.3/15.5%, carvacrol 21.216.5%, geraniol 2.3126.3%,
(Karuza-Stojakovic geranyl acetate 0.5112.0%, a-terpinyl acetate
etal., 1989)
T. 71zaly Ronn. in Hayek terpinyl acetate 4.8113.8%, a-pinene 4.3113.5%, geraniol, camphene, myrcene
(Karuza-Stojakovic
etal., 1989)
T. ~narschallzanwWilld. thymol 13.796,p-cyrnene, carvacrol, ter~inen-4-01,a-pinene
(Karuza-Stojakovic
etal., 1989)
T. pannonicus All. (syn. a-terpinyi acetate 31.396, terpinen-4-01, carvacrol, thymol, geranyl acetate
T. kosteleckyanzls Opiz)
(Karuza-Stojakovic
etal., 1989)
T. pulegioides L. (Karuza- geraniol 29.817.1%, linalool 20.0!5.4%, thymol 6.3114.2%, carvacrol
Stojakov~cet al., 1989) 4.311 1.196, a-terpinyl acetate 8.1/16.7%
T. st~*iatz~s
Vahl. (Karuza- terpinen-4-01 23.3111.096, a-terpinyl acetate 8.1111.296, linalool 6.6110.696,
Stojakovic etal., 1989) myrcene, limonene
Albania
T. serpylhnz L. (Asllani, phenols 47-7495, p-cymene 8.5-36.5%
1973)
Macedonia
T. alhnus ssp. albanus (E)-nerolidol 20.3148.496, P-car~ophyllene18.0114.896, P-pinene, geranyl
H. Braun (Kulevanova acetate, linalool
et al., 1 9 9 8 ~ )
T. pnkae Celak. var. linalool 28.1135.6%, geranial 15.3120.2%, a-terpinyl acetate 11.310.4%,
jankae (Kulevanova P-caryophyllene, thymol
et al., 1998a)
T. jankae Celak. var. linalool 30.4130.996, geranial 16.0/22.6%, P-pinene, a-terpineol, geraniol
pantotrichus Ronn.
(Kulevanova etal., 1998a)
T. pnkae Celak. var. linalool 31.296, geranial 24.996, P-pinene, borneol, (E)-nerolidol
patentipilus Lyka
(Kulevanova era/.,
1998a)
T. longidens Velen. var. a-terpinyl acetate 16.296, a-terpineol 15.696, linalool 14.896, geraniol 14.596,
da~~areticusRonn. geranyl acetate
(Kulevanova etal., 1996a)
T. longzdens Velen. var. (1) carvacrol 33.696, geraniol 3 1.596, p-cymene, y-terpinene, geranyl acetate,
lanicaz~lisRonn. and (2) thymol41.5/35.7%, geraniol 12.0118.296, a-terpinyl acetate,
(Kulevanova etal., 1996a) p-cymene, y-terpinene
T. ~UL-edunicus (Degen et geraniol 18.596, tr-sabinene hydrate 14.396, linalool 11.796, a-terpinyl
Urum.) Ronn. ssp. acetate 11.3%, 0-pinene
7izacedonicus (Kulevanova
dal., 1995)
(Kulevanova et al., (1) linalool 46.6%, (2) geraniol43.396, geranyl acetate 37.696, linalool, and
1999) (3) a-terpinyl acetate 44.1-60.696, linalool 11.7-13.696, a-terpineol
T. 7izoeriacz~sVelen. geraniol 14.8-33.396, geranyl acetate 4.1 1-16.696, linalool 8.1-25.096,
(Kulevanova et al., carvacrol 12.3-13.3%, thymol
1996b)
T. rohlenae Velen. p-cymene 33.3%, y-terpinene 11.896, thymol, geraniol, linalool
(Kulevanovaetal., 1998b)
T. tosevii Velen. ssp. (1) geraniol 21.8'35, tr-sabinene hydrate 12.7'35, a-terpinyl acetace 13.6%,
rosevi~(Kulevanova et dl., a-rerpineol, linalool, and (2) a-terpinyl acetate 19.7'35, thymol 19.496,
1995) carvacrol, linalool, neryl acetate
(Kulevanova et al., polymorphous, various combinations, maln components: thymol, carvacrol,
1997) a-terpinyl acetate, geraniol, linalool, tr-sabinene hydrate
T. torevii Velen. ssp. ( I ) thymol 33.4%,p-cymene 11.5%, y-terpinene, linalool, P-pinene,
tosev~ivar. longifrons (2) carvacrol 45.376, p-cymene, -{-terpinene, thymol, geraniol, (3) carvacrol
Ronn. (Kulevanova 3 3 . l % , thymol 21.996,~-cymene13.0%, -/-terpinene 11.1%, (4)carvacrol
et al., 1 9 9 6 ~ ) 17.4%, thymol 17.396, a-rerpinyl acetate 15.1%, 7-terpinene, (5) a-terpinyl
acetate 22.396, carvacrol 2 1.1%, 7-terpinene, thymol, p-cymene, and
(6) geraniol 37.896, linalool 25.2'35, geranyl acetate 12.096, carvacrol 10.0%
T. tosevzz Velen. ssp. (1) thymol 24. 596, carvacrol 16.496,~-cymene,y-terpinene, P-pinene,
substriatus (Kulevanova (2) thymol45.6%, linalool 13.1%, p-cymene, y-terpinene, and (3) thymol
etal., 1997) 35.8%, linalool 32.9'35, y-terpinene, p-cymene
Greece
T. capitatzir Hoffmanns. carvacrol 67%, thymol, borneol, a-pinene, linalool
et Link, today: Th~l7~zbra
capitata (L.) Cav.
(Skrubis, 1972)
Table 3.2 (Continued)

Thymus species Essentzal oil composition * (Mazn components only)

(Philianos etal., 1982) carvacrol 55.5-81.O%,p-cyrnene 5.8-15.1%, y-terpinene 2.0-15.596,

1,s-cineole, @-caryophyllene
T. lougicaulis C. Presl (1) geraniol 56.896, geranyl acetate, nerol, 0-caryophyllene, a-terpinyl acetate,
ssp. chaubardii (2) linalool 63.196, a - t e r p i n ~ lacetate 20.4%, a-terpineol, limonene, and
(Reichb.fi1.) Jalas (3) thyrnol 19.496, lirnonene 18.7%, borneol, carvacrol, terpinen-4-01
(Tzakou et al., 1998)
T. parnassicus Halicsy P-caryophyllene 4.8-14.096, spathulenol 1.0-20.9%, 1,8-cineole 1.7-11.0%,
(Tzakou and caryophyllene epoxide, carvacrol, thyrnol
Constantinidis, 1998)
T. sibtborpii Benth. (1) geraniol 31.6%, thymol 22.1%, geranyl acetate,p-cyrnene,
(Katsiotis etal., 1990) P-caryophyllene, and (2) geraniol 30.1%, l~nalool23.0%, citronellyl acetate,
P-caryophyllene, geranyl acetate
T. tosevii Velen. (Katsiotis linalool 36.596, geraniol 27.596, thyrnol, borneol, citronellol
and Iconomou, 1986)
Cyprus
T. integer Grieseb. borneol 18.7-23.0%,p-cymene 15.7-25.0%, y-terpinene 9.2-12.3%,
(Bellomaria etal., 1994) thyrnol, llnalool
Turkey
T. argaeu Boiss. et Bal. linalool 26.6%, linalyl acetate 19.5%, borneol 15.0%, geraniol, nerol
(Sezik and Bqaran, 1986)
T. aznavourii Velen. gerrnacrene D 22.8%, (E)-P-farnesene 16.1%, a-pinene 11.1%,
(Turnen et al., 1998b) P-caryophyllene, limonene
T. atticus Celak. (Turnen thyrnol 37.2%, p-cyrnene, carvacrol, P-bisabolene, borneol
etal., 1997a)
T. bornmuelleri Velen. thyrnol 45.096, p-cyrnene 12.696, carvacrol, y-terpinene, a-pinene,
(Bager et al., 1993a)
T. canoviridis Jalas carvacrol 29.5%, geraniol 13.3%, thyrnol, 0-caryophyllene, geranyl acetate
(Bager etal., 1998)
T. capitatus Hoffrnanns. camacrol49.8-60.8%, thyrnol 1.3-18.8%, p-cymene, y-terpinene,
et Link, today: Thymbra terpinen-4-01
caprtata (L.) Cav. (Tanker
and Meri~li,1988)
(Ozek etal., 1995) carvacrol 68.7-77.996,~-cyrnene 6.1-lo.>%, -/-terpinene, terpinen-4-01,
myrcene
T. carzenszs Hub.-Mor. et borneol 13.496, 1,8-cineole 12.796, a-pinene 12.2%, camphor, carnphene
Jalas (Bager etal., 1992a)
T. cilicicus Boiss. et Bal. a-terpineol 33.496, camphor, citronellol, carnphene, 1,8-cineole
(Merifli and Tanker,
1986)
(Tiimen et al., 1994) a-pinene 16.7%, 1,s-cineole 10.4%, cir-verbenol, camphor, tr-verbenol
(Akgiil et al., 1999) a-terpineol 16.496, camphor, 1,8-cineole, a-pinene, linalool
T. eigii (M. Zohary et carvacrol 75.1%, y-terpinene, P-caryophyllene,p-cyrnene, a-thujone
P.H. Davis) Jalas (Sezik
and Saracoglu, 1987)
(Bager et dl., 1996a) carvacrol64.6%,p-cyrnene, P-caryophyllene, isoborneol, -/-terpinene
T. fallax Fisch. and Mey. carvacrol 68.1%, thymol, p-cymene, P-caryophyllene, y-terpinene
(Turnen etal., 1999)
T. Jedtschenkoi var. linalool 17.2%, borneol 10.4%, thymol, carvacrol, bornyl acetate
handelii (Ronn.) Jalas
(Meri~li,1986b)
(Bager etal., 2002) linalool 12.9%, a-terpineol, 1,8-cineole, tr-sabinene hydrate, camphor
T. haussknechtii Velen. linalool 19.9%, borneol 10.4%, 1,8-cineole, camphor, P-caryophyllene
(Bager etal., 1992a)
T. kotschyanus Boiss. et carvacrol 44.2%, linalool, camphene, limonene
Hohen. var. glabrescens
Boiss. (Merisli, 1986b)
T. leucostomus Hausskn. thymol 27.096, carvacrol 22.0%, linaloo1,p-cymene, borneol
et Velen. var. argillacew
Jalas (Bager et dl.,
1992b)
T. leucostonzus Hausskn. thymol 33.2%, borneol 22.2%,p-cymene, carvacrol, camphene
et Velen. var. gypsaceus
Jalas ( B a ~ eet
r dl.,
1999~)
T. leucostomus Hausskn. (1) carvacrol 21.6%,p-cymene 17.8%, thymol 14.1%, borneol, y-terpinene,
et Velen. var. leucostomus and (2) a-terpinyl acetate 23.8%, linalool 13.7%, borneol 12.9%, thymol
(Tumen et al., 199713) 11.3%, bornyl acetate
T. longicaulis C. Presl thymol 66.5169.7%*, carvacro1,p-cymene, y-terpinene, 0-bisabolene,
ssp. chaubardii (Boiss. et * var. chaubardzilvar. alternatus
Heldr. ex Reichb.fi1.)
Jalas (Bager and
Koyuncu, 1994)
T. longicaulis C. Presl (1) thymol 52.996,~-cymene18.3%, y-terpinene, P-bisabolene, carvacrol,
ssp. longicaulis (2) a-terpinyl acetate 82.1%, limonene, a-pinene, 0-bisabolene, sabinene, and
(Bager et dl., 1993b) (3) geraniol 68.8%, geranyl acetate 16.4%, Bbisabolene, nerol
T. longicaulis C. Presl thymol 21.7%, borneol 15.8%,p-cymene 15.4%, camphene, a-pinene
var. subisophyllzlt
(Borbas) Jalas (Bager
et dl., 1992b)
T. migricu Klok. et (1) carvacrol 36.3%, thymol, (2) Thymol44.2%, carvacrol, and
Des.-Shost. (3) carvacrol 36.5%, thymol 36.3%
(Bager etal., 2002)
T. pectinatw Fisch. et thymol 35.0%, borneol 17.7%,p-cymene 11. l % , carvacrol, camphene
Mey. var. pectinatus
(Bager et al., 1 9 9 2 ~ )
(Tumen et al., 1998a; thymol 52.5%,p-cymene 14.6%, y-terpinene 12.1%, carvacrol, borneol
Bager etal., 1999d)
T. praecox Opiz ssp. thymol 26.6%,p-cymene 24.9%, a-pinene, a-terpinyl acetate,
grmheimii (Ronn.) Jalas P-caryophyllene
var. grossheivziz (Bazer
etal., 1 9 9 6 ~ )
T. praecox Opiz ssp. thymol 17.8141.4%, carvacrol 10.517.6%, 1,8-cineole, P-caryophyllene,
skorpilii (Velen.) Jalas p-cymene
var. lanzger (Borb.) Jalas
(Ba~eretal., 1 9 9 6 ~ )
T. praecox Opiz ssp. geraniol 24.2%, a-terpinyl acetate 22.7%, geranyl acetate, linalool, linalyl
skorpilii (Velen.) Jalas acetate
var. skorpilii (Bager et al.,
1996~)
Table 3.2 (Continued)

Thynzz. species Essential oil colrzposition * (Main colnpponents only)

T. psez~dopulegioides (1) thymol 23.1%, linalool 20.1%, y-terpinene,p-cymene, carvacrol,

Klokov et Des.-Shost (2) thymol 50.196, carvacrol 10.796, p-cymene 10.796, y-terpinene, methyl
(Baser et al., 1999a) carvacrol, and (3) linalool 21.696, a-terpinyl acetate 16.796, geraniol 11.2%,
p-cymene, thymol
T. pubescens Boiss. et carvacrol 17.596, p-cymene 16.496, thymol 10.896, a-pinene, borneol
Kotschy var. cratevicob
Jalas (Bager etal., 1 9 9 9 ~ )
T. reuolutus Celak. (Merifli a-terpineol 30.5%, linalool 22.596,p-cymene, Pterplneol, a-terpinene
and Tanker, 1986)
T. roegneri C. Koch thymol 58.296,~-cymene12.996, carvacrol, P-bisabolene, borneol
(Tiimen et dl., 1997a)
T. sibthorpzz Benth. thymol 34.896, y-terpinene,p-cymene, borneol, myrcene
(Bager et dl., 1992d)
T. sipyleus ssp. sipylew geranial 32.8%, a-terpineol, neral, isoborneol, 1,s-cineole
vat. davisianus (Merifli
and Tanker, 1986)
T. sipylezds Boiss. ssp. (1) geranial 25.5-37.0%, neral 13.6-25.696, 1,8-cineole, a-terpineol,
sipylew vat. sipyleus camphor, (2) l~nalool21.8%, a-terpineol, geranial, llnalyl acetate, neral, and
(Baser etal., 1995a) (3) a-terpineol + isoborneol 25.596, geranial 12.896, neral, torreyol, camphor
T. spdthuliJoliw carvacrol 47.596,p-cymene, y-terpinene, linalool, linalyl acetate
Hausskn. et Velen.
(Merifli, 1986a)
T. striatw Vahl var. (1) thymol 10.596, borneol, carvacrol, tr-sabinene hydrate, P-b~sabolene,
interruptus Jalas (Bager (2) P-caryophyllene 29.696, carvacrol 20.696, caryophyllene epoxide,
etal., 1999e) (E)-P-farnesene, germacere D, (3) thymol 34.796, 0-caryophyllene 12,796,
,8-b~sabolene,caryophyllene epoxide, carvacrol, and (4) 0-caryophyllene
56.596, germacere D 11.196, carvacrol, linalool, caryophyllene epoxide
T. subcollinus Klok. germacrene D 3 1.996, $8-caryophyllene17.696, a-pinene, 6-cadinene, limonene
(Baser et al., 1997)
T. syriacu Boiss. (Tiimen thymol49.096, carvacrol 15.996,p-cymene, borneol, y-terplnene
and Bager, 1994)
T. thracicz1~Velen. var. (1) geraniol 15.796, thyrnol 12.396,p-cyrnene 12.296, -(-terpinene 10.7%,
lougidens (Velen.) Jalas carvacrol 10.5%, and (2) geraniol 47.396, geranyl acetate 18.396, camphor,
(Baser etal., 1995b) 1,s-cineole, 0-b~sabolene
T. zj~gzoidesGriseb. vat. thymol 24.696, linalool 12.296, borneol, carvacrol, 1,s-cineole
lycao~zicus(Celak.) Ronn.
(Merisli and Tanker,
1986)
(Baser et dl., 1996b) (1) carvacrol 48.1%, y-terpinene 12.096, thymo1,p-cymene, 0-bisabolene,
(2) geraniol 68.696, geranyl acetate, P-bisabolene, nerol, 8-caryophyllene,
(3) a-terpinyl acetate 36.296, a-terpineol 19.5%, ,R-bisabolene, bornyl acetate,
lirnonene, and (4) thymol 41.8-57.296, y-terpinene 1.3-19.596, P-bisabolene
1.2-15.996, p-cymene 4.1-12.096, carvacrol
T. zygioides Griseb. vat. linalool 33.796, (E)-nerolidol 12.596, neral, geranial, camphor
zygioides (Baser et al.,
1999b)
Caucasia
T. collinus Bieb. thymol 57.096,p-cymene, 1,8-cineole, carvacrol, terpinolene
(Kasumov, 1988)
T. corilfolzw Ronn. a-pinene 11.1%, limonene, y-terpinene, linalool, p-cymene
(Kasumov, 1987)
T. dagatanicus Klok. et thymol 32.7%, 1,8-cineole 12.6%,p-cymene, carvacrol, y-terpinene
Shost. (Kasumov and
Davidenko, 1985)
T. erzophorzts Ronn thyrnol 22.0%, geraniol 1 I . > % , linalool 1 I . > % , borneol 10.7%, linalyl
(Kasumov, 1981) acetate
(Kasumov and linalool 12.2%, borneol, thymol, carvacryl acetate, limonene
Komarova, 1983)
T. Jedtschenkoi Ronn. thymol 63.4%, y-terpinene, carvacrol, a-pinene, 0-caryophyllene,
(Kasumov, 1988) terpinolene
T. fonzznzz (Kasurnov, carvacrol l5.8%, thymol 15.8%,p-cymene, y-terpinene, terpinen-4-01
1981)
T. karalnarianir7~s thymol9.1126.1/26.3%, carvacrol 9.1110.0/14.8%, citral 10.3128.6120.4%,
Klok. et Shost. a-terpineol 12.411 5.01 18.2%, linalool
(Kasumov and
Farkhadova, 1986)
T. kjapazii G . Grossh. thymol 24.3%
(Novruzova and
Kasumov, 1987)
T. kotschyanur Boiss. et carvacrol 13.7114.7%, thymol 10.111 1.1%, y-terpinene, terpinolene,
Hohen. (Kulieva et al., P-caryophyllene
1979; Kasumov and
Gadzhieva 1980;
Guseinov et al., 1987;
Kasumov, 1980)
(Kasumov, 1988) thymol 55 5 % , p-cymene 17.7%, carvacrol 11.7%, a-pinene, a-terpineol
T. nzarschallzanw Willd. geraniol 30.3%, limonene 10.1%, a-pinene, a-terpineol, P-caryophyllene
(Kasumov, 1987)
T. nzigriczts (Kasumov, carvacrol 35.7%, y-terpinene 13.3%, thymol 13.3%,p-cymene,
1981) ~caryophyllene
T. nunznzztlariw M.B. 1,8-cineole 29.496, thymoi 28.3%, borneol 13.3%, carvacrol, limonene
(Kasumov and Ismailov,
1975)
(Kasumov and 0-caryophyllene l o . > % , thymol, linalool, borneol, terpinen-4-01
Gavrenkova, 1982)
T. pustoralzs Iljin thymol 32.6%, carvacrol 2 1.1%, terpinen-4-~lacetate 19.5%, p-cymene,
(Kasumov and y-terpinene
Davidenko, 1985)
T. rarlfloru C. Koch 23 compounds, no percentages given; main compounds thymol and carvacrol
(Kasumov, 1979)
T. serpyllu77z L. (1) thymol 8 1.5176.1%,p-cymene, carvacrol, ,O-caryophyllene, a-terpineol, and
(Abetisjan etal., 1988) (2) carvacrol 49.0-62.0%, thymol 21.5-29.7%,p-cymene,
0-caryophyllene, a-terpineol
T. tiflitiensis Klok. et carvacrol 20.1%, Pcaryophyllene 18.2%, y-terpinene, linalool, geraniol
Shost. (Kasumov, 1987)
T. transcaucasiczts Ronn. thymol 36.6%, p-cymene 15.7%, carvacrol, y-terpinene, borneol
(Kasumov, 198 1;
Kasumov and
Gavrenkova, 1985)
(Kasumov and thymol 46.8%, carvacrol 10.8%, -/-terpinene, ,!?-caryophyllene, a-pinene
Komarova, 1983)
Table 3.2 (Continued)

Thymus species Essential oil co~nposition* (Main components only)

(Kasumov, 1987) 0-caryophyllene 14.3%, geraniol, geranyl acetate, carvacrol, linalool

(Kasumov, 1988) thymol 33.696, linalool 12.8%, carvacrol 11.7%, terpinolene, a-pinene
T. trautvetteri Klok. geraniol 9.8112.7%, linalool, borneol, geranyl acetate, P-caryophyllene
(Kasumov et dl., 1979)
(Ismailov etal., 1981) geraniol 10.6%, linalool, borneol, geranyl acetate
Libya
T. algeriensis Boiss. carvacrol 36.8%, myrcene 20.2%, thymol 12.5%, a-terpinene 10.7%,
(Aboutabl and a-thujene
El-Dahmy, 1995)

Egypt
T. bovei Benth. (Aboutabl thymol 68.4%,p-cymene, thymyl acetate, carvacrol, y-terpinene
etal., 1986a,b)
T. deczlssatus L. (Khodair thymol 69.796, phellandrene, carvacrol, a-terpinene, y-terpinene
etal., 1993)
Israel
T. c@itatus Hoffmanns. (1) thymol 39.3/>2.6%, carvacrol 12.7118.1 %, y-terpinene 19.416.8%,
et Link, today: Thymbra p-cymene, P-caryophyllene, and (2) carvacrol 34.8139.8%, y-terpinene
capitata (L.) Cav. (Ravid 14.7112.296,~-cymene13.6111.3%, thymol, P-caryophyllene
and Putievsky, 1985,
1986)
(Fleisher etal., 1984) (1) thymol 50.8-67.0%, carvacrol, (2) carvacrol 60.3-76.1%, thymol, and
(3) carvacrol 3 4 . 5 4 3 . 7 % , thymol 14.4-29.6%
EthiopiaIErithree
T. schimperi Ronn. (1) thymol 36.8%,p-cymene 20.5%, y-terpinene 12.9%, carvacrol,
(Nigist Asfaw etal., a-terpinene, and (2) carvacrol 63.3%, y-terpinene, thymol, linalool,p-cymene
2000)
T. serrulatzls Hochst. ex thymol 48.5%, y-terpinene 12.9%,p-cymene 12.8%, carvacrol, linalool
Benth. (Nigist Asfaw
et dl., 2000)
Saudi Arabia
T. vulgaris L. (Mossa thymol 62.2%, carvacro1,p-cymene, linalool, A-3-carene
etal., 1987)
Iran
T. carnzaniczls Jalas thymol40.8%, carvacrol 24.896, y-terpinene,p-cymene, borneol
(Rustaiyan etal., 2000)
T. kots~h~anzls
Boiss. et thymol 38.096, carvacrol 14.2%, 1,8-cineole 13.2%, linalool,p-cymene
Hohen. (Rustaiyan
etal., 2000)
(Sefidkon et al., 1999; carvacrol40.7-61.2%, thymol 7.5-26.9%, y-terpinene,p-cymene, borneol
Sefidkon and Dabiri,
1999)
T. pubescens Boiss. et thymol 37.9%, carvacrol 14.196,p-cymene 13.1%, y-terpinene, linalool
Kotschy ex Celak.
(Rustaiyan etal., 2000)
Pakistan
T. serpyllum L. thymol 42.6%,p-cymene, carvacrol, borneol, terpinen-4-01
(Sattar etal., 1991)
India
T. serpylhm L. (Razdan carvacrol49.496, p-cymene, thymol, zing~berene,eugenol
and Koul, 1975)
(Gulati and Gupta, thymol 57.696, p-cymene 20.0%, y-terpinene, zingiberene, borneol
1977)

Mongolia
T. dahurzcus Serg. a-terpineol 2996, linalool 14%, camphor, p-cymene, terpinen-4-01
(Shavarda et dl.,
1980)
T. gobicz~sTschern thymol 38%, p-cymene 20%, y-terpinene 16%, carvacrol, borneol
(Shavarda et al.,
1980)

China
T. magnus Nakai (Han thymol, carvacro1,p-cymene, y-terpinene, a-pinene; percentages not given
and Kim, 1980)
T. mongolzcus Ronn. syn. carvacrol 5 1.2%,p-cymene 11.796, borneol, 1,8-cineole, thymol
T. serpylhm L. var.
mongolicus Ronn. (Fang
Hong-ju etal., 1988)
(Luo and Song, p-cymene 30,396, thymol+carvacrol 20.0%, P-phellandrene 14.0%
1989)
(Zhang Hongli et dl., thymol 23.9%, 2,4,5-trimethyl benzyl alcohol 16.9%,p-cymene 16.3%,
1992) carvacrol 10.6%, o-tert. butylphenol
T. quinquecostatus Celak. (1) linalool45.2%, borneol 13.5%, y-cadinene, tr-menthen-1-01, camphene,
(Shyuan Qi etal., 1987) and (2) thymol 21.9, carvacrol 20.3%, sabinene 1 l.O%, borneo1,p-cymene
(Fang Hong-ju et al., (1) phenol type: see var. prazewalskii, (2) linalool 72.9129.7%, borneol
1988) 7.8/13.1%,p-cymene 0.3116.5, terpinen-4-01, camphene, and (3) ester type:
see var. asiatzcus
T. quinquecostatus Celak. geranyl acetate 18.4%, carvacrol 11.2%, geraniol, borneo1,p-cymene, myrcene
var. ariaticus (Fang
Hong-ju etal., 1988)
T. quinquecostatus Celak. carvacrol 21.296, y-terpinene 13.7%,)-cymene 10.6%, rhymol, c a m ~ h e n e
var. prazewalskii (Fang
Hong-ju et al., 1988)

Japan
T. quinquecostatus Celak. thymol 56.1%, carvacrol, p-cymene, oct-1-en-3-01, camphor
(Kameoka et al., 1973)

N e w Zealand
T. uulgaris L. (Morgan, (1) thyrnol49.796, and (2) carvacrol48.8%, further components in both types:
1989) p-cymene 8 . 8 4 1 . 4 % , y-terpinene 0.6-15.6%, linalool

Chile
T. vulgaris L. (Montes carvacrol43%,p-cymene 41%, a-pinene, limonene, borneol
Guyot etal., 1981)

Cameroon
T. uulgaris L. (Arnvam thymol 27.2%,p-cymene 23.6%, y-terpinene 22.7%, linalool,
Zollo et dl., 1998) P-caryophyllene

Note
'Main components only: first five components; components >lo% with percentages, components < l o % wwlthout
percentages, in numerical order.
adjoining Caucasian region follow. At the end, some countries of the eastern Mediterra-
nean area in Africa, the Near East, the Middle East and the Asiatic countries of the Far
East are listed, ending with New Zealand; at the very end Cameroon and the only plant
source of South America in Chile is treated. Specialities of the evaluation will be
referred to as footnotes within Table 3.2 itself.

VARIABILITY I N ESSENTIAL OIL COMPOSITION

In plants the essential oil yield and chemical composition vary considerably due to
different factors. Both intrinsic and extrinsic factors have to be considered. As intrin-
sic factors we encounter genetic and sexual variations as well as seasonal and onto-
genetic variations. Extrinsic factors are described by ecological and environmental
aspects such as altitude, soil, climate, light, etc. There are no systematic investigations
of all these circumstances influencing the oil composition for the genus Thymw.
Therefore only results gathered from individual analyses, arising from different inter-
ests of the researchers in both applied science and basic science can be summarised.
It must be stressed that there are a few early reports dealing with oil variations which
erroneously interpret them as being caused by extrinsic factors. Nowadays they are
more correctly described as being a result of chemical polymorphism. Since this
phenomenon is widespread in the genus Thymus, Chapter 4 is dedicated to this
important topic.

Infraspecific variations
In Lamiaceae the phenomenon of infraspecific variability concerning the essential oil
composition, meaning chemical differences that exist in morphologically identical species,
was examined systematically by Lawrence (1980). His concept guaranteed that only
taxonomically authenticated plants harvested at approximately the same stage of growth
were analysed, an indispensable prerequisite which has often been neglected by other
scientists. He found five species of the genus Mentha, namely Mentha arvensis, M. longzfolid,
M. pulegiurn, M . spicuta, and M. suaveolens, to be polymorphous as well as three species of
the genus Monarda, and 9 species of the genus Pycnanthenzanz.
Regarding the genus Thynzw he referred to the noteworthy publication by Adzet etal.
(1977a) who had presented an examination of the polychemism in Mediterranean
Thynzus species some years before. The authors had found some species to show a distinct
tendency towards chemical differentiation, especially T . aestivus, T. herba-barona, T. hymlis,
T . mastichina, T . nitens, T. valgaris, and T. zygis. Other southern species, such as
T . antoninae, T. longiforus, T . membranaceus, and T. piperella, only showed minor chemical
variations. Regarding the geographical distribution of the investigated species and
chemotypes, Adzet (1977a) and Passet (1979) interpreted the polychemism within
the genus Thynzus as a result of a dynamic evolution, which does not only preserve
the species, but also ensures a territorial advantage by a process of adaptation to the
environmental conditions. This very far-reaching interpretation has not been
substantiated.
Within the genus Thymus the phenomenon of polychemism, an expression coined by
TCtCnyi (1970), was described for the first time when Granger and Passet (1971, 1973)
published the results of their studies of the essential oil chemistry in T . valgaris
 E~sentzaloil chemistry of the genzls Thymus 107
collected in the south of France. Detailed analyses of populations as well as of a multitude
of individuals proved T . vulgaris to be polymorphous, showing six different chemotypes
characterised by the following main oil constituents: thymol, carvacrol, tr-sabinene
hydrateiterpinen-4-01, a-terpineol, linalool, and geraniol. A correlation between climate
and distribution could be established because the phenolic types were growing in hot
and dry regions whereas the linalool and the a-terpineol types preferred a humid
climate, the geraniol type humid and cold regions.
The six chemotypes of T . vulgaris in the south of France fulfill the requirements for
"chemical races" as defined by Hegnauer (1978) who described them as growing in
populations which are geographically separated and presenting hereditary chemical
characteristics. Contrary to that, other Thymus species such as T. baeticus, T . camphoratus,
T . hyemalis, T . praecox ssp. arcticus, T . praecox ssp. polytrichus, T . tosevii, T. willkommii,
and T. zygis ssp. sylvestris proved to be mote polymorphous, showing a higher or even an
uncertain number of chemotypes. Moreover, no restricted occurrence of a certain
chemotype can be observed and different chemotypes grow side by side within one
population. Such a non-homogeneity of populations can only be reliably proven when
a multitude of individual plants of a species are analysed. This should always be con-
sidered in the experimental concept.
Nowadays within the genus Thymw the flood of publications reporting on infraspecific
variations concerning the essential oil composition does not allow a scientifically pro-
found compilation of such data. When available they have been included in Table 3.2.
The column "Essential Oil Composition" provides a rough idea on the variability of the
oil composition by indicating there the chemotypes with (I), (2), (3). . .This enumera-
tion of chemotypes does not include any information on the quality of the experimental
concepts, which unfortunately differs considerably, nor on the authors' interpretations
if any. Further details can easily be gathered from the original publications cited in
Table 3.2. In order to establish the concept of polychemism within the genus Thymw
more thoroughly, Chapter 4 of this book is dedicated to this phenomenon and presents
representative studies in this field, in particular the results elaborated by Salgueiro etal.
from species growing in the western part of the Iberian Peninsula, by Garcia-Vallejo
etal., Arrebola, Cafiigueral etal., Blanquer etal., and Siez from species growing in the
central, eastern and southern parts of the Iberian Peninsula, as well as by Stahl-Biskup
focusing on the species of northern European latitudes. Mirtonfi contributed greatly to
the knowledge of the polychemism in the Slovenian Thymw pulegioides and Kulevanova
discussed the polychemism of the Macedonian Thymus tosevii.

Sexual variations
Within the genus Thynzw gynodioecy is widespread, revealing female and hermaphrodite
plants. Two subspecies of T. serpylloides, ssp. gadorensis and ssp. serpylloides, were investi-
gated with regard to the differences in oil yield and oil composition between female
and hermaphrodite plants. Both subspecies belong to the phenolic group containing
carvacrol as the main constituent of their essential oils. Concerning the variations of the
carvacrol content in two different phenological stages (full flowering, fruiting) of
T . serpylloides ssp. serpylloides, no significant differences in the percentages of catvacrol
between hermaphrodite and female plants were found when the mean of three years was
evaluated, although in one case a concentration of this component higher by 17 per cent
with respect to the hermaphrodites was striking (Arrebola etal., 1994). The oil content
changed reciprocally.
Differences between female and hermaphrodite plants were more distinct in
T. serpylloides ssp. gadorensis. There the highest oil content was always obtained from female
individuals in both years, as well as at all three phenological stages investigated (full
flowering, fruiting, post-fruiting). In contrast to that the carvacrol content was always
found to be higher in the hermaphrodite individuals (Arrebola etal., 1995). Sexual
differences were also discovered in T. baeticus (Cabo etal., 1990). There the contents of
1,8-cineole and terpinen-4-01 were found to be higher in the oils of female plants than
in hermaphrodite plants (21 per cent and 11 per cent resp. versus 14 per cent and 8 per
cent), whereas the contents of citral and geraniol were lower (7 per cent and 5 per cent
versus 11 per cent and 2 1 per cent, respectively). However, the results give rise to some
doubts because of the fact that T. baeticus later turned out to be a highly polymorphous
plant (Siez, 1999), and the differences then found may have been caused by having
investigated different oil types.

Seasonal and ontogenetical variations

Results published on the chemical composition of Thymw oils reveal that most of the
oils were produced from flowering plants. This period in the plants' life cycle is chosen
because the oil yield usually peaks at that time. At least this was found for T. vulgaris
and T. pulegioides in the early 1960s (Messerschmidt, 1964; Tucakov, 1964), and later
for T. herba-barona (Falchi-Delitala etal., 1983), T. granatensis (Cabo et dl., 1986a),
T. pectinatus var. pectinatus (Ba~eretal., 1999d), T. zygis ssp. sylvestris (MoldPo-Martins
etal., 1999). But this fact does not always seem to be valid because in a different year
the oil content was found to be peak at different times for T. vulgaris (Messerschmidt,
1964; Weiss and Fliick, 1970). A detailed study of T. hyemalis during its complete
vegetative cycle revealed that the oil yield varying between 0.15 and 0.58 per cent
peaks twice, the first time in April (0.52 per cent) at the flowering stage and a second
time in July (0.58 per cent) (Cabo etal., 1987). The same phenomenon was said to be
found for T. pulegioides collected from mid-April to mid-September in Campania, Italy
(Senatore, 1996). The oil increased from 0.38 per cent in April to 1.11 per cent in May
when the plant was in full flower. But studying the presented data in detail, the second
peak found in June (0.93 per cent) might be an error in the method, at least too few
collections were examined. Egyptian T. vulgaris was reported to show only minor seasonal
variations in the oil content (Karawya and Hifnawy, 1974).
With respect to the oil composition most attention was focused on the phenol
containing species due to the economic interest in these substances. Detailed results ate
documented for T. vulgaris, which shall be summarized shortly. In the two older papers
it was stated that the composition of the oils was very constant over the whole season
(Messerschmidt, 1964; Weiss and Fliick, 1970), whereas other authors could observe
considerable variations. For example Egyptian T. vulgaris showed an increase of the
total phenolic portion before the full flower with thymol and carvacrol temporarily
forming contrarotating curves (Karawya and Hifnawy, 1974). Holthuijzen (1994)
observed a clear decrease in the thymol content from the beginning of June (60.7 per cent)
to the end of July (25.5 per cent) increasing again until October (39.0 per cent). The
sum of the biogenetically related tetpenes (thymol, carvacrol, p-cymene and y-terpinene)
however varied only within limited borders (74.3-82.1 per cent). In T. pulegiozdes
 Essential ozl chenzistry ofthe genus Thymus 109
collected in Campania (Italy) the highest phenol content was observed at full flower at
the end of May (43.3 per cent) increasing from 19.2 per cent in April and decreasing to
22.4 per cent in September (Senatore, 1996). The thymol content of T . pectinutus var.
pectinutus was found to peak in the pre-flowering stage (Bager etul., 1999d).
In order to determine the optimal harvest time for T. vulgaris in New Zealand
naturalized plants were studied intensively during 13 months (McGimpsey and Douglas,
1994). It could be shown that seasonal variation has a significant effect on the yield and
composition of thyme oil. Oil yield and phenol content peaked after flowering had
finished (December). The maximum total phenol content peaked at a total of 37 per
cent after flowering in summer (De~emberIJanuar~). p-Cymene, which was an important
component of Central Otago thyme oils, ranged from 4 0 to 50 per cent in winter and
early spring (May to October), declining to 21 per cent in January. The authors conclude
that harvest after flowering in December (summer time) delivers the highest yield and
the best quality. Aiming the use of the essential oil from T . zygis ssp. sylvestrzs in Portugal
as a food ingredient, the most interesting stage is the post-flowering period, the essential
oil at this time being rich in thymol, geranyl acetate and geraniol, with p-cymene
presenting lower levels (MoldEo-Martins et dl., 1999).
Seasonal variations of compounds other than phenols were studied in various other
Thymw species. Both, the linalyl acetate content of T. pruecox ssp. arcticus (Holthuijzen,
1994) and the geraniol content of T. x citriodorus (Stahl-Biskup and Holthuijzen, 1995)
varied to different extents during the period from June to October, the first within the
limits 59-68 pet cent the latter between 62 and 7 9 per cent. A low fluctuation was
established for linalool within the oil of a linalool-chemotype of T. vulgaris in the south
of France (Granger etul., 1965). Contrary results were gained from T . leptophylhs whose
linalyl acetate content varied considerably between 35 per cent in March and 73 per
cent in September (Zafra-Polo etul., 1988a).
Marhuenda etul. (1987) focused on the borneol content in T. carnosw ranging from
38.6-50.6 per cent with one peak in April and another in August. The high content of
borneol in April coincided with a minimum of terpinen-4-01 and with a general decrease of
hydrocarbons. Very high seasonal variations are reported for T. granutensis (Cabo etal.,
1986a), T . marschalliunus (Schratz and Horster, 1970), and T. hyemulis (Cabo etal.,
1987). Referring to the latter, throughout the growth cycle of the plants, the essential
oil was rich in hydrocarbons varying from a minimum of 31 per cent in November to
a maximum of 53 per cent in July, with a secondary peak in April and May. Among
these compounds myrcene exhibits the widest fluctuations (between 9 and 31 per cent).
Variations in the alcohol-acetate fraction covered a much narrower range from 10 per cent
in August to a maximum of 20 per cent in October; 1,s-cineole peaked at 27 per cent
in August and fell to 1 3 per cent in October, camphor from 2 1 per cent in September to
11 per cent in July.
These few examples make clear that seasonal variations in the oil compositions are
encountered and have to be investigated individually. General predictions cannot be
made. Rather we learn that the oil compositions given (e.g. in Table 3.2) are not more
than momentary snaps that must be treated with caution. Especially if oils or even the
herbs are considered for commercial purposes, the decision for the time of harvest must
be made individually.
W i t h regard to ontological variations only one study is dedicated to the essential oil
of T, marschullianw (Schratz and Horster, 1970). Considerable differences in the oils
comparing young leaves and one-year-old leaves within one plant were documented, the
more if the whole season is considered. These findings reveal different biosynthetic
capacities of oil glands in statu nascendi or adult oil glands.

GLYCOSIDICALLY B O U N D VOLATILES

The existence of glycosidically bound monoterpenes became evident for the first time,
when Francis and Allcock (1969) reported on the detection of geranyl, neryl, and cit-
ronellyl glucosides in rose petals. At that time the discovery of monoterpene glycosides
focused attention on a new field of research, especially in Lamiaceae, and has led to
speculations about their role. Glycosidically bound volatiles were assumed to be transport
forms of monoterpenes or involved in monoterpene catabolism (Skopp and Horster,
1976; Croteau and Martinkus, 1979). T. vulgaris was one of the early objects which
gave rise to such hypotheses.
After acid and enzymatic treatment of fresh leaves of thyme (T. vulgaris), thymol and
carvacrol could be detected as main hydrolysis products besides minor amounts of linalool
and geraniol (Skopp and Horster, 1976). Glucose and galactose were found to be the
sugar moieties. The same hydrolysis products were found when fresh plant material of
T. vulgaris was exclusively subjected to an enzymatic hydrolysis, besides further aglycones,
namely hexan- 1-01, cis-hexen-3-01- 1, octanol-3, octen- 1-01-3, benzyl alcohol, phenethyl
alcohol and eugenol (Van den Dries and Baerheim Svendsen, 1989). In a geraniol type
of T. pulegioides geraniol was the main glycoside apart from smaller amounts of eugenol,
linalool, and 1-octen-3-01 (Mastelic etal., 1992).
These findings gave reason for a more intensive study of the composition of the
glycosidic fraction of four Thynzw taxa, T. vulgaris, T. pulegioides, T. x citriodorus,
T. pruecox ssp. polytrichus and T. praecox ssp. arcticus (Holthuijzen, 1994; Stahl-Biskup
and Holthuijzen, 1995). O n balance the result of these investigations can be summarised
as follows (Holthuijzen, 1994): a) the content of glycosidically bound volatiles usually
is much lower than the essential oil content with proportions of 1:60-100 (T. vulgaris),
1:30-120 (T. x citriodorus), and 1:400 (T. pruecox ssp. arcticus). b) After enzymatic
hydrolysis of the glycosidic fraction a multitude of compounds can be detected, a
considerable number of them occurring in all the five taxa investigated, namely cis-hexen-
3-01-1, tr-hexen-3-01, octanol-3, octen-1-01-3, linalool, terpinen-4-01, a-terpineol, geraniol,
benzyl alcohol, phenyl ethyl alcohol, and eugenol. c) Nevertheless, besides these ubi-
quitous compounds each species had its individual pattern correlated to the composi-
tion of its free volatiles, at least in T. vulgaris (thymol and carvacrol), T. pulegioides
(thymol and linalool), and T. x citriodorus (geraniol). In T. pruecox ssp. polytrichus and
ssp. arcticus a structural equivalence between glycosidically bound volatiles and free
volatiles was much less distinct.
Comparing the variation (development, course) of the compositions of the glycosidically
bound volatiles and the free volatiles during one vegetation period, a difference between
T. vulgaris and T. x cztriodorus on the one hand and T. arcticus on the other hand could
be observed. In T. vulgaris and T. x cztriodorus the curves of main components of the
aglycone fraction run parallel to the free volatiles, thymol or geraniol, respectively,
always being the main component within both fractions (Stahl-Biskup and
Holthuijzen, 1995). In contrast to that, the composition of the aglycone fraction of
T. pruecox ssp. arcticus varied irregularly without any structural correlation to the essential
oil constituents (Holthuijzen, 1994).
 Essential oil chenzistry ojthe genus Thymus 11 1
Glycosylation in vegetable tissue is quite common and in essential oil-bearing plants it
might be a protective mechanism to prevent the lipophilic volatiles such as phenols or
alcohols from destroying membranes. Therefore it is of little wonder that in essential oil
plants the key intermediates of the terpene biosynthesis such as geraniol, nerol, linalool,
terpineol, terpinen-4-01 or intermediates of other pathways, such as aliphatic alcohols,
benzyl alcohol, phenylethyl alcohol are widespread in their glycosidic forms. From
observations in Thynzus (Skopp and Horster, 1976; Holthuijzen, 1994; Stahl-Biskup
and Holthuijzen, 1995) or in Mentha (Stengele and Stahl-Biskup, 1993, 1994), it can
be derived that if the essential oils mainly consist of hydroxylated terpenes, e.g. thymol
in T, valgaris, geraniol in T. x citriodorus, or menthol in M. xpzperita, the corresponding
glycosides are present in the same plant. This is the case although in Lamiaceaes special
accumulation sites exist in the form of glandular trichomes where such membrane
destroying compounds are assumed to be stored safely. Electron microscopic observa-
tions of the secretory cells of glandular trichomes of young Mentha piperita leaves make
it plausible that glycosylation takes place when the subcuticular space is full (Stahl-
Biskup etal., 1993).

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4 Essential oil polymorphism
in the genus Thymus
Francisco Skex and Elisdbeth Stahl-Biskup

INTRODUCTION

The complexity and diversity of living or even of extinct organisms have always attracted
man, since basic differences are recognized as essential for understanding the evolutionary
development of life. Scientists studying this variability have used the knowledge and
techniques available at their time to enhance their knowledge of the living creatures. Early
taxonomists interested in structuring the increasing complexity of the plant kingdom
initially discussed whether a certain form was or was not a true species. From this
debate the question developed if this form was sufficiently constant and distinct from
other forms, and whether the differences were sufficiently important to deserve a specific
name. Within the classical taxonomy based on plant morphology this happened many
times, as long as a genus was imperfectly known, until the limits among various species
were considered to be clearly established. The enhancement of this knowledge resulted
in the fact that more individuals were put into intermediate positions, thus demanding
a revision of previous concepts.
In the plant kingdom chemical polymorphism is well known and is seen in an
infraspecific variability of the chemical patterns of individuals or even of populations.
TCtCnyi (1970) coined the term 'polychemism' for this phenomenon, and it was he, in
the early 1970s, who showed 750 plant species of 106 families which were known to be
chemically polymorphous (TetCnyi, 1970). In species containing essential oils the
phenomenon of polychemism seems to be widespread; in 1975 TCtenyi had already
estimated 360 species of 36 families, and it was Lawrence (1980) who gave a first report
on the infraspecific differences in several genera of the Labiatae. In his publication, he
mentioned four genera which were found to be polymorphous, namely Mentha, Monarda,
Pycnanthemztm, and Thymus. Nowadays, as a result of the understanding that it is
a fundamental requirement to analyse infraspecific differences, we encounter a flood of
reports on polychemism, the genus Thymw being one of the most frequently investigated
and the most detailed research regarding this phenomenon.
The studies on the polymorphism of the genus Thymzts can be said to start with the
publications by Granger and Passet (1971, 1973), who reported 6 chemotypes for
T. vulgarzs after studying several populations and many individuals in the south of
France. In contrast, the majority of other initial efforts in this field must be compared
with the first steps of morphological taxonomy, mentioned above. As scientists tried to
characterise the essential oil of a species after analysing only a few samples, it was
impossible to know where in the 'cloud' of chemical variability these samples should be
placed.
126 Francisco Sdez and Elisabeth Stahl-Biskup
During the next decade some variations in essential oil composition were published
by authors such as Adzet etul. (1976), Bellomaria etul. (1981), Benjilali etal. (1987),
and it became evident that the real limits for this variability were still unknown. At the
same time new questions and doubts were arising, namely, whether a correlation between
classical taxonomy and chemical taxonomy could be found at the genus level; or, to
which extent one chemotype described for one species was exclusive or widely spread
over a group of them, or even over the whole genus.
This way the phenomenon became more and more interesting, and in the 1990s,
taking advantage of the technical improvements made during the previous decades,
numerous studies discussed the problem of polychemism in Thynzus. It was accepted
that a greater number of samples taken under homogeneous ecological conditions had
to be analysed and that the flood of data obtained needed to be examined with the help
of specific statistical techniques. Thus, Salgueiro, dedicated to the study of Thymus in
western Iberia, Garcia-Vallejo, Arrebola, Cafiigueral, Blanquer, SBez processed samples
from central, eastern and southern Iberia, Stahl-Biskup focused on the variability in
northern European latitudes.
When one looks at the taxonomical diversity described in Chapter 1 and sees how few
of these taxa have been intensively studied from the chemical point of view, the feeling
arises that still much research is necessary before we can develop a realistic impression
about chemical polymorphism in Thymus. Thus, the present chapter will focus on the
highlights found by the researchers interested in the phenomenon of polychemism so far,
with the conviction that the years to come will significantly improve our understanding
of the matter. The methods, techniques, and procedures used by the research teams
during the last 30 years or so have produced a bewildering variety of data that cannot
easily be brought together with at least a minimum of scientific confidence in the accur-
acy of the conclusions. It does not make any sense to mix in the same pot data obtained
from a few samples and a few compounds identified in the essential oil with studies whose
conclusions are based on statistical analysis of a representative number of individuals
and whose essential oils components have been adequately established.

GENETIC ORIGIN OF CHEMICAL POLYMORPHISM

Based on the distribution of different Thymw species in northern Africa, the Canary Islands,
the Iberian Peninsula and the Balearic Islands, and using today's knowledge of the
geological evolution of these areas during the last 5 million years from late Miocene
onwards, Morales (1986) explains the possible early evolution of the genus saying that
the diversification and expansion occurred mainly after the separation of the Iberian
peninsula from Africa. The origin would be the Tertiary xerophytic flora, with a great
evolutionary success achieved as new arid periods were encountered, especially during
the Pliocene and onwards up to now. The section Sevpyllum would play an important
role during the periods that showed a withdrawal of the ice cover with great diversification
during the cold phases of the Quaternary.
Genetic differentiation within a population may basically have developed in three
ways. (a) It may be a result of the isolation of individuals from a genetically variable
parental pool at the periphery of a population, with any new contact producing a clinal
intergradation. Individuals of different genotypes may have selective advantage in different
places within the total area of a population, forming a pattern of genetic polymorphism
 Essential ~ilpol~nzorphism
i n the germs Thymus 127
in a patchy but stable environment. (b) Where intrinsic barriers to gene exchange arise
in an environment that is changing in a particular direction (for instance, warming-up),
individuals whose genotype provides a better adaptation to the new circumstances are
selected, producing a sorting of variability. (c) Stabilizing or normalizing selection
produce uniformity in an already genetically variable population which is well adapted
to its environment. When this is not changing directionally and fundamentally, new
individuals that deviate significantly from the mean have less chance to survive than
the ones better adapted.
Apart from these gradual processes of separation from ancestral species, there is
another possibility for developing new taxa, namely abrupt speciation, by which new
species suddenly arise. Within the plant kingdom this is mostly due to polyploidy, and
Thymus owes a good part of its variability to this phenomenon, since several species
have been found to be polyploid.
All these events mentioned above may have occurred profusely along the Mediterranean
and adjacent areas, affected by a quite variable environment, resulting in today's diversity
of morphological and chemical taxa in Thymus. This speciation process cannot be regarded
as concluded, with precise and well-developed barriers among the different species.
Several reports from Morales and SBez on Spanish Thynzus and from Stahl-Biskup for
northern species show that intense interspecific relationships can be noticed in regions
where distribution areas for different species overlap and climatic conditions allow
simultaneous blooming and interchange of genetic material between them. This is
especially achievable for species included in the section Thymw.
In this chapter we provide a review of studies published on chemical polymorphism
of Thymus. They have been grouped regionally, reflecting both species distribution and
the different approaches that several authors have made to the problem.

THE SITUATION OF THYMUS I N SOUTHEASTERN SPAIN

The chemical polymorphism of the genus Thymus in southeastern Spain was studied in
detail by SBez (1996), who sampled 13 species living there, showing high taxonomical
diversity, perhaps influenced by the high variability in ecological conditions for such
a small area. These species are classified in the following four sections: (a) Section Thynzus:
T . hye~izalis,T. zygis, T. vulgaris, T. baeticus, T . orospedanw, T. serpyllaides ssp. gadorensis.
(b) Section Pseudothymbra: T . nzenzbranacezls, T . longzflorz~s,T.funkii, T. moroderi, T. antoninae.
(c) Section Piperella: T . piperella (monospecific), (d) Section Mastichina: T. vzastichina
(monospecific).
The essential oils of a total number of 327 individual plants of southeastern Thymzls
were analysed using gas chromatography (GC), and the results were studied with different
statistical methods to detect similaritiesldissimilarities among them. In order to quan-
tify the chemical polymorphism realised at the species, section and genus level, three
different sets of extensive statistics were put together: (a) Analysis of many individual
plants of each species to find out the infraspecific variability; the number of samples
investigated per species is directly related to its abundance and size of distribution area.
(b) Cluster analysis including all individual plants belonging to the same section dis-
regarding their affiliation to a distinct species. The results are presented in the form of
tables, one for the section Thynzw (Table 4.1) and another for the section Psez~dothymbra
(Table 4.2). (c) Principal component analysis of the complete data set of the genus.
 Essential oilpolymorphzsm i n the genus Thymus 129

Table 4.2 Section Pseudothymbra - the result of a cluster analysis of 85 individual samples

Chemotypes Number qi Species

individuals
T. antoninae T . junkii T. longrflorzs T . membranaceus T . moroderi

1,8-cineole(50-72%) 24 - 6 5 10 3
1,8-cineole (33-50%)1 18 1 3 7 3 4
camphor (10-27%)
1,s-cineole (22-5 1%) 15 3 4 1 5 2
camphor/borneol 12 2 2 4 3 1
1,8-cineole (73-82%) 5 - 3 1 1 -
several* 11 - 4 1 1 5
total 85 6 22 19 23 15

Note
* This group contains samples chemically quite different, with unusual combinations of compounds.

70
I Section thymus i iGeraniol
jAA j
'
.......... :...... ....... ..... ..... 4 .....
.......;~. ~~~~ ~ 4~~~ ........
....;.............. ~~ >~~ .....~ . ~ ~ . ~ .

-50 8 , m ; m + , m i m m , i r 8 ' ; m m m i , m m i m ' *

8 0 -60 -40 2 0 0 20 40 60 80
Factor 1

Figure 4.1 Principal component analysis of essential oils from southeastern Spain. Section Thymus.

Figures 4.1-4.4 reflect the results with respect to each section, section Thymw (Figure
4.1), section Psezdothymbra (Figure 4.2), section Mastichina (Figure 4.3), and section
Piperella (Figure 4.4).
Some aspects related to the whole genus are highlighted here before focusing on the
peculiarities of the species: (1) The homogeneity of the monospecific sections Mastichina
and Piperella contrasts with the more polymorphic sections Thymzs and Psezdothymbra.
From the Figures 4.1-4.4, it can be derived that there is not only more variability in
the latter two at the section level but also at the species level since T . mastichim or
T. piperella are not so widely dispersed. (2) The location of the samples containing
130 Francisco Sa'ez and EIisabeth Stahl-Bi~kup

70 -
-
- Section ~ i e u d o t h ~ m b r b
.. . . . .
50 ~~

T. longifloms
0 T. funkii
A T. moroderi

-50 -0
-80 -60 4 0 -20 0 20 40 60 80
Factor 1

Figure 4.2 Principal component analysis of essential oils from southeastern Spain. Section Pseztdothymbru.

70 -

Section Gastichina j
50 .. : . ; ....~~:
....... ; .. .... ~'~~~ .... ...... : ..... ...... . .

30 . . . . . . . . . . . . : ....... ~~~ ..,

'. ~ ~~~~~ ~~ ~ ~ . ..........
. ~ .....
~~~ ~ ~ ~ ....
~~~ ~ . . ..~
~ ~.
~~~ ~~ ~~

i linalool i
-
N

d
10 ;
..... ~~~ .... '~ ...... ...
~~ ' i
i o
.... ...
0 i o
i
...:. ....... ..:. . . :.... . ....
~ ;. . ; . . ~

i o i
1 0 .. . . . . . ..,...~~ ..... ....~~1
l ...........
o '
........... 1 . . . ..A ..... ' - - - ---- ' ---- - - -
~~ ---

1,8-cineole 8
. . . . .
r
s@o . i . . . . . ~~~'....
.....~' . . . .~I
....... ... ~'~ ............. . .
30
~
~~~~~~~
-

0 0
01

- 5 0 1 ; , 2 , , m 1 , , ,
-80 -60 4 0 -20 0 20 40 60 80
Factoi- 1

Fzgwe 4.3 Principal component analysis of essential oils from southeastern Spain. Section Mastichind

phenolic chemotypes can be clearly determined by comparing the sections Thynzw

(Figure 4.1) and Psezldothynzbra (Figure 4.2). Indeed, most samples from T. hyenzalis and
T. zygis plus some from T. serpylloides ssp. gadorensis, are concentrated in a relatively
small area and they mainly differ only in the concentration that phenols reach in the
 Essential oilpolymo~phzsmin the genus Thymus 131

70

I Section piperella
50 : :~~~~~
............ 4 . ~ .~ .~ ~ ~ ~ ~ .~ 4.. . ... . . :.... ........

1- 1
~~ ~~

. ..~~~...~: thjrnol. i.
30 . . . '-~~~......~......d.

cirvacrol
.

~ ~.....~
...~~~L~... ~ ~~ ~.
~~~~~ ~ ~ ,

N
...;....... ......i....~~~~~~~~~~~~~;~
............... 4
: o . . Q ;~
. . . O ~ ~ ~ ~ L... ......~ ~ .~ ~
~ ~ ~
~ ~ ~
~ ~
g
+ 10
0
d
-10 ..... + ~ .......... i.... 1 . . ' ' :...~.. .. .
.......... .... ....... .~...... . '... ...........

-30 . ....~~~:
. . . . .~ ~ : ~ .~ ~ .
. ~ ........ ~' :...... ~~~~~~~ .... '.. ....... :. . ........... :....~ .
- ~ ~

- 5 0 8 , 8 ; , r 8 ; * ' m ; , , , i , , , ; 8 8 8 ; z ' 7 ; m , ,
-80 -60 -40 -20 0 20 40 60 80
Factor 1

Figzlre 4.4 Principal component analysis of essential oils from southeastern Spain. Section Piperella.

essential oils. (3) In the section Thymus a small group of samples with multiple
taxonomical adscription is characterized by the presence of linalool. (4) The geraniol
chemotype realized in three samples from T. serpylloides ssp. gadore?uis is represented in
the most positive values for Factor 2, thus reflecting the low frequency for this com-
pound in the essential oils studied. (5) The deviations from the main trends in the
essential oils of a distinct species can easily be detected. This is the case of the two sam-
ples of T. zygis that were placed in the area for high 1,s-cineole, reflecting the influence
of T. vulgaris. (6) T. orospedanw shows an interesting pattern of chemotype distribution,
with highly dispersed samples through the variability defined by the genus. Something
similar happens to all the species in the section Pseudothymbra: there is no area in the
'cloud' of samples which is exclusive to one of the species, but all contribute in a similar
way to develop its shape.

Description of chemical polymorphism o n t h e species level

T. antoninae is a tetraploid species that is found in a very small area. SBez (1996)
described all the essential oils as being characterized by the presence of 1,s-cineole
(max. 44.3 per cent) and camphor (max. 35.8 per cent) with similar proportions.
Camphene and borne01 show lower levels, from 10 to 20 per cent, and myrcene reached
up to 8.2 per cent. Cafiigueral etal. (1994) only found a little chemical polymorphism
with respect to p-elemol, P-eudesmol and an unidentified sesquiterpene. Figure 4.2
shows this species in about the center of the diagram for the section Psendothymbra,
meaning comparatively that not very high percentages for 1,s-cineole or camphor were
found.
T. baeticus is spread along the central and eastern parts of southern Spain. It is an erect
shrub characterised by the greyish colour of the leaves and a globular inflorescence and
132 Francisco S& and Elzsabeth Stahl-BisRup
is of some economical importance. SBez (1999) studied the eastern-most communities
and found the remarkable presence of terpinen-4-01 (rnax. 28.8 per cent) appearing
together with a-terpineol, linalool or geranyl acetate. Borneo1 (rnax. 53.6 per cent) and
1,8-cineole (rnax. 56.2 per cent) characterise other groups of samples, and it is worth
mentioning a content of 14.9 per cent geranial in one sample, and 20.1 per cent
tr-sabinene hydrate in another. The presence of the precursors of thymol or carvacrol
(p-cymene, y-terpinene) was recorded in quantities up to 37 per cent, but the phenols
themselves did not appear in high quantities. This high variability can be observed in
Figure 4.1, although in the cluster analysis performed for the section Thymw (Table
4.1), most of the samples for this species are included in one main group characterised
by either myrcene or terpinen-4-01.
T . funkii is characterised by the small size of the bracts at the inflorescence and a high
morphological variability compared with other species from the section Pseudothymbra.
SBez (1996) studied 22 individuals whose essential oils were mostly characterised by
1,s-cineole (rnax. 78.8 per cent) present in all the samples. Camphor was also found
frequently, but in lower percentages, up to 19.1 per cent. Myrcene (max. 20.4 per cent)
appeared sporadically, and camphene and borneol showed even lower levels. The per-
centage of 21.4 per cent thymol in one sample is remarkable and explained as an intro-
gression with T. zygis. Figure 4.2 shows the samples for T . fankii mostly situated at the
1,8-cineole end. Vila etal. (1995) studied two populations using both collective and
individual samples. They noticed chemical differences between the two communities, but
only with respect to some compounds that presented low percentages in the essential
oils, such as a-eudesmol or caryophyllene oxide. No significant chemical polymorphism
was found in this study.
T.hyemalis grows in areas with moderate winter temperatures, near sea shores. It is of
very high economical importance due to the absolute predominance of phenols in the
essential oil. Thus, thymol (rnax. 36.7 per cent) and carvacrol (max. 41.3 per cent)
chemotypes are the most important ones, but the high ability to interbreed with other
species from the section Thymw leads to the appearance of compounds such as 1&cineole
(rnax. 25.7 per cent) and borneol (20.4 per cent) in restricted areas (SBez 1995a). The
case of one population with some individuals presenting a linalool chemotype and some
others a thymol chemotype is interesting, given the tendency of the linalool chemotype to
appear in colder areas. Other compounds with less significance with regard to the percent-
age in the essential oil (about 10 per cent) are a-pinene, camphene and a-terpineol.
Figure 4.1 and Table 4.1 clearly reflect this separation between phenolic and linalool
chemotypes in T . hyemalis.
The essential oil of T.longzflwus was found to be characterised mainly by either 1,8-cineole
(rnax. 74.7 per cent) or camphor (max. 46.6 per cent). Camphene and borneol reached
maximum percentages of about 20 per cent. These four compounds dominated, in different
combinations, in all the essential oils studied. Phenols or their precursors were practi-
cally absent, with a maximum of 3.5 per cent thymol in one sample. Morales (1986)
described one sample containing 26.2 per cent of terpinen-4-01. Figure 4.2 shows the
samples clearly split into two groups, and the one characterised by camphotiborneol in
Table 4.2 is the more common.
T. mastichina (Spanish marjoram) has attracted economical interests and has a wide
distribution area across Spain. In southeastern Spain SBez (1996) found its essential oil
characterised by 1,s-cineole (max. 79.9 per cent) and linalool (max. 56.6 per cent).
The chemotypes detected were either characterised by 1,s-cineoleilinalool, or - less
 Essential oil polymorphism in the genus Thymus 133
frequently - by 1,s-cineole alone, but no individual containing linalool without
1,s-cineole was found. Sabinene, tr-sabinene hydrate, camphor and borneol reached
percentages up to 5 per cent in different samples. Linalyl acetate scored 35.1 per cent in
one sample due to the early stage of development achieved at the time of collection.
The situation in T.mastichina is reflected in Figure 4.3 with the 1,8-cineole chemotype
represented more frequently. Garcia-Vallejo etal. (1984) studied a wider area, with 228
samples and described three chemotypes: a 1,8-cineole-type, a linalool-type, and a mixed
1,s-cineolellinalool-type.
T. membranaceus is recognised by an inflorescence with broad, pale-yellow bracts. SBez
(1996) described the essential oils as mostly characterised by 1,s-cineole (rnax. 82.2 per cent;
min.13 per cent), with camphene (max. 15.7 per cent), camphor (max. 35.6 per cent)
and borneol (max. 23.6 per cent) usually presenting lower levels. The oil of one sample
was described to contain 39.9 per cent linalool. It was collected in an area where also
T.zygis grows showing the same chemotype, thus suggesting a relation with this
species. Phenols were found to be almost absent (rnax. 1.5 per cent for both thymol and
carvacrol), but Zarzuelo etal. (1987) reported quantities of about 3.5 per cent for these
compounds. Studying T.membranaceus on the section and genus level (Table 4.2 and
Figure 4. I), these observations are supported.
T.moroderi is characterised by its deep purple bracts, and is of some economic interest
as a raw material for producing a liqueur. The essential oil is characterised by 1,s-cineole
(rnax. 68.1 per cent), camphor (rnax. 34.6 per cent) and borneol (rnax. 23.3 per cent).
Camphene reached percentages of about 20 per cent in two individuals from the same
population. Thymol (14.4 per cent) and carvacrol (19.0 per cent) appeared in two
different populations where this species may have introgreded with T.hyemalis. Previous
studies by Cafiigueral etal. (1994) revealed the existence of chemical polymorphism
based on the presence of either an unidentified oxygenated sesquiterpene or on the
simultaneous occurrence of /3-eudesmol, ledol, a-elemol and /3-elemol. But it is not
clear if this species presents a well-defined set of chemotypes. Figure 4.2 and Table 4.2
show this species, placing its samples all over the range of diversity for the section
Pseudothy mbra.
T. orospedanus presents an erect habit, with dense and short branching to protect itself
from low winter temperatures in the medium-high mountainous areas where it grows,
in a relatively small area. Twelve individuals have been chemically studied, and they
show the important presence of myrcene (rnax. 27.4 per cent) and 1,s-cineole (max.
34.3 per cent). The highest score obtained for linalool was 87.6 per cent. Caryophyllene
oxide, P-caryophyllene, borneol, camphene and tr-sabinene hydrate presented percentages
from 10-20 per cent in different samples. No significant quantities of phenols or their
precursors were found by SBez (1996). Although Table 4.1 collects most of the samples
studied into one chemical group, their situation within Figure 4.1 supports the idea of
a chemically variable species.
T,piperella presents broad-round to heart-shaped leaves and other morphological
features that makes this species quite distinct from the rest. The essential oil is typically
phenolic, and SBez (1996) described, for southern populations of the species, maximum
values of 14.4 per cent thymol and 10.2 per cent carvacrol, with higher percentages
of p-cymene (65.1 per cent). The presence of a-pinene, limonene, terpinen-4-01 and
1,s-cineole in quantities from 5-10 per cent did not represent important alternatives
to the phenolic character of the essential oil. A similar situation was found by Blanquer
etal. (1998), who described three chemotypes based on 31 individuals investigated:
134 Francisco Skez and Elisabeth Stahl-Kirkup
One p-cymenelthymol-type, one p-cymenelcarvacrol-type, and one p-cymenelcarvacroll
y-terpinene-type. They also found geographical separation among the chemotypes.
Figure 4.4 shows this species as the least variable one when studied within the whole
genus context.
T. serpylloides ssp. gadorensis can be found in the upper-most mountain ranges, exposed
to very low winter temperatures, thus showing a procumbent habit. It is of little
economic interest. The studies from SBez (2001) show that it is a mainly phenolic species,
with both thymol (max. 56.9 per cent) and carvacrol (max. 27.7 per cent) chemotypes,
usually excluding each other but sometimes present at the same time in one plant.
A linalool chemotype is frequently found, with percentages up to 79.7 per cent, and
linalyl acetate reached 39.4 per cent. With concentrations up to 30.4 per cent and 79.9
per cent respectively myrcene and geraniol chemotypes were determined in restricted
areas. tr-Sabinene hydrate (28.9 per cent) and caryophyllene oxide (14.1 per cent)
characterised some samples but cannot be regarded as common within the species.
Arrebola etal. (1995) found thymol levels up to 15.8 per cent and carvacrol up to 73.5
per cent. The idea of a quite variable species is supported by Figure 4.1 and Table 4.1
(T. gadorenszs), both also showing the clear separation of a geraniol chemotype.
T. vnlgarzs is well known and was the first Thymw species to receive attention on its
chemical variability. Six chemotypes have been described in the south of France:
thymol, carvacrol, tr-sabinene hydratelterpinen-4-01, linalool, a-terpineol and geraniol.
Another chemotype has been found vastly dispersed in Spain, 1,s-cineole, reported by
several authors (Adzet etal., 1977). The highest levels in the essential oils of plants
from southeastern Spain were about 70 per cent, usually accompanied by camphene
(max. 24.2 per cent), camphor (rnax. 38.6 per cent) and borneol (max. 34.2 per cent) as
secondarily important compounds after 1,s-cineole. But mixed chemotypes camphor1
camphene with 1,s-cineole almost absent were also found. A linalool chemotype was
recorded only at a few points in the area studied, with up to 41.5 per cent. The absence
of phenols is noticeable (max. 1.1 per cent thymol), although a 32.2 per centp-cymene
was recorded in one sample, perhaps reflecting some introgression with T. hyemalzs
growing nearby. Comparing Figures 4.1 and 4.2 it can be concluded that the distribu-
tion of the samples from T. vnlgarls is similar to that of the whole section Pseudo-
thyvzbra, thus demonstrating how both are highly characterised by 1,s-cineole. It is
remarkable that no well-defined groups can be seen for T. vnlgarls (Figure 4.1), but
rather a progressive substitution of 1,s-cineole by other compounds such as camphor or
borneol.
T. zygzs is represented by two subspecies in southeastern Spain. The ssp. graczlis is
widespread and presents a more erect habit than ssp. sylvestris which grows far away
from the coast in wetter and colder environments (SLez, 1995b). Thymol (rnax. 72.9
per cent) and carvacrol (rnax. 22.8 per cent) chemotypes are the ones most frequently
found in the area. A linalool chemotype (rnax. 91.4 per cent) is predominant in places
of high altitudes and low temperatures, where individuals show smaller and denser
habit than those living in the foothills, which present phenolic chemotypes. Geranyl
acetate was found to reach 24. 3 per cent in the oil of one sample, and limonene,
camphene, camphor and terpinen-4-01 registered maximum percentages between 10-20
per cent. The remarkable presence of 1,s-cineole in the oils of some samples seems to be
connected with the presence of T. vzllgaris within the population. The distribution
pattern of T. zygis in Figure 4.1 reflects the important chemical partitions commented
on here.
 Essential oil polynzorphzsm in the genus Thymus 135
THE SITUATION OF THYMUS I N WESTERN IBERIA

The essential oil of Thymus species from Portugal and the western part of Spain has
been studied by Salgueiro and her co-workers during the last two decades. Special
attention has been given to the chemical polymorphism, having even been established
in experimental cultures for some species. The area shows a high taxonomical variability,
with eight species from four different sections studied: (a) Section Thynzus: T. zygis,
T. carnosus, T. camphoratus; (b) Section Pseudothymbra: T. lotocephalus, T. villosw; (c) Section
Mastichina: T. mastichina, T. albicans; (d) Section AIIicantes: T. caespititzw. These species
were studied from collective samples that provided the general characteristics of the
populations, and from individual samples, to determine the chemical variability.
Section Thymw presents two subsections in the area, mainly differing in the presence1
absence of floral bracts. T. canzphoratus is spread along the southern-most part of Portugal
and belongs to the subsection Thymastra, thus presenting floral bracts. The analysis of
72 individual plants (Salgueiro etal., 1997a) showed four main groups characterised by
(a) linalool (max. 2 1.0 per cent in the oil), (b) borneol (max. 24.0 per cent), (c) 1,8-cineole
(max. 20.0 per cent), d ) 1,s-cineolelborneol. Terpinen-4-01 and tr-sabinene hydrate
were found in lower concentrations with maximum values of 10.2 per cent and 10.8 per
cent respectively. All these percentages were achieved from collective samples obtained at
the same localities as the individual ones. The absence of phenols or their precursors in
significant quantities should be noticed.
T. capitellatus is also endemic in southern Portugal. The presence of ovate floral bracts
place this species within the subsection Thymastra. The chemical composition of the
essential oils resembles that of T. canzphoratus. Three chemotypes could be described,
a 1,s-cineole-type, a camphenel1,S-cineolelborneol-typeand a linaloolllinalyl acetate-type
(Salgueiro, 1992).
T. carnosus and T. zygzs do not present floral bracts, which puts them in the subsection
Thynzus. The essential oil of T. carnosw was studied; on analysis of 83 samples (Salgueiro
etal., 1995) showed a division into three main groups: (a) a large group of individuals
was characterised by borneollcis-sabinene hydratelterpinen-4-01; (b) a group with lin-
aloolltr-sabinene hydrate, (c) a small group of samples was characterised by borne011
camphene. The highest percentages obtained from collective samples were 32.0 per
cent for borneol, 25.5 per cent for linalool, 17.0 per cent for tr-sabinene hydrate, 13.0
per cent for camphene, 1 1.2 per cent for cis-sabinene hydrate and 1 1.1 per cent for
terpinen-4-01. Phenolic chemotypes were absent.
T. zygis ssp. zygis and ssp. sylvestris differ mainly in their indumentum and the
distribution area, ssp. sylvestris being hairier and found farther south than ssp. zygis.
The latter proved to be polymorphous showing five chemotypes (Salgueiro and Proenfa
da Cunha, 1989), a linalool-type (max. 87.0 per cent linalool), a thymol-type (49.2 per
cent thymol), a geraniollgeranyl acetate-type (52.5 per cent and 38.0 per cent respect-
ively), a 1,s-cineolellinalool-type(32.5 per cent and 42.3 per cent respectively), and
a 1,s-cineolelthymol-type (29.2 per cent and 25.6 per cent respectively).
In western Iberia there are two species in the section Pseudothynzbra, T. lotocephalus
and T. villosus, the latter with the ssp. villosus and ssp. lusitanicus. Salgueiro et al. (1997b)
studied the essential oil of T. villosw ssp. villosus, finding four groups of individuals
characterised by (a) p-cymene/camphorllinalool, (b) p-cymenelborneol, (c) linalooll
geraniollgeranyl acetate, (d) a-terpineollcamphorimyrcene. They reported maximum
percentages in collective samples as follows: 40.0 per cent for p-cymene, 19.0 per cent
136 Francisco SAez and Elisabeth Stahl-Biskztp
for camphor, 23.7 per cent for linalool, 30.2 per cent for a-terpineol, 18.5 per cent for
myrcene, 15.5 per cent for geraniol and 12.9 per cent for geranyl acetate. Maximum
percentages for thymol and carvacrol were only 11.7 per cent and 5.5 per cent, despite
high levels ofp-cymene.
The essential oils of T. villuszls ssp. lzlsitanicus were studied by Salgueiro etal.
(2000a), who found that the samples could be classified into five main groups, with
either (a) linaloollterpinen-4-olh-sabinene hydrate, (b) linaloolll,8-cineole, (c) lin-
alool, (d) geranyl acetatelgeraniol, e) geranyl acetate/geraniolll,8-cineole.T. lotucephalzls
presented 1,8-cineole, camphor, linalool, linalyl acetate and a-pinene as the main
constituent(s) in one sample (Salgueiro, 1992). The analyses of four populations of
this species demonstrated the existence of groups characterised by either linalool,
1,8-cineole, linaloolll ,8-cineole, linalyl acetatellinalool, or geranyl acetate (Salgueiro
etal., 2000b).
In Western Iberia the section Mastichina shows two species: T. albicans and T. mastichina,
the latter with two subspecies, ssp. mastichina and ssp. donyanae. The chemical variabil-
ity of these three taxa is described in Salgueiro etal. ( 1 9 9 7 ~ )They
. identified 77 com-
pounds in 304 individuals of T. mastichina ssp. mastichina, in 15 individuals of
T. mastichina ssp. donyanae and in 43 plants of T. albicans. The ssp. mastichina presents
individuals arranged in three clusters, characterised by either 1,8-cineole (rnax. 64.2
per cent), linalool (rnax. 45.0 per cent), or plants with similar quantities of both
1,8-cineole and linalool. With respect to ssp. donyanae, the main characteristic is a
higher level of borne01 (max. 15.3 per cent) than in ssp. mnastichina, although 1,8-cineole
predominates (rnax. 38.4 per cent), and no clear chemical polymorphism was detected
among individuals. Finally, for T. albicans they found a chemical pattern similar to
T. mastichina ssp. mastichina, with three groups of individuals differing in the relative
proportions of 1,8-cineole (rnax. 42.9 per cent) and linalool (rnax. 22.0 per cent). All
these percentages were obtained in population samples, although the chemical groupings
were detected in individual samples. Trace quantities of phenols or their precursors
were reported.
Section Micantes presents one species in the Iberian Peninsula, T. caespititiw, studied
by Salgueiro etal. (1997d) by means of 91 plants and collective samples, provenant
from northwest Portugal and the Azores. These two locations showed important chemical
differences since the samples collected on the mainland were characterized by a-terpineol
(rnax. 40.5 per cent), p-cymene (max 9.1 per cent) and T-cadinol (max 8.7 per cent),
while the Azores sample registered carvacrol (36.3 per cent), thymol (16.1 per cent),
carvacrol acetate (8.3 per cent) andp-cymene (6.8 per cent). It is worth mentioning that
the presence of tr-dihydroagarofuran (rnax. 3.0 per cent), an oxygenated sesquiterpene,
was recorded in all samples, this compound not having been previously described
within the essential oils of the genus Thymus.

THE SITUATION OF THYMUS I N NORTHERN EUROPE

The essential oil chemistry of Thymw species of northern Europe and Greenland has
been studied intensively by Stahl-Biskup and her co-workers as revealed in a series of
publications which appeared from 1984 to 1998. Their papers also contain detailed
studies on the chemical polymorphism of the species concerned. The experimental
concepts fulfilled all requirements for studies of the chemical variation of a taxon, the
 Essential oil polymorphism in the genus Thymus 137

Table 4.3 T h y m u species of northern Europe and Greenland investigated concerning their chemical
polymorphism
-
Thynzus species Country Number of individuals Rejerences
analyzed

T . praecox ssp. arcticus Norway Stahl-Biskup, 1986a

Iceland Stahl, 1998
Greenland Stahl, 1984
Scotland Bischof-Deichnik, 1997
South of England Schmidt, 1998
Ireland Schmidt, 1998
T. serpyllzlm ssp. serpyllum Finland Stahl-Biskup and
Laakso, 1990
T . serpyllum ssp. tanaensis Finland Stahl-Biskup and
Laakso, 1990
T . pulegioides Norway Stahl-Biskup, 1986b
South of England Schmidt, 1998

analysis of individual plants being the most important. Table 4.3 presents a list of the
investigated species, the regions, where the plant material was collected, and the number
of individuals analysed.
The Thymus species studied belong to the section Serypyllum which contains the
largest number of species (see Chapter 1) and is the most extensive section covering
Central and North Europe, the eastern Mediterranean region and the Middle East
extending over eastern Asia. Whereas the southern species of the section Serpyllzlm
grow as small subshrubs and ate woody at the base, the northern species are hardly
lignified, procumbent and herbaceous. In general all species of the section Serpyllum
are charactetised by a high morphological variety. Intrasectional as well as intersectional
hybridisation is common, which makes the taxonomy of this phylogenetically young
section more difficult, this being one of the reasons why this section has often been
revised.
The chemical characteristics of the essential oils of the northern species can be defined
as exclusively terpenoid including mono- and sesquitetpenes. The quantitatively most
important compounds of T . praecox ssp. arcticus are the monoterpenes linalyl acetate and
linalool, accompanied by some sesquiterpene alcohols more exceptional in Thymus species
namely hedycaryol, nerolidol, T-cadinol, a-cadinol, germacra-l(10),4-dien-6-01, and
germacta-1(10),5-dien-4-01 (Stahl, 1984b). The essential oil of T . serpyllum ssp. tanaensis
resembles that of the former with high contents of linalool andlor linalyl acetate and
the two germacradienols as minor compounds (Stahl-Biskup and Laakso, 1990). In
T . serpyllum ssp. serpyllum the monoterpenes 1,8-cineole and myrcene are dominant and
are accompanied by germacrene D and the more exclusive sesquiterpene alcohols
hedycaryol, germacra- 1( I 0),4-dien-6-01, and getmacra- 1(10),5-dien-4-01 (Stahl-Biskup
and Laakso, 1990). The oil of T . pulegioides (Stahl-Biskup, 1986b; Schmidt, 1998) differs
from the other oils, containing the terpene phenols, thymol and carvacrol, which occur
only sporadically in T . praecox ssp. arcticus and which are lacking in T . serpyllum ssp.
serpyllum and ssp. tanaensis. Further components of importance are linalool and geraniol.
The variation of the essential oil composition within the species will be described in
the following paragraphs.
138 Francisco Sa'ez and Elisabeth Stahl-BisRup
T h y m u s praecox ssp. arcticus
T. praecox Opiz ssp. arcticus (E. Durand) Jalas is a tetraploid species with chromosome
numbers 2 n = 50-56 (Jalas and Kaleva, 1967), 2 n = 50-58 (Pigott, 1955), 2n= 56, 58
(Schmidt, 1968), and 2n=50, 51, 54 (Jalas, 1972). In literature various synonyms
exist; the one most commonly used is T. drucei which was established by Ronniger for
the population of the British Isles. It is mainly indigenous to the European North
Atlantic region reaching from Iceland, the Faeroes via the British Isles to the west coast
of Norway and Greenland. It has been supposed that this subspecies survived the Ice
Age on ice-free areas. Only in the south of England it is associated with T. pulegioides,
whereas in the other regions it grows apart from other Thymw species. T. praecox ssp.
arcticus is a plant with long, somewhat woody, creeping branches, non-flowering or
with a terminal inflorescence, flowering stems are born in rows (Jalas, 1972).
A compilation on the early findings concerning the chemical polymorphism of
Thymuspraecox ssp. arcticus was published in 1984b when Stahl presented 8 chemotypes
of this species evaluating essential oil data from 177 individual plants. Seven chemotypes
contained essential oils with high percentages of linalyl acetate (about 70 per cent).
The chemotype characterising compounds were the sesquiterpene alcohols nerolidol,
hedycaryol, and T-cadinol which occurred in different combinations within the oils
(2-1 1 per cent). One type was totally lacking these sesquiterpenoids. One chemotype
did not contain linalyl acetate but hedycaryol in high percentages (42 per cent).
Two further facts were remarkable: (a) in Iceland all the 8 chemotypes were present, in
Norway 6 with the hedycaryol- and nerolidol-type lacking, in Greenland only 4
chemotypes could be found; (b) within all the populations the different chemotypes
were growing side by side.
In the 1990s, T. praecox ssp. arcticus was studied again. There were 377 individuals
analysed from Scotland (Bischof-Deichnik, 1997), 303 individuals from South England,
and 52 individuals from Ireland (Schmidt, 1998). At that time multivariate statistical
analysis was accessible and applied to evaluate the flood of quantitative GC data
obtained. The oils of the Scottish population proved to be chemically similar to the oils
of Iceland, Norway and Greenland with some further compounds, e.g. neral, geranial,
citronellol, thymol, carvacrol, 7-terpinene, and tr, tr-farnesol. As a result of a multi-
variate statistical evaluation of the individual oils, 22 chemotypes could be established,
with a linalyl acetatellinalool-type as the most frequent one (105 individuals), followed by
a hedycaryol type (35), a germacra-1(10),4-dien-6-olllinaloolllinalylacetate type (34),
a tr-nerolidol-type (39), a germacra-1(10),4-dien-6-ol-type(21), a germacra-l(l0),
5-dien-4-ollgermacra-1(10),4-dien-6-ol-type (20), a monoterpene hydrocarbon-type
(17), a tr-ocimene-type (l7), and a borneol-type (1 1). The further chemotypes occurred
only in 10 individuals or fewer.
New aspects concerning the number of chemotypes arose when Schmidt (1998)
evaluated the essential oil data from 52 individuals from Ireland and 303 individuals
from the south of England. Her aim was to present a compilation of all the oil data
available for T. pruecox ssp. arcticus in the North Atlantic region, applying neuronal nets
for the formation of groups. In comparison to the multivariate statistical analysis this
method produced more plausible results because a greater value was placed on the
quantitative presence of substances, not only on the fact that it was present. The results
are more compatible with our subjective perception by including all 909 individuals in
the calculation with the neuronal nets; 17 chemotypes of T. praecox ssp. arcticus were
 Essential oil polynzorphism i n the genus Thymus 139
found. Again the linalyl acetateilinalool-type (36 per cent of the individuals), the
hedycaryol-type (20 per cent), and a germacra-1(10),4-dien-6-ol-type(14 per cent)
were the most abundant types, followed by the tr-nerolidol-type (5.3 per cent), a
T-cadinolihedycaryol-type (5 per cent), a P-caryophyllene-type (4.7 per cent), and
a linalool-type (3 per cent). All the other chemotypes were represented by fewer than
2 per cent of the individuals, of which a thymol-type is worth mentioning.
As a result one can say that chemical polymorphism in the northern T. praecox ssp.
arcticus with 17 chemotypes is more highly developed than in the southern species. The
revised definition of the oil types revealed the following frequency of oil types within
the countries: Greenland 2, Iceland 5 , Norway 1, Scotland 13, Ireland 11, south of
England 17. A north-south gradient of the linalyl acetateilinalool-type with higher
frequency in the north and a contrary pattern of the thymol-type with higher frequency
in the south is striking. The existence of a thymol-type in Thymw pruecox ssp. arcticus
gives reason to discuss a relation with the also polymorphous T . praecox ssp. polytrichw
of the Tyrolean Alps which was proved to show 12 chemotypes, one of them a thymol-
type which 33 per cent of the investigated plants of this region belonged to (Bischof-
Deichnik etul., 2000).

T . SERPYLLUM SSP. SERPYLLUM AND T. SERPYLLUM SSP.

TANAENSIS

T. serpyllum L. ssp. serpylhm (syn. ssp. ungustifoliw (Pers.) Arcangel) is widespread in the
sandy soils in southern and central Finland but rare north of the 62nd parallel, whereas
T . serpyllam ssp. tanaensis grows north of the polar circle in two areas of Lapland: in the
northeastern part of Finland and the area around Kuusamo as well as in the north on
either bank of the Tana river, which forms the border to Norway. The chromosome
number for both subspecies is 2n=24. The polymorphism of both taxa was investigated
by Stahl-Biskup and Laakso (1990) evaluating the oil composition of 52 and 133
individual plants respectively. At that time neither multivariate statistical analysis nor
neuronal nets were available; therefore the group-forming method was based on the
subjective evaluation of the peak patterns.
The pattern of the above-mentioned sesquiterpene alcohols again gave reason to
distinguish four different chemotypes of T . serpylhm L. ssp. serpyllum in Finland, namely a
hedycaryol-type, a germacra-1(10),4-dien-6-01-type,a germacra-1(10),5-dien-4-01-type
and one type lacking those alcohols. It must be stressed that these chemotype charac-
terising compounds were not the main constituents of the oils. 1,s-cineole and myrcene
were the main compounds. T . serpylhm ssp. tanaenszs also showed 4 chemotypes, two of
them characterised again by the germacradienols, the two further by high percentages
of linalool and linalyl acetate, respectively.
A chemical overlap of the chemotypes of these two Finnish subspecies is remarkable
and one can speak of about 6 chemotypes of Thymus serpyllum s.1. in Finland if the
subspecies level is not considered. Once more a certain north-south gradient is notice-
able, again with the linalyl acetate type only present in the north (northern Lapland)
and the linalool-type only present in southern Lapland. The germacradienol-types as well
as the hedycaryol-type become more abundant from the north to the south, rhe latter
totally lacking in-Lapland. The assumption disc-ussed in the past that T, sevpyIIztm ssp.
tunaensis belongs -ta a group of plants which immigrated from the Northeast, the
140 Francisco Sa'ez and EIisabehh Stahl-Biskup
Eurasian Taiga, whereas the other northern Thymus species originated from the Medi-
terranean center of the genus (Meusel etal., 1978), cannot be derived from the chemical
patterns which are similar in both subspecies.

T h y m u s pulegioides
T.pulegioides L. (syn. T . chmaedrys Fries) is widely distributed on the European contin-
ent south to the Mediterranean isles. Chromosome numbers are 2n= 28 (Pigott, 1955;
Schmidt, 1968), 2n=28, 30 (Jalas, 1972). Northern occurrences are in the south of
Sweden, in the south of Norway near the Oslo Fjord, and in the south of England. It
is a more upright growing Thymus species, 25-40cm high, somewhat woody at the
base, branched flowering stems, long creeping branches absent (Jalas, 1972). The
populations of Norway (Stahl-Biskup, 1986b) and the south of England were studied
(Schmidt, 1998).
Contrary to the other northern Thymus species the essential oil of T . pulegioides
contains monoterpene phenols, thymol and carvacrol, as well as their precursors
p-cymene and y-terpinene. These chemical characteristics make it resemble the southern
Thymw where the phenols are abundant and characteristic compounds of many species.
With regard to polymorphism, when analysing 7 9 individual plants, T . pulegioides
turned out to be less polymorphous than T . arcticus. In Norway only two chemotypes
could be found, a carvacrol-type with average percentages of about 37 per cent caravcrol
in the oils, and a thymol-type with 35 per cent thymol. Three quarters of the plants
belong to the carvacrol type, one quarter to the thymol type (Stahl-Biskup, 198613).
O n evaluating the oil data of 85 individual plants collected in southern England
(Schmidt, 1998), T . pulegioides was found to comprise 4 chemotypes, a thymol-type, a
linalool-type, a geraniol-type and a carvacrolly-terpinene-type. The thymol-type was
the most abundant including 65 per cent of the individuals followed by the linalool-
type with 29 per cent of the plants. The geraniol- and the carvacrolly-terpinene-type
occurred only sporadically with 4 per cent and 2 per cent of the plants respectively.
Within the populations the different chemotypes grew side by side as was found within
the other northern Thymus species. The chemotypes found agree with those detected in
T . pulegioides ssp. chumaedrys in Slovakia (Mirtonfi, 1992). He analysed 181 samples
using a principal component analysis which resulted in 5 chemotypes: a thymol chemo-
type (20.8 per cent thymol in average), a carvacrol chemotype (32.9 per cent carvacrol
in average), a citrallgeraniol chemotype (29.1 per cent citral and 22.4 per cent geraniol),
a linalool chemotype (54.8 per cent linalool), and a fenchone chemotype (33.9 per cent
fenchone). The latter had never been found before in a Thymus species. In a further
paper the chemotype pattern differentiation on different substrates is studied (MBrtonfi
etal., 1994).

Despite some reservations concerning the comparability of the experimental concepts

of the polymorphism studies it can be postulated that in Thymw two forms of poiymor-
phism are manifested: some species occur with only few chemotypes, such as T . pzllegioides,
T. vulgaris, T.mastichina, other species show more than seven or even an uncertain number
of chemotypes. The latter may include T . praecox ssp. arcticus, T . baeticus, T.cumporatzls,
 Essential oil polymorphism in the genus Thymus 141
T. herba-barona, T. tosevii ssp. tosevii and T. zygis ssp. sylvestris. Some species have been
found to be tetraploid, such as T. herba-barona, T. zygis ssp. sylvestris, T. praecox ssp.
arcticus or T. mastichina ssp. mastichina among others (Jalas and Kaleva, 1967; Morales,
1986), but it is still unclear whether in some species chemical polymorphism and high
ploidy levels are related. The highest chemical variability seems to concentrate in species
from section Serpyllum and section Thymus.

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5 Flavonoids and further polyphenols
in the genus Thymas
Roser Vila

INTRODUCTION

Flavonoids constitute one of the main groups of natural phenolic compounds, being
widely extended among green plants, where they can be found in different organs:
leaves, flowers, barks, fruits, etc. Flavonoid aglycones may have several types of struc-
tures, all of them with a 15 carbon nucleus arranged in a C6-C3-C disposition, that is:
two aromatic rings linked by a three carbon chain, that may or may not form a third
ring. The main flavonoid aglycone structures are related by a common biosynthetic
pathway that involves precursors from both the shikimic and polyketide route (Ebel
and Hahlbrock, 1982; Grisebach, 1985; Hahlbrock and Grisebach, 1975). The first
flavonoid is formed immediately after the confluence of the two ways, and it seems to
be the chalcone, from which all the other structures derive. During the biosynthetic
process, several reactions of either addition or loss of hydroxyl groups, methylation or
isoprenylation, dimerization, bisulphate formation, and, what is more important, glyc-
osylation of either the hydroxyls or the flavonic nucleus, may occur at different levels.
All this, will lead to a great diversity of structures (about 2000 flavonoids are known
at present) compiled in several revisions (Harborne and Mabry, 1982; Harborne etal.,
1975; Wollenweber and Dietz, 1981).
Flavonoids can be found as aglycones or, more frequently, as glycosides either 0 - or
C-glycosides. Although any flavonoid position may be glycosylated, some have more
probabilities, such as 7-OH of flavones, flavanones and isoflavones, 3-OH and 7-OH of
flavonols and dihydroflavonols, and 6- andlor 8-C in C-glycosides, being glucose the
most usual sugar found in them. Glycosylation, as well as methylation, occurs in
the latest stages of biosynthesis and is catalyzed by high specific enzymes. Sometimes
glycosides may have acyl-substituents linked to one or more hydroxyl groups of the
sugars by an ester bond, or more rarely directly linked to the flavonoid molecule
(Wollenweber, 198513). Among these acyl-substituents there are aliphatic acids such as
acetic, malonic, or succinic acid, and aromatic acids like benzoic, gallic, p-coumaric, and
caffeic acid (Aguinagalde and Pero Martinez, 1982; Harborne, 1986; Wollenweber etal.,
1978).
The role of flavonoids and, in general, polyphenolic compounds in plants is not com-
pletely established. Their pigmentary function, responsible for the attraction of
zoopollinators and zoodispersors, is well known. Other functions as antioxidants,
antimutagenics, on plant growth regulation and on resistance to plant diseases have
also been attributed to this group of natural polyphenols (Harborne, 1985; McClure,
1975).
 Flavonoids and further polyphenols in the genus Thymus 145
Flavonoids, for their structural variability, their physiological and chemical stability,
their wide distribution among plants and their relatively easy detection, constitute one
of the main chemotaxonomic markers. Their importance in this respect has been the
object of a large number of publications (Bate-Smith, 1962, 1963; Harborne, 1966,
1967). The more or less restricted distribution of specific types of flavonoids or substitu-
tion patterns in certain systematic groups is what determines their chemotaxonomic
and possibly phylogenetic application (Harborne, 1975; Harborne and Turner, 1984;
Swain, 1975). The latter is based on accepting the fact that plants which are able to
synthesize structures placed in advanced stages of the biogenetic pathways will have a
superior and more complex enzymatic supply. In general, evolution involves an increase
of the number of flavonoid types present in each systematic group, and concomitantly
their structural complexity increases.
Among Lamiaceae the presence of flavonoids is well known (Adzet and Martinez,
1981a; Barberin, 1986; Harborne, 1967; Hegnauer, 1966, 1989; Semrau, 1958;
Zinchenko and Bandyukova, 1969). Especially during the last two decades several
authors have revealed a wide range of substitution patterns with chemotaxonomic
significance from both supra- and infrageneric point of view (Tom&-Barberin and
Wollenweber, 1990; Tom6s-BarberBn etal., 1988a,b). Particularly, the genus Thymas
has been shown to have a noteworthy flavonoid composition of valuable taxonomic
importance (Adzet and Martinez, 1981b; Adzet etal., 1988; HernBndez etal., 1987;
Litvinenko and Zoz, 1969; Martinez, 1980; Simonyan, 1972; Simonyan and
Litvinenko, 1971; Vila, 1987). Thus, flavonoid aglycones play an important role to
separate T . capitatas (now Thymbra capitata) from other species of this genus (Adzet and
Martinez, 1981a,b; Barberin etal., 1986); luteolin and 6-hydroxyluteolin, two impor-
tant taxonomic markers (Semrau, 1958; Hegnauer, 1966; Harborne and Williams,
1971), have been found in several Thymus taxa (Adzet and Martinez, 1980a), while
Section Pseudothymbra is characterized by a high content of methoxylated flavones
(Adzet etal., 1988).

FLAVONOID DISTRIBUTION A N D OTHER POLYPHENOLS

I N THE GENUS THYMUS

Within the genus Thymas, many more flavonoids, especially aglycones, have been
described, than other polyphenols, which only include phenolic acids. In Tables 5.1,
5.2 and 5.3 the substitution patterns and frequency of flavonoid aglycones, flavonoid
glycosides and phenolic acids found in the genus Thymus are shown, respectively. As it
can be seen, the former have been largely investigated in thyme plants, while glycosides
and phenolic acids have been studied to a lesser extent. A brief discussion of each table
follows below.

Flavonoid aglycones
Among the aglycones (Table 5.1), 32 flavones, 4 flavanones, 2 flavonols and 2 dihydro-
flavonols have been described, luteolin and apigenin being, by far, the more frequently
found (in 100 and 99 taxa, respectively), followed by scutellarein (in 55 species). U p to
now, no isoflavonoids have been reported in Thymas taxa.
146 Roser Vila
Flavones

Concerning the substitution pattern of these compounds (Table 5. l ) , flavone aglycones

found in the genus Thymas may have a 5,7-, 5,6,7-, 5,7,8- or 5,6,7,8- substituted A-ring,
the C-5 position being always hydroxylated and the C-8 methoxylated, while the C-6 and
C-7 positions may have both types of substitution. Most of the flavones found in Thymus
species have methoxyl groups in the A- and/or B-rings. Some of them are highly methoxy-
lated flavonoids, as for example: 5-de~meth~lnobiletin with five methoxy groups in 6, 7,
8 , 3' and 4' (reported in 28 taxa), 8-OMe-cirsilineol with a 6,7,8,3'-(OMe)4-substitution
(reported in 31 taxa), 5-desmethylsinensetin with a 6,7,3',4'-(OMe)4-substitution (found
in 28 taxa), gardenin B with a 6,7,8,4'-(OMe)4-substituted structure (reported in 5 taxa),
and 5,6-(0~)~-7,8,3',4'-(0~e)~-flavone (described in only one taxon, T. piperella). The
5,7,8-trisubstituted A-ring has only been found once in 5,4'-(OH),-7,s-(OMe),-
flavone (8-OMe-genkwanin), in T. moroderi (Vila, 1987).

Table 5.1 Substitution pattern and frequency of flavonoid aglycones found in Thynzzls sp

-OH -0Me Name N o qftaxa

(A) Flavones and flavonols

Acacetin
Apigenin
Chrysoeriol
Cirsilineol
Cirsiliol
8-OMe-Cirsilineol
Cirsimaritin
5-Desmethylnobiletin
5-Desmethylsinensetin
Diosrnetin
Gardenin B
Genkwanin
Kaempferol
Ladanein
Luteolin
6-OH-Luteolin
7-OMe-Luteolin
Pilloin
Pebrellin
Quercetin
Salvigenin
 Flavonoids andfurther polypheno in the genus Thymus 147

Scutellarein
Sideritoflavone
Sorbifolin
Thymonin
Thymusin
Xanthomicrol
4'-OMe-Genkwanin
8-OMe-Genkwanin

(B) Flavanones and dihydroflavonols

1 3,5,7,4' - Dihydrokaempferol
2 5,7,3',4' - Eriodictyol
3 5,7,4' - Naringenin
4 5,4' 7 Sakuranetin
5 3,5,7,3',4' - Taxifolin
6 5,4' 6,7,8 Dihydroxanthomicrol
(C) Anthocyanidins

With respect to the B ring of thyme flavones, it can be 4'-monosubstituted or 3',4'-

disubstituted, either by hydroxy andlor methoxy groups.
Other flavones that are widely reported within the Thymw taxa investigated are
(Table 5.1): xanthomicrol (in 34 taxa), cirsimaritin (in 33 taxa), cirsilineol (in 32 taxa),
sideritoflavone (in 30 taxa), thymusin (in 29 taxa) and thymonin (in 24 taxa), whereas
148 Roser Vila
chrysoeriol, cirsiliol, ladanein, pilloin and pebrellin have only been described in one
taxon.

Flavonols
Kaempferol and quercetin are the only flavonols described for Thymw species They have
only been found once in T . moroderi (Vila, 1987) and T . vulgaris (Morimitsu etal., 1995),
respectively.

Flavanones and dihydroflavonols

Concerning to flavanones and dihydroflavonols, they are less widespread within the genus
Thymus than flavones (Table 5.1). Among the former, eriodictyol, naringenin, sakuranetin
and dihydroxanthomicrol have been reported in 25, 23, 11 and 8 taxa, respectively,
while only dihydrokaernpferol and taxifolin have been described among the latter, in
8 and 7 taxa, respectively. The A- and B-rings of these flavonoids are mainly characterized
by having a 5,7,3'- andlor 4'-substitution pattern, these substituents usually being
hydroxy groups, except in sakuranetin, which has a methoxy group at C-7 position, and
in dihydroxanthomicrol, which represents a 5,4'-(0~)~-6,7,8-(OMe)3-flavanone.

Acylated flavonoid aglycorzes

Although acylated flavonoid aglycones have been rarely described in the Labiatae family,
unusual 8-C-p-hydroxybenzyl-derivativesof several flavone (apigenin, luteolin, diosmetin)
and flavonol (quercetin, kaempferol) aglycones have been identified in T . hirtus, an
Algerian taxon (Merghem etal., 1995). This is the only report of acyl substituents directly
linked to the flavonoid skeleton in the genus Thymus.

Anthocyanidins
Anthocyanidins and other flavonoid-related structures have scarcely been found in the
genus Thymus, the anthocyanidin cyanidin (Table 5.1) being the only one reported in
two species: T . pulegioides and T . vulgaris (Stoess, 1972).

Flavonoid glycosides
Flavonoid glycosides of Thymus have been less intensively investigated than aglycones.
Only 16 different structures of this group of flavonoids, particularly hydroxylated
flavone-glycosides, have been reported in the reviewed literature (Table 5.2), all of
them being O-glycosides with one exception. Those derived from luteolin and apigenin
ate the most widespread, especially luteolin-7-0-glucoside and apigenin-7-O-glucoside,
which have been found in ten and eight species, respectively. All the other flavonoid-
O-glycosides included in Table 5.2 have been reported in only one or two species of
Thymus, mainly T . menzbranaceus (Tom6s etal., 1985), T. moroderi (Vila, 1987) and
T . se~pyllum(Olechnowicz-Stepien and Lamer-Zarawska, 1975). Flavonol and isoflavonoid
glycosides have not been previously reported in this genus.
The flavonoid aglycone more frequently found in its glycosidic form is luteolin,
from which nine different 7-O-glycosides have been described in Thynzw. More rarely,
 Flavonoids and fzrther polypheno in the genzs Thymus 149

Table 5.2 Substitution pattern and frequency of flavonoid glycosides in T h y m u sp.

Structures Aglyrone N O taxa

Apigenin
Apigenin
Diosmetin
Luteolin
galactoarabinoside Luteolin
7-O-glucoside Luteolin
7-O-diglucoside Luteolin
7-O-glucuronide Luteolin
7-O-neohesperidoside Luteolin
7-O-rutinoside Luteolin
7-O-sambubioside Luteolin
7-O-xyloside Luteolin
7-O-glucoside 6-OH-Luteolin
glucosylglucuronide Scutellarein
7-O-glucosyl(1- 4)rhamnoside Scutellarein
6,8-di-C-glucoside (Vicenin-2) Apigenin

6-OH-luteolin-, scutellarein- and diosmetin-derived glycosides have been also identi-

fied. Glycosides from A-ring methoxylated flavones have not been reported in thyme
plants.
Sugars found in Thymus flavonoid glycosides are usually linked to the aglycone through
the hydroxy group in the C-7 position of the flavonoid skeleton, being either a mono-
saccharide (glucoside, xyloside, alloside, glucuronide) or a disaccharide (diglucoside,
galactoarabinoside, neohesperidoside, rutinoside, sambubioside, glucosylglucuronide,
glucosylrhamnoside). Sometimes the linkage data of the sugar chain are not completely
described by the authors. The presence of acyl-substituents in the sugar moiety has
been reported only once, particularly apigenin-4'-0-p-cumaroyl-glucoside, in T . serpyllam
(Washington and Saxena, 1983).
Concerning C-glycosides, vicenin-2 (apigenin-6,s-di-C-glucoside) is the only one
found in Thymw sp. It is the flavonoid glycoside most frequently reported in this
genus, particularly in 20 taxa (Table 5.2). It has been observed that while flavone
O-glycosides occur ubiquitously among several Labiatae genera, vicenin-2 occurred
only in certain taxonomic groups, for instance, in the sections Pseudothymbra and Thymus
of the genus Thymus (Husain and Markham, 1981).
Extraction procedures used as well as difficulties in structure elucidation, particularly
sugar linkages, may have been the cause of the too few results reported on flavonoid
glycosides in Thymas compared with those on aglycones.

Phenolic acids
Nine different phenolic acids have been reported in the genus Thymw (Table 5.3), caffeic
and rosmarinic acids being those more frequently found (in 29 and 20 species, respect-
ively). The others have only been detected in one or two taxa. Particularly, chlorogenic,
p-cournaric, 3,5-dicaffeoylquinic, protocatechuic and syringic acid have been identified
in T. webbianw (B16zquez etal., 1994), while caffeic, p-coumaric, p-hydroxybenzoic,
syringic and vanillic acid have been found in T. carnosus (Marhuenda etal., 198710).
 15 0 Roser Vila

Table 5.3 Frequency of phenolic acids in Thymm sp.

Arartze N O taxa

Caffeic acid
Chlorogenic acid
p-Coumaric acid
3,5-Dicaffeoylquinic acid
p-Hydroxybenzoic acid
Protocacechuic acld
Rosmarinic acid
Syringic acid
Vanillic acid

Compilation of flavonoids and phenolic acids in Thymus

Table 5.4 includes a review of the literature published on the polyphenols found in
Thymw species between 1969 and 1999, with some significative preliminary references
from 1958 and 1959. This wide group of secondary metabolites has been investigated
in 120 Thymus taxa. Usually, leaves are the part of the plant investigated, although in
some cases the plant material is not well indicated by the authors.
It is important to consider that the information given in this table comes from very
heterogeneous sources. First, it has to be taken into account that the authors have used
different extraction methods which determine the compounds to be found in the final
extracts. Thus, in some cases extraction was performed with solvents of increasing polar-
ity or more frequently, after removing the more lipophilic compounds with petroleum
ether or hexane, a methanolic or hydroalcoholic extract was obtained and successiveiy
partitioned by solvents of increasing polarity. In addition, the more polar extracts were
sometimes submitted to acidic hydrolysis, consequently the free flavonoid aglycones
found probably occur in their glycosidic form in the plant. Sometimes only the flavonoid
aglycones from leaf surfaces (exudate flavonoids) were investigated, in these cases the
plant material was rinsed with lipophilic solvents such as chloroform. As a matter of
fact, the extraction procedure obviously restricts the compounds to be found, meaning
that those flavonoids which are not described in a species are not necessarily absent.
Second, while some authors isolated the flavonoids or other polyphenolic constitu-
ents and determined their structures from their spectroscopic data (UVlVis, MS andlor
nuclear magnetic resonance (NMR) data), others analysed the extracts by high-perfor-
mance liquid chromatography (HPLC) and/or several thin-layer chromatography (TLC)
systems, comparing retention indices with those of reference substances previously iso-
lated from other extracts.

EXUDATE FLAVONOID AGLYCONES: INFLUENCE OF THE

HABITAT A N D GENETIC FACTORS O N THEIR PRODUCTION

The exudate flavonoid aglycones from leaves and stems surfaces have been shown to play
an important ecological role. Particularly, a correlation between the preferred habitat of
the plant and the production of excreted flavonoids has been reported, the species from
(semi-)arid habitats being those which generally accumulate external flavonoids
(Tom&-Barberiin and Wollenweber, 1990; Wollenweber, 1985a). These are usually
 151
Table 5.4 Polyphenols in Thynzus sp.

Speczes Polyphenol~ References

T. aestivz~sReut. ex Willk. Apigenin Adzet etal., 1988

Cirsilineol Adzet etal., 1988
8-OMe-Cirsillneol Adzet etal., 1988
Cirsimaritln* Adzet etal., 1988
5-Desmethylnobiletin Adzet etal., 1988
2,3-Dihydrokaempferol Adzet etal., 1988
2,3-Dihydroxanthomicrol Adzet etal., 1988
Eriodictyol Adzet etal., 1988
Genkwanin Adzet etal., 1988
Luteolin Adzet etal., 1988
Naringenin Adzet etal., 1988
Sakuranetin Adzet etal., 1988
Salvigenin Adzet et al., 1988
Sideritoflavone Adzet etal., 1988
Taxifolin Adzet et al., 1988
Xanthomicrol Adzet et al., 1988
Caffeic acid Adzet et al., 1988
Rosmarinic acid Adzet et al., 1988
T . albanus H . Braun Apigenin Kulevanova et al., 1997
Luteolin Kulevanova et al., 1997
Caffeic acid Kulevanova et dl., 1997
T . algeeriensis Boiss. Eriodictyol El-Domiaty etal., 1997
Taxifolin El-Domiaty etal., 1997
5,6-(OH),-7 ,3',4'-(0Me)3- El-Domiaty etal., 1997
flavone
5,6,4'-(OH),-7,3'-(0Me)2- El-Domiaty et al., 1997
flavone
T . alsaerenstJ- Ronn Apigenin Kulevanova et al., 1997
Eriodictyol Kulevanova et al. , 1997
Luteolin Kulevanova et al., 1997
Caffeic acid Kulevanova et al., 1997
Rosmarinic acid Kulevanova et al., 1997
Apigenin Litvinenko and ZOZ,1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T . anzzctus Klok. Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellare~n Litvinenko and Zoz, 1969
T . antoninae Rouy et Colncy Apigenin Adzet et dl., 1988
Cirsilineolt Adzet etal., 1988; Hernindez etal., 1987
8-OMe-Cirsilineol$ Adzet etal., 1988; Hernindez etal., 1987
Cirsimaritin Adzet etal., 1988; Hernindez etal., 1987
5-De~meth~lnobiletin Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylsinensetin Hernindez et al., 1987
2,3-Dihydroxanthomicrol Adzet et al., 1988
Eriodictyol Adzet etal., 1988
Genkwanin Adzet etal., 1988
Luteolin Adzet etal., 1988
Naringenin Adzet etal., 1988
Sakuranetin Adzet et al., 1988
Sideritoflavone Adzet etal., 1988; Hernindez etal., 1987
Taxlfolin Adzet etal., 1988
Table 5.4 (Continued)

Species Polyphenols References

T . antoninae Rouy et Coincy Thymusin Hernindez et al., 1987

(Continued) Xanthomicrol Adzet et al., 1988; Hernindez et al., 1987
Vicenin-2 Husain and Markharn, 1981
Caffeic acid Adzet etal., 1988
Rosmarinic acid Adzet etal., 1988
T . aranjueziz Jalas Cirsilineol Hernindez etal., 1987
( T . lacaitae Pau) 8-OMe-Cirsilineol Hernindez etal., 1987
Cirsimaritin Adzet and Martinez, 1981b;
Hernindez etal., 1987
5-De~meth~lnobiletin Hernindez et al., 1987
5-Desmethylsinensetin Hernindez etal., 1987
Luteolin Adzet and Martinez, 1981b
6-OH-Luteolin Adzet and Martinez, 1981b
Sideritoflavone Hernindez et al., 1987
Thymonin HernPndez etal., 1987
Thymusin Hernindez et al., 1987
Xanthomicrol Adzet and Martinez 1981b;
Hernindez etal., 1987
T. araratz-mznorzs Klok. et Apigenin Simonyan and Litvinenko, 1971
Schost. Luteolin Simonyan and Litvinenko, 1971
Scutellarein Simonyan and Litvinenko, 1971
T . attenuatus Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. baeticus Boiss. ex Lacaitae Apigenin Adzet and Martinez, 1981b;
Adzet etal., 1988
Cirsilineol Adzet etal., 1988; Hernindez etal., 1987
8-OMe-Cirsilineol Adzet etal., 1988; Hernindez etal., 1987
Cirsimaritin Adzet and Martinez 1980b, 1981b;
Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylnobiletin Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylsinensetin Hernindez et al., 1987
2,3-Dihydrokaempferol Adzet etal., 1988
Eriodictyol Adzet etal., 1988
Genkwanin Adzet and Martinez, 1980b, 1981b;
Adzet etal., 1988
Luteolin Adzet and Martinez, 1980a, 1981b;
Adzet etal., 1988
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b
Naringenin Adzet etal., 1988
Sakuranet~n Adzet etal., 1988
Sideritoflavone Adzet etal., 1988; Hernindez etal., 1987
Taxifolin Adzet etal., 1988
Thymonin Hernindez et dl., 1987
Thymusin Hernindez et al., 1987
Xanthomicrol Adzet and Martinez, 1980b, 1981b;
Adzet etal., 1988; Hernindez etal., 1987
Vicenin-2 Husain and Markham, 1981
Caffeic acid Adzet et al., 1988
Rosmarinic acid Adzet etal., 1988
T . balcanus Borb Apigenin Kulevanova et al., 1997
Luteolin Kulevanova et dl., 1997
Caffeic acid Kulevanova et al., 1997
T. bashkiriensis Klok. et Apigenin Kurkin et al., 1988
Schost. Luteolin Kurkin etal., 1988
Caffeic acid Kurkin et dl., 1988
Rosmarinic acid Kurkin etal., 1988
T. borysthenicu Klok. et Apigenin Litvinenko and Zoz, 1969
Schost. Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. bracteatus Lange ex Cirsilineol Hernindez etal., 1987
Cutanda 8-OMe-Cirsilineol HernPndez et al., 1987
Cirsimaritin Hernindez et al., 1987
5-Desmethylnobiletin Hernindez etal., 1987
5-Desmethylsinensetin Hernindez et dl., 1987
Sideritoflavone Hernindez etal., 1987
Thymonin Hernindez et al., 1987
Thymusin Herndndez et dl., 1987
Xanthomicrol Hernindez etal., 1987
T. caespititius Brot. Apigenin Adzet and Martinez, 1981b
Cirsilineol HernPndez etal., 1987
8-OMe-Cirsilineol Hernindez et dl., 1987
Luteolin Adzet and Martinez, 1980a, 1981b
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b
Thymonin Hernindez et al., 1987
Thymusin Hernindez et dl., 1987
Xanthomicrol Hernindez et al., 1987
7'.calcareus Klok. et Schost. Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. callieri BorbPs ex Velen. Acacetin Litvinenko and Zoz, 1969
Apigenin L~tvinenkoand Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. canzphoratm Hoffmanns. Apigenin Adzet and Martinez, 1981b;
et Link Adzet etal., 1988
Cirsilineol Hernindez etal., 1987
8-OMe-Cirsilineol Hernindez et al., 1987
Cirsimaritin Hernindez etal., 1987
J-Desmethylnobiletin Hernindez etal., 1987
5-Desmethylsinensetin HernPndez etal., 1987
Luteolin Adzet and Martinez, 1981b;
Adzet et al., 1988
Adzet and Martinez, 1981b
Naringenin Adzet etal., 1988
Sideritoflavone HernPndez etal., 1987
Taxifolin Adzet et dl., 1988
Th~monin Hernindez etal., 1987
Thymusin HernPndez et al., 1987
Xanthomicrol Adzet etal., 1988; Hernindez etal., 1987
Vicenin-2 Husain and Markham, 1981
Caffeic acid Adzet et al., 1988
Rosmarinic acid Adzet etal., 1988
T. capitellatus Hoffmanns. Apigenin Adzet etal., 1988
et Link Cirsilineol Hernindez etal., 1987
8-OMe-Cirsilineol Hernindez et dl., 1987
Cirsimaritin Hernindez etal., 1987
5-Desmethylnobiletin HernPndez etal., 1987
Table 5.4 (Continued)

Species Polyphenols ReJerences

-

T. capitellatus Hoffmanns 5-Desmethylsinensetin Hernindez et al., 1987

et Link (Continued) Eriodictyol Adzet et dl., 1988
Luteolin Adzet etal., 1988
Naringenln Adzet etul., 1988
Sideritoflavone Hernindez et al., 1987
Taxifolin Adzet etal., 1988
Thymonin Hernindez et dl., 1987
Thymusin Hernindez et al., 1987
Xanthomicrol Adzet etul., 1988; Hernindez etal., 1987
Vicenin-2 Husaln and Markham, 1981
Caffeic acid Adzet etal., 1988
Rosmarinic acid Adzet etal., 1988
T. camosw Boiss. Apigenin Marhuenda etal., 1987a
Cirsilineol Hernindez et dl., 1987;
Marhuenda et dl., 1987a
8-OMe-Cirsilineol Hernindez et dl., 1987
Cirsimaritin HernLndez et dl., 1987
5-Desmethylnobiletin Hernindez etal., 1987
5-Desmethylsinensetin Hernindez et dl., 1987
Luteolin Marhuenda etal., 1987a
6-OH-Luteolin Marhuenda etal., 1987a
Sideritoflavone Hernindez etal., 1987
Thymonin Hernindez et dl., 1987
Thymusin Hernindez et dl., 1987
Xanthomicrol Hernindez et dl., 1987
Caffeic acid Marhuenda etal., 198713
p-Coumaric acid Marhuenda et al., 1987b
p-Hydroxybenzoic acid Marhuenda et dl., 1987b
Syringic acid Marhuenda et al., 1987b
Vanillic acid Marhuenda etal., 1987b
T. caucasicus Willd Apigenin Simonyan and Litvinenko, 1971
Luteolin Simonyan and Litvinenko, 197 1
Scutellarein Simonyan and Litvinenko, 1971
T. cephulotos L. Husain and Markham, 1981
T. cberlerioides Vis. Husain and Markham, 198 1
T. cilzatissmnzw Klok Apigenin Litvinenko and Zoz, 1969
Luteol~n Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T . cirzerascens Apigenin Semrau, 1958
T. circ?~nzcinctusKlok Apigenin Lltvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Lltvinenko and Zoz, 1969
T. collininw Bieb. Apigenin Simonyan and Litvinenko, 1971
Luteolin Simonyan and Litvinenko, 197 1
Scutellarein Simonyan and Litvinenko, 1971
Apigenin-7-0-,!To-glucoside Simonyan, 1972
Luteolin-7-0-P-D-glucoside Simonyan, 1972
T. cretacezds Klok. et Schost Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
 Scutellarein Litvinenko and Zoz, 1969
T. czernajeuii Klok. et Apigenin Litvinenko and Zoz, 1969
Schost. Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. dagestanzcus Klok. et Apigenin Litvinenko and Zoz, 1969
Schost. Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. decussatus L. Apigenin Khodair et dl., 1993
Thymonln Khodair et dl., 1993
5,6,4'-(0~)~-7,3'-(0Me), Khodair etal., 1993
flavone
T. desjatouae Ronn. Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. dolopicus Form. Vicenin-2 Husain and Markham, 1981
T. dimorphus Klok. et Schost. Acacetin Litvinenko and Zoz, 1969
Apigenin Litvinenko and Zoz, 1969;
Simonyan and Litvinenko, 1971
Luteolin Litvinenko and Zoz, 1969;
Simonyan and Litvinenko, 197 1
Scutellarein Litvinenko and Zoz, 1969;
Simonyan and Litvinenko, 1971
T. dzeuanovskyi Klok. et Apigenin Litvinenko and Zoz, 1969
Schost. Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. elzsabethae Klok. et Schost. Apigenin Simonyan and Litvinenko, 197 1
Scutellarein Simonyan and Litvinenko, 197 1
T. eupatoriensis Klok. et Scutellarein Litvinenko and Zoz, 1969
Schost.
T. fontqueri (Jalas) Molero Cirsilineol Hernindez etal., 1987
et Rovira 8-OMe-Cirsilineol Hernindez et dl., 1987
Cirsirnaritin Hernindez etal., 1987
5-Desmethylnobiletin Hernindez et dl., 1987
5-Desmethylsinensetin Hernindez et dl., 1987
Diosmetin Hernindez etal., 1987
Gardenin-B Hernindez etal., 1987
Salvigenin Hernindez et al., 1987
Sideritoflavone Hernindez etal., 1987
Thymonin Hernindez etal., 1987
Thymusin Hernindez et dl., 1987
Xanthomicrol Hernindez etal., 1987
T. fof7zi7zii Klok. et Schost. Apigenin Simonyan and Lltvinenko, 1971
Luteolin Simonyan and Litvinenko, 1971
Scutellarein Simonyan and Litvlnenko, 1971
T. fj,nkii Coss. Apigenin Adzet etal., 1988
Cirsilineol Adzet etal., 1988; Hernindez etal., 1987
8-OMe-Cirsilineol Adzet et al., 1988; Herniindez et dl., 1987
Cirsimaritin Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylnobiletin Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylsinensetin Hernindez et al., 1987
2,3-Dihydroxanthornicrol Adzet etal., 1988
Eriodictyol Adzet etal., 1988
Table 5.2 (Continued)

Species Polyphenols References

T . funkii Coss. (Continued) Genkwanin Adzet et al., 1988

Luteolin Adzet etal., 1988
Naringenin Adzet etal., 1988
Sakuranetin Adzet et al., 1988
Salvigenin Adzet etal., 1988
Sideritoflavone Adzet etal., 1988; Hernindez etal., 1987
Thyrnusin Hernindez etal., 1987
Xanthomlcrol Adzet etal., 1988; Hernindez etal., 1987
Caffeic acid Adzet etal., 1988
T. gbndulosus Lag. ex Apigenin Adzet etal., 1988
H . Del Villar Cirsilineol Adzet et al., 1988
8-OMe-Cirsilineol Adzet etal., 1988
Cirsimaritin Adzet et dl., 1988
2,3-Dihydroxanthornicrol Adzet et al., 1988
Eriodictyol Adzet et al., 1988
Luteolin Adzet etal., 1988
Naringenin Adzet etal., 1988
Sakuranetin Adzet etal., 1988
Sideritoflavone Adzet etal., 1988
Taxifolin Adzet etal., 1988
Xanthornicrol Adzet etal., 1988
Caffeic acid Adzet etal., 1988
Rosrnarin~cacld Adzet etal., 1988
T. granatensis Boiss. Apigenin Adzet and Martinez, 1981b
Luteolin Adzet and Martinez, 1981b
6-OH-Luteolin Adzet and Martinez, 1981b
T. graniticus Klok. et Schost. Acacetin Litvinenko and Zoz, 1969
Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. herba-barona Loisel Apigenin Corticchiato etal., 1995
Cirsiliol Corticchiato etal., 1995
Cirsilineol Corticchiato etal., 1995
8-OMe-Cirsilineol Corticchiato etal., 1995
Cirsimaritin Corticchiato etal., 1995
Eriodictyol Corticchiato etal., 1995
Genkwanin Corticchiato et al., 1995
Luteolin Corticchiato etal., 1995
Naringenin Cort~cchiatoet al., 1995
Sideritoflavone Corticchiato etal., 1995
Sorbifolin Corticchiato etal., 1995
Thyrnusin Corticchiato etal., 1995
Xanthornicrol Corticchiato etal., 1995

T. hirsutus Bleb. Acacetin Litvinenko and Zoz, 1969

Apigenin Litvinenko and Zoz, 1969;
Semrau, 1958
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. hirtellu Acacetin Litvinenko and Zoz, 1969
Apigenin Litvinenko and Zoz, 1969
 Luteolin Litvinenko and Zoz, 1969
Scutellarein Lirvinenko and Zoz, 1969
T. hirtus Willd. Apigenin Merghem etal., 1995
Diosmetin Merghem etal., 1995
Luteolin Merghem etal., 1995
8-C-p-Hydroxybenzyl- Merghem etal., 1995
apigenin
8-C-p-Hydroxybenzyl- Merghem et al., 1995
diosmetin
8-C-p-Hydroxybenzyl- Merghem et dl., 1995
kaernpferol
8-C-p-Hydroxybenzyl-luteolin Merghem etal., 1995
8-C-p-Hydroxybenzyl- Merghem etal., 1995
quercetin
Vicenin-2 Husain and Markham, 1981
T. hyemalzs Lange Apigenin Adzet and Martinez, 1981b
Cirsilineol Hernindez etal., 1987
8-OMe-Cirsilineol Hernindez etal., 1987
Cirsimaritin Adzet and Martinez, 1981b;
Hernindez et al., 1987
5-Desmethylnobiletin Hernindez etal., 1987
5-Desmethylsinensetin HernPndez etal., 1987
Genkwanin Adzet and Martinez, 1981b
Luteolin Adzet and Martinez, 1980a, 1981b
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b
Sideritoflavone Hernhndez etal., 1987
Thymonln Hernindez et al., 1987
Thymusin Hernindez etal., 1987
Xanthornicrol Adzet and Martinez, 1981b;
Hernindez etal., 1987
T. jajlae (Klok. et Schost.) Acacetin Litvinenko and Zoz, 1969
Starkov Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
Vicenin-2 Husain and Markham, 198 1
T. jankae var. jankae Celak. Apigenin Kulevanova et al., 1997, 1998
Diosmetin Kulevanova etal., 1997, 1998
Luteolin Kulevanova et al., 1998
Naringenin Kulevanova et al., 1998
Caffeic acid Kulevanova etal., 1997, 1998
T. jankae var. pantotrzchza Apigenin Kulevanova et al., 1997, 1998
Ronn. Diosmetin Kulevanova et dl., 1997, 1998
Luteolin Kulevanova et al., 1998
Naringenin Kulevanova et al., 1998
Caffeic acid Kulevanova et dl., 1997, 1998
T. jankae var. patentzpilzs Apigenin Kulevanova et dl., 1998
Lyka Diosmetin Kulevanova etal., 1998
Eriodyctiol Kulevanova etal., 1998
Luteolin Kulevanova et dl., 1998
Naringenin Kulevanova et al., 1998
Caffeic acid Kulevanova et al., 1998
T. kdlmiussicus Klok. et Schost. Acacetin Litvinenko and Zoz, 1969
Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
Table 5.4 (Continued)

Species Polyphenols References

T. karanzarjanicus Klok. et Apigenin Simonyan and Litvinenko, 197 1

Schost. Luteolin Simonyan and Litvinenko, 1971
T . kostcbyanus Boiss. et Apigenin Simonyan and Litvinenko, 1971
Hohen. Luteolin Sirnonyan and Litvinenko, 1971
Scutellarein Simonyan and Litvinenko, 1971
T. latifolius (Besser) Andrz. Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. leptophyllus Lange Cirsilineol Hernindez et al., 1987
8-OMe-Cirsilineol Hernindez et dl., 1987
Cirsimaritin Hernindez etal., 1987
5-Desmethylnobiletin Hernindez etul., 1987
5-Desmethylsinensetin Hernindez etal., 1987
Sideritoflavone Hernindez et dl., 1987
Thymonin Hernindez et al., 1987
Thymusin Hernindez et nl., 1987
Xanthomicrol Hernindez etal., 1987
T . lezlrotrzchus Halicsy Husain and Markham, 1981
T. lzttorulis Klok. et Schost Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. loevianus Opiz Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Litvinenko and Zoz, 1969
T. longidens var. dassareticus Apigenin Kulevanova et al., 1997
Ronn. Diosrnetin Kulevanova et dl., 1997
Eriodictyol Kulevanova etal., 1997
Naringenin Kulevanova et dl., 1997
Caffeic acid Kulevanova et al., 1997
T . longidens var. Iunicuulis Apigenin Kulevanova etal., 1997, 1998
Ronn. Erlodyctiol Kulevanova et dl., 1997, 1998
Luteolin Kulevanova et dl., 1997, 1998
Naringenin Kulevanova et dl., 1997, 1998
Caffeic acid Kulevanova et dl., 1997, 1998
Rosmarinic acid Kulevanova etul., 1997, 1998
T. longfirus Boiss. Apigenin Adzet etal., 1988
Cirsilineol Adzet etal., 1988; Hernindez etal., 1987
8-OMe-Cirsilineol Adzet et al., 1988; Hernindez et al., 1987
Cirsimaritin Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylnobiletin Adzet etul., 1988; Hernindez etul., 1987
5-Desmethylsinensetin Hernindez et dl., 1987
2,3-Dihydrokaempferol Adzet et dl., 1988
2,3-Dihydroxanthornicrol Adzet etal., 1988
Eriodictyol Adzet et dl., 1988
Genkwanin Adzet etul., 1988
4'-0-Me-Genkw anin Adzet etal., 1988
Luteolin Adzet etal., 1988
Naringenin Adzet etul., 1988
Sakuranetin Adzet etal., 1988
 Slderitoflavone Adzet etal., 1988; Hernindez etal., 1987
Taxifolin Adzet etal., 1988
Thymusin Hernindez et al., 1987
Xanthomicrol Adzet etal., 1988; Hernindez eta/., 1987
Vicenin-2 H u s a ~ nand Markham, 1981
Caffeic acid Adzet eta/., 1988
Rosmarinic acid Adzet eta/., 1988
T. loscosii Willk Luteolin Adzet and Martinez, 1980a, 1981b
6-OH-Luteolin Adzet and Martinez, 1980a, 198 1b
Luteolin-7-0-P-D-glucoside Adzet et al., 1982
6-OH-Luteolin-7-0-,!-D- Adzet etal., 1982
glucoside
T. nzacedoniczls (Deg. et Ur.) Apigenin Kulevanova et dl., 1997
Ronn. Eriodictyol Kulevanova et dl., 1997
Luteolin Kulevanova et al., 1997
Caffeic acid Kulevanova et dl., 1997
Rosmarinic acid Kulevanova et dl., 1997
T. nzarschallianzls Willd. Apigenin Litvinenko and Zoz, 1969;
Simonyan and Litvinenko, 1971
Luteolin Litvinenko and Zoz, 1969;
Simonyan and Litvinenko, 197 1
Scutellarein Lltvinenko and Zoz, 1969
Apigenin-7-0-P-D-glucoside Simonyan, 1972
Luteolin-7-0-P-D-glucoside Simonyan, 1972
T. mastichina L. Apigenin Adzet and Martinez, 1981b
Cirsimaritin Herniindez etal., 1987
5-Desmethylnobiletin Hernindez et al., 1987
5-Desmethylsinensetin Hernindez et al., 1987
Genkwanin Adzet and Martinez, 1981b
Luteolin Adzet and Martinez, 1980a, 1981b
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b
Thymusin Hernindez etal., 1987
Xanthomicrol Hernindez et al., 1987
T. 17zastzgophorz~sLacaita Cirsilineol Hernindez et al., 1987
8-OMe-Cirs~lineol Hernindez etal., 1987
Cirsimaritin Hernindez eta/., 1987
5-Desmethylnobiletin Hernindez etal., 1987
5-Desmethylsinenserin Hernindez et al., 1987
Gardenin-B Hernindez eta/., 1987
Sideritoflavone Hernindez et al., 1987
Thymonin Hernindez et al., 1987
Thymusin Hernindez eta/., 1987
Xanthomicrol Hernindez et al., 1987
Vicenin-2 Husaln and Markham, 1981
T. nzembranaceus Boiss. Aplgenin Adzet and Martinez, 1981b;
Adzet eta/., 1988; Ferreres etal., 1985a
Cirsilineol Adzet eta/., 1988; Hernindez etal., 1987
8-OMe-Cirsilineol Adzet etal., 1988; Ferreres eta/., 1985a;
Hernindez etal., 1987
Cirsimaritin Adzet and Martinez 1981b; Adzet eta/.,
1988; Ferreres etal., 1985a; Hernindez
eta/., 1987
Chrysoeriol Ferreres et dl., 1985a
5-De~meth~lnobiletin Adzet eta/., 1988; Ferreres etal., 1985a;
Hernindez eta/., 1987
Table 5.4 (Continued)
-

Species Polyphenols References

5-Desmethylsinensetin Ferreres et al., 1985a;

Hernindez etal., 1987
2,3-Dihydrokaempferol Adzet et al., 1988
2,3-Dihydroxanthomicrol Adzet etal., 1988
Eriodictyol Adzet et al., 1988
Eupatorin Ferreres et al., 1985a
Genkwanin Adzet and Martinez, 1981b; Adzet etal.,
1988; Ferreres etal., 1985a
Luteolin Adzet and Martinez 1980a, 1981b;
Adzet etal., 1988; Ferreres etal., 1985a
6-OH-Luteolin Adzet and Martinez 1980a, 1981b
7-0-Me-Luteolin Ferreres etal., 1985a
Naringenin Adzet etal., 1988; Ferreres etal., 1985a
Sakuranetin Adzet etal., 1988
Sideritoflavone Adzet etal., 1988; Ferreres etal., 1985a;
Hernindez etal., 1987
Taxifolin Adzet etal., 1988
Thymusin Ferreres et al., 1985b; Hernindez etal.,
1987
Xanthomicrol Adzet and Martinez 198 1b; Adzet et al.,
1988; Ferreres etal., 1985a; Hernin-
dez etal., 1987
Tomis et dl., 1985
Tomis etal., 1985
Tomis et al., 1985
T o m b etal., 1985
neohesperidoside
Luteolin-7-0-P-D-rutinoslde Tomis etal., 1985
Luteolin-7-0-8-D- Tomis etal., 1985
sambubioside
Luteolin-7-0-xyloside Tomis et al., 1985
6-OH-Luteolin-7-0-0-~- Tomis et al., 1985
glucoside
Vicenin-2 Husain and Markham, 1981;
Tomis et al., 1985
Caffeic acid Adzet etal., 1988
Rosmarinic acid Adzet et al., 1988
T. migricus Klok. et Schost. Apigenin Simonyan and Litvlnenko, 1971
Luteolin Simonyan and Litvinenko, 1971
Scutellarein Simonyan and Litvinenko, 197 1
T. nzoesiacus Velen Apigenin Kulevanova et dl., 1997
Luteolin Kulevanova et dl., 1997
Caffeic acid Kulevanova et al., 1997
T. nzoldavicus Klok. et Apigenin Litvinenko and Zoz, 1969
Schost. Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. moroderi Pau ex Martinez Apigenin Adzet etal., 1988
Cirsilineol Adzet et dl., 1988; Hernindez etal., 1987
8-OMe-Cirsilineol Adzet etal., 1988; Hernindez etal., 1987
Cirsimaritin Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylnobiletin Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylsinensetin Hernindez et al., 1987
 2,3-Dihydrokaempferol Adzet et al., 1988
2,3-Dihydroxanthomicrol Adzet etal., 1988
Eriodictyol Adzet etal., 1988
Genkwanin Adzet etal., 1988
8-OMe-Genkwanin Vila, 1987
Kaempferol Vila, 1987
Luteolin Adzet etal., 1988
6-OH-Luteolin Vila, 1987
7-0-Me-Luteolin Vila, 1987
Naringenin Adzet etal., 1988
Pilloin Vila, 1987
Sakuranetin Adzet et dl., 1988
Sideritoflavone Adzet etal., 1988; Hernindez etal., 1987
Sorblfolin Vila, 1987
Taxifolin Adzet etal., 1988
Thymusin Hernindez etal., 1987; Vila, 1987
Xanthomicrol Adzet etal., 1988; Hernindez etal., 1987
Luteolin-3'-0-alloside Vila, 1987
Luteolin-7-0-glucoside Vila, 1987
Luteolin-7-0-glucuronide Vila, 1987
Luteolin-7-0-xyloside Vila, 1987
Vicenin-2 Vila, 1987
Caffeic acid Adzet etal., 1988
Rosmarinic acid Adzet etal., 1988
T. neruosw Gay ex Willk. Apigenin Adzet and Martinez, 1981b
Cirsilineol Herndndez et dl., 1987
8-OMe-Cirsilineol Hernindez et dl., 1987
Cirsimaritin Hernindez et al., 1987
5-Desmethylnoblletin Hernindez et dl., 1987
5-Desmethylsinensetin Hernindez etal., 1987
Diosmetin Hernindez et al., 1987
Gardenin-B Hernindez, etal., 1987
Luteolin Adzet and Martinez, 1981b
Salvigenin Hernindez etal., 1987
Sideritoflavone HernPndez etal., 1987
Thymonin Herndndez et dl., 1987
Thymusin Hernindez et al., 1987
Xanthomicrol Hernindez et al., 1987
T. nummularius Bieb. Apigenin Slmonyan and Litvinenko, 1971
Luteolin Simonyan and Litvinenko, 1971
Scutellarein Simonyan and Litvinenko, 1971
Apigenin-7-0-P-D-glucoside Simonyan, 1972
Luteolin-7-0-,L?-D-glucoside Simonyan, 1972
T. orospedanus H . Del Villar Apigenin Adzet etal., 1988
Cirsilineol Adzet etal., 1988; Hernindez etal., 1987
8-OMe-Cirsilineol Adzet etal., 1988; Hernindez etal., 1987
Cirsimaritin Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylnobiletin Adzet et al., 1988; Hernindez etal., 1987
5-Desmethylsinensetin Hernindez et al., 1987
Diosmetin Hernindez etal., 1987
Eriodictyol Adzet et al., 1988
Genkwanin Adzet et al., 1988
Luteolin Adzet etal., 1988
Naringenin Adzet etal., 1988
Sakuranetin Adzet et al., 1988
Table 5.4 (Continued)

Species Polyphenols R&ences

T . orospedanus H . Del Villar Sideritoflavone Adzet etal., 1988; Hernindez etal., 1987
(Continued) Taxifolin Adzet et dl., 1988
Thymonin Hernindez et al., 1987
Thymusin Hernindez et dl., 1987
Xanthomicrol Adzet etal., 1988; Hernindez etal., 1987
Caffeic acid Adzet etal., 1988
Rosrnarinic acid Adzet et al., 1988
T.pallasianus H . Braun Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T . pannonicus All. Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. pastoralic Turrill, non Iljn Apigenin Simonyan and Lltvinenko, 197 1
Luteolin Simonyan and Litvinenko, 1971
Scutellarein Simonyan and Litvinenko, 197 1
Apigenin Adzer and Martinez, 1981b;
Barberin et al., 1985
Eriodictyol Barberin etal., 1985
Ladanein Barberin etal., 1985;
Hernindez et al., 1987
Luteolin Adzet and Martinez, 1980a, 1981b
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b
Naringenin Barberin et dl., 1985
Pebrellin Barberin etal., 1985;
Hernindez et dl., 1987
5-OH-7,4'-(OMe),-flavone Barberin et dl., 1985
5,6-(OH),-7,3',4'-(0Me)j- Barberin etal., 1985;
flavone Hernindez et al., 1987
5,6-(OH),-7,8,3',4'-(OMe),,- Barberin et dl., 1985;
flavone Hernindez etal., 1987
Apigenin-7-0-P-D-glucoside Barberin etal., 1985
Luteolin-7-0-b-D-glucoside Barberin et al., 1985
Vicenin-2 Barberin etal., 1985
T. pbtyphyllus Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. podolicus Klok. et Schost. Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. polessiczls Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
?: praecox Opiz Apigenin Adzet and Martinez, 1981b
Cirsilineol Hernindez et al., 1987
8-OMe-Cirsilineol Hernindez etal., 1987
Cirsimaritin Hernindez et al., 1987
5-Desmethylnobiletin Hernindez etal., 1987
5-Desmethylsinensetin Hernindez etal., 1987
Luteolin Adzet and Martinez, l 9 8 l b
 T. pruerox Opiz 6-OH-Lureolin Adzet and Martinez, 1981b
(Continued) Sideritoflavone Hernindez etal., 1987
Thymonin Hernindez et dl., 1987
Thymusin Hernindez et dl., 1987
Xanthomicrol Hernindez et dl., 1987

T. psezldograniticus Klok. et Acacetin Litvinenko and Zoz, 1969

Schost. Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969

T.pseadohzlnzillimas Klok. et Apigenin Litvinenko and Zoz, 1969

Schost. Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969

T. pse~~donanzmalurias
Klok. Apigenin Simonyan and Litvinenko, 197 1
et Schost. Luteolin Simonyan and Litvinenko, 197 1
Scutellarein Simonyan and Litvinenko, 197 1
Apigenin Adzet and Martinez, 1981b;
Stoess, 1972; Van den Broucke etal.,
1982a
Cyanidin Stoess, 1972
Cirsilineol Hernindez et dl., 1987
8-OMe-Cirsilineol Hernindez et dl., 1987
Cirsimaritin Hernindez et dl., 1987
5-Desmethylnobiletin Hernindez etul., 1987
5-Desmethylsinensetin Hernindez et al., 1987
Luteolin Adzet and Martinez, 1980a, 1981b;
Stoess, 1972; Van den Broucke etul.,
1982a
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b
Sideritoflavone Hernindez etul., 1987
Thymonin Hernindez et dl., 1987
Thymusin Hernindez et dl., 1987
Xanthomicrol Hernindez etal., 1987

T. rurifirz~sC. Koch Apigenin Simonyan and Litvinenko, 197 1

Luteolin Simonyan and Litvinenko, 197 1
Scutellarein Simonyan and Litvinenko, 1971

T. richurdii Pers. ssp. Apigenin Adzet and Martinez, 1981b

ebzlsitunus (Font Quer) Jalas Luteolin Adzet and Martinez, 1980a, 1981b
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b

T. rzchurdzi Pers. ssp Apigenin Adzet and Martinez, 1981b

richurdzz Luteolin Adzet and Martinez, 1980a, 1981b
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b

T. satarezoides Coss Apigenin Van den Broucke etul., 1982a

Cirs~lineol Van den Broucke etul., 1982a;
Voirin etul., 1985
Van den Broucke etul., 1982a;
Voirin etal., 1985
Cirsirnaritin Voirin etul., 1985
5-Desmethylsinensetin Voirin etal., 1985
Luteolin Van den Broucke etal., 1982a
Thymonin Van den Broucke etal., 1982a;
Voirin etal., 1985
Table 5.4 (Continued)

Speczes Polypbenols Refrences

T. satureioides Coss. Xanthomicrol Voirin etal., 1985

(Continued) 5,6,4'-(OH);-7,3'-(OMe), Voirin etal., 1985
flavone
T. serpylloides Bory Apigenin Adzet and Martinez, 1981b
Luteolin Adzet and Martinez, 1980a, 1981b
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b
T . serpylloides Bory ssp. Cirsilineol Hernindez et al., 1987
serpylloides 8-OMe-Cirsilineol Hernindez et al., 1987
5-Desmethylnobiletin Hernindez etal., 1987
Sideritoflavone Hernindez et dl., 1987
Thymonin Hernindez et al., 1987
Thymusin Hernindez etal., 1987
Xanthomicrol Hernindez etal., 1987
T . serpylloides Bory ssp. Cirsilineol Hernindez et al., 1987
gadorensis (Pau) Jalas 8-OMe-Cirsilineol Hernindez et al., 1987
Cirsimaritin Hernindez et al., 1987
5-Desmethylnobiletin HernLndez et al., 1987
5-Desmethylsinensetin HernLndez et al., 1987
Sideritoflavone HernLndez etal., 1987
Thymonin Hernindez etal., 1987
Thymusin HernLndez etal., 1987
Xanthomicrol Hernindez et dl., 1987
Apigenin Litvinenko and Zoz, 1969;
Olechnowicz-Step~enand
Lamer-Zarawska, 1975; Semrau,
1958; Van den Broucke etal., 1982a
Diosmetin Olechnowicz-Stepien and
Lamer-Zarawska, 1975
Luteolin Litvinenko and Zoz, 1969;
Olechnowicz-Stepien and
Lamer-Zarawska, 1975; Van den
Broucke etal., 1982a
Scutellarein Litvinenko and Zoz, 1969;
Olechnowicz-Stepien and
Lamer-Zarawska, 1975
Olechnowicz-Stepien and
Lamer-Zarawska, 1975
Apigenin-~'-O-P-D-~- Washington and Saxena, 1983
cumaroyl-glucoside
Diosmetln-7-O-P-D- Olechnowicz-Stepien and
glucuronide Lamer-Zarawska, 1975
Luteolin-galactoarabinoside Olechnowicz-Stepien and
Lamer-Zarawska, 1975
Olechnowicz-Stepien and
Lamer-Zarawska, 1975
Olechnowicz-Stepien and
Lamer-Zarawska, 1975
Scutellarein-glucosyl Olechnowicz-Stepien and
glucuronide Lamer-Zarawska, 1975
Scutellarein-7-O-P-~-glucosyl Washington and Saxena, 1986
(1-4)a-L-rhamnoside
T. sosnowskyi Grossh Apigenin Simonyan and Licvinenko, 197 1
Luteolin Simonyan and Litvinenko, 1971
Scutellarein Simonyan and Litvinenko, 1971

T. striatus Vahl. Apigenin Van den Broucke et al., 1982a

T. subalpestric Klok Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. tauricus Klok. et Schost Apigenin Litvinenko and Zoz, 1969
Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T. tiflsiensis Klok. et Apigenin Simonyan and Litvinenko, 197 1
Schost. Luteolin Simonyan and Litvinenko, 197 1
Scutellarein Simonyan and Litvinenko, 1971
T. toseuii ssp. substriatu Apigenin Kulevanova et al., 1997
(Borb.) Matevski Eriodictyol Kulevanova et al., 1997
Luteolin Kulevanova et al., 1997
Caffeic acid Kulevanova et al., 1997
Rosmarinic acid Kulevanova et al., 1997
T. toseuii ssp. toseuii var. Apigenin Kulevanova etal., 1997
degenii Ronn. Eriodictyol Kulevanova et al., 1997
Luteolin Kulevanova et al., 1997
Caffeic acid Kulevanova et dl., 1997
Rosmarinic acid Kulevanova et al., 1997
T. toseuzi ssp. toseuii var. Apigenin Kulevanova et al., 1997
longifrons Ronn. Eriodictyol Kulevanova et dl., 1997
Naringenin Kulevanova et dl., 1997
Luteolin Kulevanova et al., 1997
Caffeic acid Kulevanova et al., 1997
Rosmarinic acid Kulevanova et al., 1997
T. toseuii ssp. toseuii var. Apigenin Kulevanova etal., 1997, 1998
toseuii Velen. Eriodictyol Kulevanova et dl., 1997, 1998
Naringenin Kulevanova et al., 1997, 1998
Luteolin Kulevanova etal., 1997, 1998
Caffeic acid Kulevanova etal., 1997, 1988
Rosmarinic acid Kulevanova etal., 1997, 1998
T. transcaucasicus Ronn Apigenin Simonyan and Litvinenko, 1971
Luteolin Simonyan and Licvinenko, 1971
Scutellarein Simonyan and Litvinenko, 1971
T. Zrautuettet*zKlok. e t Apigenin Simonyan and Litvinenko, 197 1
Schost. Luteolin Simonyan and Litvinenko, 1971
Scutellarein Simonyan and Litvinenko, 1971
T. ucrainicus Klok. et Apigenin Litvinenko and Zoz, 1969
Schost. Luteolin Litvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
Apigenin Adzet and Martinez, 1981b
Cirsilineol Hernindez etal., 1987
8-OMe-Cirsil~neol Hernindez et al., 1987
Cirsimaritin Hernindez et al., 1987
5-Desmethylnobiletin Hernindez et al., 1987
5-Desmethylsinensetin Hernindez et al., 1987
Table 5.4 (Continued)

specie^ Polyphenols References

T . villosus L. (Continued) Gardenin-B Hernindez et al., 1987

Luteolin Adzet and Martinez, 1981b
6-OH-Luteolin Adzet and Martinez, 1981b
Salvigenin Hernindez et dl., 1987
Sideritoflavone HernLndez etal., 1987
Thymusin Hernindez et dl., 1987
Xanthomicrol Hernindez et al., 1987
Vicenin-2 Husain and Markham, 1981

Apigenin Adzet and Martinez, 1981b;

Adzet etal., 1988; Awe etal., 1959;
Kiimmell, 1959; Olechnowicz-
Stepien and Lamer-Zarawska, 1975;
Semrau, 1958; Stoess, 1972;
Van den Broucke etal., 1982a
Cyanidin Stoess, 1972
Cirsilineol Adzet et al., 1988; Hernindez etal.,
1987; Miura and Nakatani, 1989;
Morimitsu etal., 1995; Van den
Broucke etal., 1982b
Adzet et al., 1988; Hernindez et al.,
1987; Miura and Nakatani, 1989;
Van den Broucke etal., 1982b
Cirsimaritin Adzet and Martinez, 1981b;
Adzet et dl., 1988; HernLndez etal.,
1987; Miura and Nakatani, 1989
5-Desmethylnobiletin Adzet etal., 1988; Hernindez etal., 1987
5-Desmethylsinensetin Hernindez etal., 1987
2,3-Dihydrokaempferol Adzet etal., 1988
2,3-Dihydroxanthomicrol Adzet etal., 1988
Eriodictyol Adzet etal., 1988; Morimitsu etal., 1995
Gardenin-B Hernindez etal., 1987
Genkwanin Adzet etal., 1988; Miura and
Nakatani, 1989
Luteolin Adzet and Martinez, 1980~1,1981b;
Adzet etal., 1988; Awe etal., 1959;
Kiimrnell, 1959; Olechnow~cz-
Stepien and Lamer-Zarawska, 1975;
Semrau, 1958; Stoess, 1972;
Van den Broucke etal., 1982a
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b
Naringenin Adzet etal., 1988; Stoess, 1972
Quercetin Morimitsu etal., 1995
Sakuranetin Adzet etal., 1988
Salvigenin Hernindez et dl., 1987
Sideritoflavone Adzet etal., 1988; Hernindez etal., 1987
Taxifolin Adzet etal., 1988
Thymonin Hernindez etal., 1987; Morlmitsu et al.,
1995; Van den Broucke etal., 1982b
Thymusin Hernindez et al., 1987
Xanthomicrol Adzet and Martinez, 1981b;
Adzet et al., 1988; Hernindez etal.,
1987; Miura and Nakatani, 1989
 Miura and Nakatani, 1989
Olechnowicz-Stepien and
Lamer-Zarawska, 1975
Olechnowicz-Stepien and
Lamer-Zarawska, 1975
Olechnowicz-Stepien and
Lamer-Zarawska, 1975
Vicenin-2 Husain and Markham, 1981
Caffeic acid Adzet etal., 1988
Rosmarinic acid Adzet etal., 1988
T.~ulgarisL. ssp. erycoide~ Cirsilineol Herndndez etal., 1987
8-OMe-Cirsilineol Hernindez etul., 1987
Cirsimaritin Hernindez etal., 1987
5-Desmethylnobiletin Herndndez et al., 1987
5-Desmethylsinensetin Hernindez etal., 1987
Siderltoflavone Hernindez et dl., 1987
Thymonin Herndndez et dl., 1987
Thymusin Herndndez et al., 1987
Xanthomlcrol Hernindez et dl., 1987
T. we6bianus Rouy Apigenln Bldzquez etal., 1990, 1994
Cirsimaritin Bldzquez et dl., 1990
Eriodictyol Blizquez et dl., 1994
Genkwanin Blizquez et dl., 1990
Luteolin Bldzquez etal., 1990, 1994
Naringenin Bldzquez etul., 1994
Thymonin Bldzquez et al., 1990
5-OH-7,4'-(OMe)2-flavone Bldzquez et al., 1990
Apigenin-7-0-glucoside Bldzquez etul., 1994
Luteolin-7-0-glucoside Blizquez et al., 1994
Vicenin-2 Bldzquez etal., 1994
Chlorogenic acid Bldzquez etul., 1994
p-Coumaric acid Blizquez etal., 1994
J,5-Dicaffeoylquinic acid Bldzquez et dl., 1994
Protocatechuic acid Bldzquez et al., 1994
Syringic acid Blizquez etal., 1994
T.willkonzii Ronn Apigenin Adzet etal., 1988
Clrsilineol Adzet et dl., 1988
8-OMe-Cirsilineol Adzet etul., 1988
Cirsimaritin Adzet etal., 1988
2,J-Dlhydrokaempferol Adzet etul., 1988
Eriodictyol Adzet etal., 1988
Luteolin Adzet etal., 1988
Naringenin Adzet etal., 1988
Sakuranetin Adzer et dl., 1988
Sideritoflavone Adzet etal., 1988
Taxifolin Adzer etul., 1988
Xanthomicrol Adzet etal., 1988
Caffeic acid Adzet etal., 1988
Rosmarinic acid Adzet etal., 1988
T. zeleneitzkyi Klok. et Schost. Luteolin Lltvinenko and Zoz, 1969
Scutellarein Litvinenko and Zoz, 1969
T.ziaratinw Klok. et Schost. Apigenin Simonyan and Litvinenko, 1971
Luteolin Simonyan and Litvinenko, 1971
Scutellareln Simonyan and Litvlnenko, 1971
168 Roser Vila
Table 5.4 (Continued)

Species Polyphenols Rehrences

T . zygis L. Apigenin Adzet and Martinez, 1981b

Cirsimaritin Adzet and Martinez, 1981b
Luteolin Adzet and Martinez, 1980a, 1981b
6-OH-Luteolin Adzet and Martinez, 1980a, 1981b
Xanthomicrol Adzet and Martinez, 1981b
Vicenin-2 Husain and Markham, 1981
T. zygis L. ssp. syluestris Cirsilineol Hernindez etal., 1987
(Hoffmanns. et Link.) 8-OMe-Cirsilineol Hernindez etal., 1987
Brat. ex Coutinho Cirsimaritin Hernindez etal., 1987
5-Desmethylnobiletin Hernindez etal., 1987
5-Desmethylsinensetin Hernindez etal., 1987
Sideritoflavone Hernindez et dl., 1987
Thymonin Hernindez et al., 1987
Thymusin Hernindez etal., 1987
Xanthomicrol Hernindez et al., 1987
T . zygu Loefl. ex L. ssp. zygis Cirsilineol Hernindez et al., 1987
8-OMe-Cirsilineol Hernindez et dl., 1987
Cirsimaritin Hernindez et al., 1987
5-Desmethylnobiletin Hernindez et dl., 1987
5-Desmethylsinensetin Hernindez et al., 1987
Sidericoflavone Hernindez et dl., 1987
Thymonin Hernindez et al., 1987
Thymusin Hernindez et dl., 1987
Xanthomicrol Hernindez et al., 1987

Notes
* Cirslmaritin coeluted together wlth 8-OMe-Genkwanin In the TLC and HPLC systems used by Adzet etal. (1988).
t C~rsllineolcoeluted together w ~ t hEupatorin in the TLC and HPLC systems used by Hernindez etal. (1987).
i 8-OMe-Cirs~l~neol coeluted together with Gardenin D in the TLC and HPLC systems used by Hernindez etal. (1987).

constituted by apolar methoxylated aglycones, with different A-ring substitution patterns

of taxonomic importance.
In particular, the genus Thymw is rich in exudate flavonoids, especially the sections
Pseudothymbra, Thymus, Piperella and Mastichina, which grow in semi-arid habitats. In this
way, it has been found that species of the section Pseztdothymbra (T. membranaceus, T. moroikri,
T.funkii and T . longiflorus) from southeastern Spain, or T.herba-barona (section Serpylhm)
from Corsica, produce higher levels of excreted flavonoids than species growing in alpine
habitats, as it has been reported for some other taxa of the section Serpyllum (T. praecox,
T . pulegioides and T . nervosus) growing in the Pyrenees. This fact supports the influence of
ecological factors on the excretion of flavonoids. Although T . herba-barona and the species
of the section Pseudothymbra have been found to produce external flavonoids with different
A-ring susbstitution patterns, none of them being thymonin, the latter can be character-
ized by the presence of 4'-,6- and 6,s-di-substituted flavones which are absent from
T . herba-barona (Corticchiato etal., 1995; Hernindez etal., 1987).
Furthermore, Corticchiato etal. (1995) also studied the distribution of the exudate
flavonoids in seven essential oil chemotypes of T . herba-barona. The presence of three
different flavonoid patterns among them confirms the inherent chemical variability in
this species.
 Flavonoids a n d further polypheno i n the genus Thymus 169
Not only the habitat but also the genetic factors may influence the production of
external flavonoids, as it has been reported by HernBndez etal. (1987). The authors found
that thyme species growing under the same ecological conditions show very different
amounts of exudate flavonoids, which must be explained on the basis of the metabolic
capability for their production. That was the case of T . camphoratus, T . capitellatus and
T . carnosus, originally included in the section Thymm subsection Thymastra, which grow
in sandy areas near the sea in Portugal. While the former two species contained only
trace amounts of these compounds, the latter showed a very high concentration.
In general, although the exudate flavonoids in thyme taxa are quite different in structure,
the most remarkable feature is the presence of 5,6-dihydroxy-7,8-dimethoxyflavones
(thymonin, thymusin or pebrellin) and the nearly absence of 5,6-dihydroxy-7-methoxy-
flavones, which are usually co-occurring with the former in related genera (TomBs-
Barberin and Wollenweber, 1990). These two types of flavonoids have been only found
together in four Thymus species: in T . piperella (Barber& etal., 1985) and T. herba-barona
(Corticchiato etal., 1995) as exudate flavonoids, and in T . satureioides (Voirin etal.,
1985) and T . moroderi (Vila, 1987) as constituents of lipophilic extracts of ground plant
material (Table 5.5).

FLAVONOID PATTERN: INFLUENCE OF THE ENVIRONMENTAL

CONDITIONS

Within the genus Thymus the influence of the environmental conditions on the pattern
of flavonoids has been investigated. Analysis of the flavonoid composition of individuals
belonging to the same species coming from different localities, as well as studies of
seasonal variations, have been performed in order to establish whether a genetic control
is directly related to it or not (Gil, 1993).
The fact that the flavonoid composition of different thyme species is clearly different,
although they grow under the same environmental conditions, supports a direct relation
of the flavonoid pattern with the genetic features of these plants.
Furthermore, thyme plants of the same species coming from different geographical
localities showed no variations of their flavonoid composition but of their total amount.
It has been found that even though differences in altitude may influence the total
flavonoid production as well as the relative amount of each one, they did not cause any

Table 5.5 Co-occurrence of 5,6-(OH),-7,8-(OMe),- and 5,6-(OH),-7-OMe-flavones in some Thymus taxa

Thymus taxa Flavones according substitz~tionputtern References

T . piperella Pebrellin 5,6-(OH),-7 ,3',4'-(0Me)3- Barberin et al.,

flavone 1985
5,6-(OH),-7,8,3'4'- Ladanein
(OMe)4-flavone
T . suturezoides Thymusin 5,6,4'-(OH) j-7 ,3'-(OMe),- Voirin et dl., 1985
flavone
T . moroderi Thymusin Sorbifolin Vila, 1987
T . herba-barona Thymusin Sorbifolin Corticchiato etal.,
1995
170 Roser Vila

Table 5 . 6 Flavonoid Composition of Thymbra capitata (syn. Thymus capztatas)

Flaoonoids Refirences

Acacetin Adzet and Martinez, 1981b

Apigenin Adzet and Martinez, 1981b
Diosmetin Barberin et al., 1986
Luteolin Adzet and Martinez, 1981b; Barberin etal., 1986
5,6-(OH),-7,3',4'-(0Me)Gavone Barberin et dl., 1986
5,6,4'-(OH)j-7,1'-(0Me)2-flavone Barberin et dl., 1986
Luteolin-7-O-rutinoside Barberin et al., 1986
Vicenin-2 Barberin etal., 1986; Husain and Markham, 1981

changes in the pattern of flavonoids, as it occurs in T . serpylloides. In this species, the

total flavonoid content is much lower in plants growing at a high altitude than in those
growing in a low altitude, the same flavonoids being found in all of them (HernBndez,
1985).
The monthly evolution of the flavone pattern from several thyme species growing
under different climatic conditions (alpine and xeric habitats) has also been evaluated in
order to establish the influence of the seasonal variations on the flavonoid composition.
The results showed that the pattern of flavonoids did not undergo any changes during
the year in any of them, as for example in T . serpyllaides, which showed the same pattern
in winter when it was covered by snow as in summer during flowering time (Gil, 1993;
HernBndez, etal., 1987).

CHEMOTAXONOMIC VALUE OF THYME FLAVONOIDS

Five papers (Adzet and Martinez, 1981b; Adzet etal., 1988; Hernindez etal., 1987;
Kulevanova et al., 1997, 1998) are devoted to a comparative TLC andlor HPLC analysis
of flavonoids of several Thymus species from different sections: Pseudothymbra, Thymus,
Mastichina, Micantes, Piperella, Hyphodromi and Serpyllum, mostly Iberian or Balearic
endemisms, and Marginati, from Macedonia. The results obtained give valuable inform-
ation from the chemotaxonomic point of view, which allow us to arrive at interesting
conclusions that support recent re-classifications of this genus.
In this sense, T . capitatus (Thymbra capitata), a Mediterranean plant which constitutes
a taxonomic problem extensively discussed (Adzet and Martinez, 1981b; Elena-Rossell6,
1976; Morales, 1985), was found to lack 6-OH-luteolin, present in almost all the species
of the subgenus Thymus, but to contain acacetin, a flavonoid of restricted distribution
within Thymw species (Adzet and Martinez, 1981b). In addition, Barbedn etal. (1986)
found that this taxon externally accumulated the same unusual 5,6-dihydroxy-7-methoxy-
flavonoids (Table 5.6) previously isolated from Thymbra spicata (Miski et dl., 1983) and
occasionally reported in the genus Thymus. This fact supports the separation of this
taxon from the genus Thymus and its inclusion in the genus Thymbra on the basis of
morphological and caryological data (BarberBn etal., 1986; Morales, 1985). The absence
of 6-hydroxyflavone glycosides, universally present in Thymw species, from T . ~apitatus
(TomBs-BarberBn etal., 1988b), also supports its inclusion in the genus Thymbra as has
been proposed also on the basis of its flavonoid aglycones.
 Flavonoids andfzrtberpolypbenols in the genw Thymus 17 1
Excluding T . capitatus, HernBndez etal. (1987) distinguished two well-defined groups
among the sections of the genus Thynzw according to their flavonoid pattern, particu-
larly to the presence or absence of the highly methoxylated 5,6-dihydroxyflavones
thymusin andlor thymonin, which are the most characteristic flavones of the genus.
One group is constituted by the section Piperella (T.piperella), characterized by the presence
of pebrellin, ladanein, 5,6-(OH),-7 ,8,3',4'-(OMe)4-flavone and 5,6-(OH),-7,3',4'-(OMe)?-
flavone and by the lack of thymusin and thyrnonin. The second group includes all the
other sections of the genus, in which thymusin and/or thymonin are always present.
Although some sections (i.e. Piperella, Mastichina, Micantes) do not pose taxonomic
problems, their caryological and morphological data being in accordance with their
flavonoid pattern, in several species of other sections, like Pseudothymbra and Thymus,
the flavonoid composition may help to resolve some taxonomic uncertainties.
Section Pseudothymbra is divided into two subsections: Anomalae, which only includes
T . antoninae, and Pseudothymbra (Morales, 1985). The most remarkable feature of the
flavonoid pattern of this section is the high content of apolar methoxylated flavones,
especially xanthomicrol, and the presence of flavanones, mainly naringenin, and dihydro-
flavonols (Adzet etal., 1988). Thymusin was detected in T . membranaceus, T . longiflorus,
T . moroderi, T . fankii, T . antoninae and T . villosus, which however lacked thymonin.
Significant amounts of the latter were found in T . mastigophorus, its flavonoid compo-
sition being very similar to that of T . lacaitae, supporting the inclusion of T . mastigopho-
rus, originally included in section Pseudothymbra (Jalas, 1972), in section Hyphodromi as
suggested by Morales (1985). Furthermore, similarities between the flavonoid pattern
of T . villosw and T . antoninae might allow to join them in the subsection Anomalae of
this section, being clearly differentiated from the rest of the species of the subsection
Pseudothymbra (Herngndez etal., 1987).
Section Thymus, which is morphologically very heterogeneous, comprises two sub-
sections: Thymustra, with T . carnosus, T , camphoratas and T . capitellatus, and Thymus,
which includes the rest of the species of the section (Jalas and Kaleva, 1970). The
analysis of their flavonoid composition allows to differentiate two groups of taxa. One
of them with practically no methoxylated flavonoids (neither flavones nor flavanones),
only traces of xanthomicrol, that includes T . camphorutus and T . cupitellatus belonging to
the subsection Thymastra. The other one, which comprises taxa of the subsection Thymw
(T. vulgaris, T . vulgaris ssp. aestivus, T . glandulosw, T . baeticus, T . orospedanus, T . hyemalis,
T . zygis and T . serpylloides)as well as T . carnosus, turned out to contain a great variety of
non-polar flavones, flavanones and dihydroflavonols (Adzet etal., 1988; HetnBndez
etal., 1987). These findings are in accordance with morphological results reported by
Morales (1985), which separated T . carnosas from the subsection Thymastra.
Within section Serpyllam, the largest one of the genus Thymus (Jalas, 1971), only the
flavonoid pattern of few taxa has been investigated, particularly of T . nervosas and
T . praecox (subsection Pseudomarginati), T . pulegioides (subsection Alternantes), T . willkonzii
(subsection Insulares) and T . herba-barona (subsection Pseudopiperellue). In general, those
of alpine occurrence, the first four species, show low levels of high methoxylated fla-
vones, thymusin and thymonin being reported in all of them except in T . willkomii
(HernBndez etal., 1987). The latter, an Iberian northeastern endemism which grows in
restricted areas, shows a flavonoid pattern mainly characterized by flavanones and dihy-
droflavonols, the most important being naringenin and sakuranetin (Adzet etal., 1988).
In contrast, T . herba-barona growing in xeric habitats produces several methoxylated
flavones among which thymusin but not thymonin was found (Corticchiato etal., 1995).
172 Roser Vila
The main features of the flavonoid pattern of the sections Pseudothymbra, Thymus, and
Serpyllum, according to the results provided by Adzet etal. (1988), Corticchiato etal.
(1995) and Hernfindez etal. (1987), are summarized in Table 5.7.
The flavonoid composition of 14 taxa belonging to the subsections Verticillati and
Marginati of the section Marginati (A. Kerner) A. Kerner (nowadays included in the
section Serpyllum (Jalas, 197 1 ) ) from the Macedonian flora has also been investigated
(Kulevanova etal., 1997, 1998). Apigenin and luteolin are present in each taxa, the lat-
ter being the major one in all of them, except in T . jankae var. jankae and T . jankae var.
pantotrichus. They both contain diosmetin instead of luteolin as the principal flavone.
No methoxylated flavones other than diosmetin were detected in the taxa investigated.
Furthermore, the distribution of phenolic acids shows that caffeic acid is present in
the taxa of the two subsections, whereas rosmarinic acid is only detected in those of the
subsection Verticillati. In addition, the authors found great resemblance between the
flavonoid pattern of T . moesiacus, T . albanw and T . balcanus from Macedonia, and
T . zhegulzensis and T . bashkiriensis from Russia, all of them closely related from a bota-
nical point of view.
Finally, although in general flavonoid glycosides are not considered to have as much
chemotaxonomic significance as aglycones, some interesting results on the distribution
of 6-hydroxy-, 6-merhoxy- and 8-hydroxyflavone glycosides in the Labiatae, Scrophu-
lariaceae and related families have been reported (Tomb-Barberfin etal., 1988a). Particu-
larly, in the genus Thymus, only 6-hydroxyflavone glycosides (mainly 6-hydroxyluteolin
glycosides) were found in twenty-two taxa belonging to several sections (Mastichina,
Micantes, Piperella, Pseudothymbra, Thymus, Hyphodrami, Serpylhm).
All these findings support the fact that flavonoids are valuable taxonomic markers
that particularly in the genus Thymw have provided worthy information that together
with those obtained from caryological and morphological studies, allow one to resolve
taxonomic doubts and improve the classification of the genus. Despite the great work
carried out, much more research should be done in order to complete the characteriza-
tion of the genus.

Table 5.7 Main features of the flavonoid pattern of the sections Psezldothymbra, Thymw, and Serpyllunz
(Adzet etal., 1988; Corticchiato et dl., 1995; Hernindez etal., 1987)

Section Subsection Highly Thymusin Thynzonin Exzldateflavonoids

nzethoxylated level
flavones

Psezldothymbra Anomalae T Yes No L

Psezldothymbra T Yes No 1
Thymu Thymastra 1 Yes Yes 1
Thy~nu 1 Yes Yes 1
Serpyllnm Alternantes 1 Yes Yes 1
Psezldomarginati L Yes Yes 1
Pseudopipe~ellde T Yes No 1
Insulares 1 -* - -

* Not reported
 Flavonoids and further polyheols in the genus Thymus 173
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17 6 Roser Vila
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6 Field culture, in vitro culture
and selection of Thymas
Charles Rey and Fruncisco Sdex

INTRODUCTION

People have known and used plants from the Labiate family for many centuries, Thymw
being one of these, due to its medicinal and flavouring properties that have long been
recognised. Poetic descriptions of early Persian gardens include Thymw among the
plants that they cultivated, showing their interest in the plant. The demand has always
increased with the growth of the human population, especially in the last decades with
the investigation of its pharmacological properties. Lawrence (1992) reports
a production of 25 tons of essential oil of T. zygis in 1989, mostly provenant from
collections of wild material.
A continually growing demand for thyme products is not likely to be supported by
natural populations, which are threatened by destructive gathering methods and insuf-
ficientiitregular rainfall in traditional source areas. Additionally, the interests of the
pharmaceutical/food industries do not focus on all chemotypes available in nature, but
only on a few, namely thymol-, carvacrol- and linalool-types. Therefore an increase in
the demand of thyme of cultivated origin must be expected with standardised com-
position and yield of the essential oils and with uniform organoleptic properties of
the leaves.
In this chapter the recent efforts made by the different researchers to meet the
demands of growers and markets will be reviewed, focusing on the selection and culti-
vation techniques used both under natural conditions and under a controlled environment
in the laboratory. Special attention is given to the organic culture of thyme, which is of
great interest in order to produce plant material for the food industry. Switzerland is a
well-known centre for the cultivation of thyme, and the experiments performed there
will serve to illustrate a number of procedures involved in the development of new varieties
with commercial interest in thyme.

ORGANIC CULTURE OF COMMON THYME (THYMUS VULGARIS

L.) I N SWITZERLAND

Organic culture of common thyme is developed basically in marginal areas (characterised

by worse climatic, edaphic and accessibility conditions than traditional agricultural areas)
by motivated and conscientious growers. Today it has a small market growing progres-
sively year by year. In Switzerland about 30 tons of dry plants were produced under
contract during 1999, in mid-range mountain areas between 600 and 1 2 0 0 m altitude
178 Charle~Rey and Francisco Sa'ez
(Gammetter, 2000). This production basically feeds local markets related to the food
industry and the phytotherapy.
The Swiss organic label by the Association Suisse des Organisations d'ilgriculture
Biologique (ASOAB) can only be obtained under stricter conditions than in the normal
European directives for cultivation (prohibition of synthetic chemical fertilisers, herbi-
cides, insecticides and fungicides). The industries interested in the organic products
usually develop their own pricing system taking into consideration the higher production
costs, which must be calculated as the sum of a salary about 15-20 Swiss francs (9-12
US$) per hour plus the specific costs for the cultivation.
Today, based on the selection activities developed by the 'Station f6dgrale de recherches
en productions vCg6tales de Changins B Conthey', the growers have developed thyme
varieties available that are adapted to the continental climate of mountain ranges. These
varieties are thymol chemotypes (Rey, 1993a, 1994a,b). They stand out due to their
homogeneity, productivity, and good yield in essential oil (>3.5 per cent). It should be
noted that the Swiss Pharmacopoeia VII had fixed the minimum essential oil content at
1.5 per cent, in the dry herbs of thyme. Today the European Pharmacopoeia demands
a minimum of 1.2 per cent oil content for thyme herb.
For the success of the culture of this thermophyllous species it is of great importance
to choose a convenient place. In mountain ranges between 700 and 1200 m altitude,
warm, sheltered places guarantee good productivity and good quality. Under these
conditions, avoiding harmful frosts, the cultivation may continue for 3-5 years. These
qualities have made the Valais and Poschiavo valleys (Grisons) the preferred places for
culture.

Cultivation
Common thyme (T. vulgaris) prefers a light and permeable soil, somewhat rich in organic
matter and mineral fertilising elements (ITEIPMAI, 1983; Rey, 1990a; Anonymous,
1992, 2000). Its culture is preferably established on land previously cultivated with
cereals or legumes. Bovine manure (0.5-1 m3/are) constitutes the basic fertilizer.
SRVA's directives for thyme are that the usual fertilisation is 80 nitrogen, 80
phosphorus and 100 potassium units. The establishment of the culture is done by
mid-May at the usual plant density of 57 000 plantslha (70 x 25 cm). Direct sowing by
early September is more advantageous. This technique, recently fine-tuned, is only
possible in proper soils (Rey, 1993b). It is important to note that plants bearing only
few pairs of leaves at the beginning of the winter (2-5 cm height) are quite frost-resistant.
Weeds must be removed 4 or 5 times during the season to maintain the culture
adequately. Although selective herbicides are properly used for industrial culture, they
are strictly prohibited in organic cultures. Thus, in order to reduce this unpleasant work,
culrivation with black plastic is possible (My-Pex plastic). Regular watering enhances
the response after spring harvest and guarantees a second harvest by mid-August.
Concerning parasites, one must expect to find cicadas (Eupterix decemnotata Rey) which
may cause damage in hot summers. But this damage is usually of little importance, and
antiparasitic protection based on natural authorised insecticides (Parexan, Biocide. . .) is
sufficient.
Provided that cultivation starts in spring, the yield for T. vulgaris is from 2000 to
2500 kglha of dry plant material in the first year after the August harvest. From
the second year on, the harvests in June and August provide an annual yield of
 Field culture, in vitro czlture and selection ofThymus 179

Figz~re 6.1 Culture of common thyme in full bloom before harvest

3500-5000 kglha. A culture started by seeds directly sown on the fields in September
allow two harvests the following year. If the spring harvest (Figure 6.1) is made at full
bloom (depending on the kind of market), the August harvest is always performed on
the branches bearing leaves, since the plants do not produce new flowers, or just a little
bloom. The harvest is carried out during dry and sunny weather using motorized
shears or a harvester with an adequate cutting bar (Figure 6.2). Cutting at a height of
10-15 (20)cm above the soil level is advisable in order to avoid problems with frost.
If harvesting takes place too late at the end of the summer, problems with cold temper-
atures may arise (Rey, 1991).

Qualitative and economical aspects

There are two aspects that influence the quality: (a) The proportion of leaves and stems,
leaves presenting a higher quality, and (b) The content of essential oil. In order to
obtain a proportion of leaves higher than 50 per cent, it is important to harvest before
blooming. Contrarily, to obtain an optimal yield in essential oil, harvest during full
bloom is preferable. The yield in essential oil is dependent on local climatic conditions
and on seasonal variations. Thus, in mid-range mountain cultures where two harvests
per year are obtained, a 50-100 per cent improvement in the yield of the summer harvest
with respect to the spring harvest can be achieved.
The drying of the material must be done carefully in dry places and protected
from light. The plants are placed in stratified beds of 1-2 m height, which are venti-
lated with warm air at 30 and 45 "C. Under these conditions drying takes 2-3 days
180 Charles Rey and Franczsco Sdez

Figure 6.2 Harvest of common thyme in a mountain field

(10-12 per cent moisture as a maximum at the end of the process) depending on
the density of the beds, thus keeping all the intrinsic properties as well as a good
appearance. With regard to organic cultures the market for the pure leaf is small.
If demanded, separation of stems and leaves is done using sieves.
Cultivation in the mountains, developing patches at different altitudes, notoriously
increases the production costs because it is not possible to provide a mechanised
response to all needs of natural cultivation. The culture of thyme needs about 1 500-
2 000 hours of work per hectare, more than half of the time due to the elimination of
weeds.

SELECTION OF COMMON THYME (THYMUS VULGARIS L.)

FOR MARGINAL AREAS

In marginal areas such as the mid-range mountain, only the common thyme from the
German race, or 'German thyme' (AL), could be cultivated with success. With respect
to the Mediterranean thyme or the French thyme, it is better adapted to lower tempera-
tures. However, it has quite a heterogeneous phenotype, and this results in less
regularity of the culture and lower quality of the final product. The yield in essential
oil is insufficient and does not reach the minimum of 1.5 per cent required by the Swiss
Pharmacopoeia VII or 1.2 per cent by the European Pharmacopoeia. People growing
thyme in the mountains have demanded a homogeneous variety better adapted to the
peculiarities of a mountainous climate that can be grown from seed. W i t h the aim of
 Field culture, in vitro cultz~reand selection of Thymus 181
fulfilling this demand, a selection program was started at the end of the 1980s at the
'Station f6dCrale de recherches en productions vCg6tales de Changins' at 'Centre des
Fouggres de Conthey' (Valais).

Plant material
Common thyme (T. vztlgaris) is of Mediterranean origin. The natural distribution area
is from Italy to Spain. It is mostly erect, with lignified stems of 10-40 cm height. The
leaves, with involute margins, are linear to lanceolate and of variable size. They have an
acute tip and bear glands. On the upper side they are greyish-green to greyish-blue,
while being whittish in the lower face. The flowering stems bear capitated or verticil-
lated inflorescences. The flowers, with pink petals, bloom from April to June depend-
ing on the altitude. Hermaphrodite flowers are bigger than female ones. The seeds
become mature 1 month after the bloom. One thousand seeds weigh about 0.25-0.298.
The leaves, flowers and herbaceous stems bear glandular hairs that contain the essential
oil. The tector hairs that constitute the hairy characteristic of the leaves and stems
protect the plant from evaporation of water. In natural conditions a thyme plant may
live for 15 years.
The floral biology of common thyme has been described in detail by Assouad and
Valdeyron (1975). To maintain the vigour of the species, the crossed fertilisation
between male fertile (hermaphrodites, MF) and male sterile (MS), enhanced by protandry
(development of anthers before pistils), is more frequently found, although auto-
fertilisation is possible among MF individuals. The use of male-sterility of common
thyme for breeding and production of new varieties was first proposed by Key (1990a).
Another aspect to keep in mind during the selection activities is that the European
Pharmacopoeia focuses on the thymol chemotype, postulating 30-70 per cent thymol
and 3-1 5 per cent carvacrol. The linalool and carvacrol chemotypes are preferred by the
condiment market (ITEIPMAI, 1983).
The commercial varietieslpopulations of German common thyme and a natural
population of common thyme in the Aoste valley in northern Italy have served as starting
material for the selection. German thyme is a result of an empiric selection managed by
generations of farmers. Its frost-resistance allows it to be cultivated even in northern
Europe, e.g. Holland and Finland (Aflatuni etal., 1994; Simojoki etal., 1994) or in
Canada (Laflamme etal., 1994) (at least in annual culture), but its quality is not as good
as when produced in more southern latitudes. Its foliage is greyish-green, and it blooms
later (about 10 days) than the thyme from the Aoste valley. Its habitus is mainly erect.
German thyme is generally quite heterogeneous with respect to its vigour and the colour
and dimension of the flowers.
The thyme from the Aoste valley or 'ValdBtain thyme' (here abbreviated as VA)
corresponds to a more typical Mediterranean thyme. It is more sensitive to frost than
German thyme, but its quality with regard to essential oils is superior. Its foliage is
mainly greyish-blue and the stems are more lignified, resulting in a more erect habitus. The
ValdBtain ecotype represents the most northern occurrence for this species. In this internal
valley of the Alps, at subcontinental climate, thyme is placed in the warmest areas and
the driest places, at a maximum of altitude 1 600m observed in this region. As a pioneering
species in these limiting conditions common thyme presents a wide variability of the
phenotypic characters such as habitus, vigour, colour and size of the leaves and flowers,
and its sex, and therefore the plants are interesting elements for selection. In contrast to
182 Charles Rey and Francisco Skez
that the chemical variation is low, only the thymol chemotype being found in this area.
This chemotype, preferring the warmer and drier areas of the Mediterranean climate,
represents a good bioclimatic indicator of such conditions in the area (Rey, 1989,
1990b). Although being a stable chemotype, a certain variability is observed in the
yield of essential oil among the individuals from this population.

Method of selection
A large number of essays for comparison of German thyme and Vald8tain strains were
made at the mountain sites of Arbaz valley (920m altitude, sunny exposure) and Bruson
(1 1 0 0 m altitude, ombrous exposure) and have preceded and oriented the selection
activities (Rey, 1988, 1993a, 1994b). As an example, Table 6.1 shows the differences of yields
in dry matter and active matter, as well as the differences in flowering and frost-resistance
obtained from German thyme and Vald8tain thyme.
The complementary characters of these two races of common thyme suggested the
idea of making crosses between them, in the hope of obtaining more regularity, vigour
and quality in the hybrids. A study of the variability within the best provenances has
been made. Thus, it was possible to isolate and multiply by classic cutting or in vitro
culture (L@,1989) the elite plants from both sexes MS and MF. After 1989 many cross-
ings have been made each year using the best clones. The cultivation of parental clones
with the aim of obtaining hybrids, as well as the method itself, have been described by
Rey (1993a). Briefly, the scheme for selection is as follows:

(a) Localisation of elite plants, male-sterile and male-fertile, in the natural populations
of the Aoste valley and in the varietiesipopulations of German thyme.
(b) Vegetative multiplication by spring cuttings or micropropagation of these initial
clones to verify their performance under field conditions (agronomic test, laboratory
analysis and annotation of flowering period). Only the best are retained to test their
crossing value.
(c) Isolation and crossing of the best clones using bees, by pairs MS-MF. Only the
seeds from male-sterile parents are collected.
(d) This hybrid seed is sown and the experimental hybrid judged for performance and
homogeneity. The parents of the best hybrid are multiplied on a large scale. The
production of seeds of the commercial hybrid may thus start under isolation.

More than 120 different crossings have been made up to now. The results presented
here concern crossings performed in 1989 up to 1991 (Figure 6.3; see also Rey, 1993a, b)
for which we have 3 years of harvests, this being the normal life span for a culture of
German thyme. These results are based on a mean of 50 plants.

Productivity
With respect to productivity we recognise four criteria to characterise the yields and
quality for each variety. These are:
Yield in dry matter. In the 1989 experiment, with 3 years accumulated and comprising
5 harvests in total, the mean yield in dry matter from hybrids VAXAL was 1.95 times
higher than the reference, 1.45 times higher than the polycross VAXAL and 1.72
 T 1 2 3 4 5 6

dry matter leaves g/rn'

Figure 6.3 Productivity of hybrids of thyme in 1989 up to 1991 (5 harvests in 3 years). Upper graph:
cumulative yields of essential oil (EO) in llha and mean percentages of EO. Lower graph:
cumulative yields of dry matter and leaves in g/m2. T = control AL; 1 = Hybrids VA x AL;
2 = Polycross VA x 4 AL; 3 = Hybrld VA x VA; 4 = Clones VA MS; 5 = Clone AL MF;
6 = Clone VA MF
 Field cztlture, in vitro culture and selection ofThymus 185
times higher than the hybrid V A x V A (Figure 6.3). This advantage was observable
from the first harvest in the second year of culture. The yields from the Vald8tain and
German parents is quite different from those presented by the hybrid itself. In the
experiment in 1991, the higher mean yield of the four hybrids VAXAL with respect
to the reference was confirmed. The five retrocrossed hybrids (VAXAL) x VA and
(VA xAL)xAL lose all advantages with respect to FI cross. Four autofecundations VA
and AL showed the depressing effect attached to consanguinity.
Yield in leaves. Calculated from portions of dry plant, with 100g, the leaves separated
manually, these yields show the same patterns as the dry matter. In the 1989 experiment,
the yield in leaves of the hybrids VAXAL was superior (Figure 6.3) to the control, the
polycross and the hybrid VAXVA, respectively. The hybrids were also superior to the
parents here.
Pevcentage of essential oil (EO). It was obtained from samples of 1 0 0 g of dry leaves,
mentioned in Figure 6.3, and are means for the 5 harvests. The hybrids VAXAL have
surpassed 1.3 times the value of the control. Their level anyway was 1 0 per cent lower
than the 2 MS Vald8tain parents and also the hybrid VAXVA. In the 1991 experiment,
the superiority of hybrids VAXAL with respect to the control was confirmed. The
hybrids ALXVA, VAXVA, ALx AL, (VAx AL) XVA and (VAXVA) x AL showed
comparable results with a mean of more than 4 per cent. One should note the high level
of 4.55 per cent of hybrid ALxAL that results from the careful selection of elite plants
within a population of German thyme. Plants obtained from autofecundation VA and
AL have proven to be low in essential oil.
Yield in llha of essential oil. For this parameter the hybrids VAXAL in the 1989
experiment for example, produced a mean of 362 liha, confirming their superiority with
tespect to the control (Figure 6.3). The polycrosses, with 2601iha, showed an inter-
mediate value. The yields of the German and Vald8tain parents are respectively between
170 and 245 liha.
During these two crossing trials, the hybrids VAXAL and ALXVA have largely
surpassed the AL controls, concerning the yields in dry matter, in leaves and in liha of
essential oil. Considering the genetical distance between these two races of thyme, the
effect of heterosis observed is not surprising. For the first experiment the medium level
of hybrids VAXAL represents an appreciable enhancement of 30 per cent with tespect
to the AL control. The quality of hybrids VAXAL and ALxVA from the second experi-
ment was also very satisfactory.

Homogeneity of the phenotype

In the 1989 experiment, the hybrids of clones VAXAL showed a very good regularity
in the morphological characters with respect to the German control and the polycross
VAXAL. This homogeneity concerned the habitus and dimension of the plants, the
size and colour of the leaves and the flowers (Table 6.2). This was not comparable to the
one obtained with the clonal culture. Similar results were reproduced in the second
trial with certain hybrids VAXAL as well as some hybrids in the inverted way
ALxVA. For the transmission of sex for example, 98-100 per cent MS individuals
were obtained if the female parent was homozygous for this character. In the retro-
crossed hybrids the sex is usually 50 per cent of MS and 50 per cent of MF, thus dimin-
ishing the phenotype regularity.
186 Charles Rey a n d Francisco Sd'ez

Table 6.2 Mean percentage for the transmission of floral characteristics and the colour of the leaves
depending on the plant type

Pblzt-t3'pe Flowers Leaves

Sex % Size % Colour %

MS MF S??zallto nzediuvz Mediuvn to bzg Grey-blue Grey Grey-greelz

Control AL
1. Hybrid VA 1 x AL
4. Clone VA 1 MS
1. Hybrid VA 2 x AL
4. Clone VA 2 MS
3. Hybrid VA x VA
2. Polycross VA X 4 AL
5 . Clone AL MF
6. Clone VA MF

Notes
AL, German.
VA, ValdBtain.
MS, Male sterile.
MF. Male fert~le.

The homogeneity obtained by the clone hybrids is itself a great advantage with
respect to the heterogeneity of the initial varieties1populations. Since the hybrid thyme
is produced by crossing two heterozygous parents, its homogeneity is comparable to
that of a double hybrid (from 4 parents). Only the simple hybrid with homozygous parents
allows a rigorous homogeneity. The simple hybrids are easily obtained from autogamous
species, spontaneously homozygous, but more difficult from allogamous species like
thyme. For the polycross VAXAL, where the same parents MS VA are crossed with
4 parents MF AL, the sexual characters have the tendency to level off to a 1:1 proportion.
This method of selection is thus not the best with respect to homogeneity matters.

Table 6.3 with the results obtained in the winter of 199011991 presents the damages due
to frost sorted according to their severity. Common thyme, when frozen about 20-25
per cent, recovers completely and does not yield significantly less. O n the other hand,
plants strongly affected, about 30-90 per cent, show a considerably lower yield. The
hybrids VAXAL were almost as resistant to winter frost as the German control plants.
The polycross VA XAL were also the same. O n the contrary, much lower resistance was
noted for the hybrid VAXVA and its Vald8tain parents. The plants obtained by
autofecundation were found more sensitive to frost than their respective parents. After
3 years of cultivation most of the hybrids showed a very high resistance to winter ftost
with respect to the German control, with more than 70 per cent ftost damage.

Production of seed$
The crossings between Valdctain and German clones produced a variable quantity of
seed depending on the size of the plants, the flowering stadia1 and the quantity of
 Field cultare, in vitro culture and selection of T h~ m u s 187

Table 6.3 Mean percentage of frost damages per plant, by the plant type in winter 199011991

Pbnt-tj@e Level of ice damage per plant

0-2 5 % 30-90%

control AL 100 0
1. Hybrids VA x AL 98 2
2. Polycross VA x 4 AL 100 0
3. Hybrid VA x VA 82 18
4. Clones VA MS 60 40
5 . Clone AL MF 95 5
6. Clone VA MF 79 21

Notes
AL, German.
VA, ValdBtain.
MS, Male sterile.
MF, male fert~le

bees around. For the crossings studied in 1989 the production of seeds varied from
1.2 g for the VA x VA hybrid, to 5.5 g for a VA x AL polycross. The autofecundation of
MF clone VA produced only 0 . 3 g of seeds, that is 4 times less than the V A X V A
hybrid. The same tendencies were noted during the crossings developed afterwards,
with a maximum production of 8.1 g for a VAXVA crossing. The mean production
was 2-3g per plant, or 10-15g/m2, and this is 30-100 per cent higher than the
reports by Heeger (1956).

Germinative ability
Only allogamy can warrant the quality of the seeds, its germinative ability and
strength. A low germination percentage and weak plantlets from autofecundation.

Cultural considevations
The heterogeneity of common thyme has so far been a problem for its rational culture
by seeds. That is the reason why the culture of French clones (Mediterranean race) by
lignified cuttings is strongly recommended for the Mediterranean countries. For Nordic
countries the culture by seeds from German thyme, which is more frost-resistant, is
desirable. In mountain areas such as the Valais and Poschiavo valleys, the limiting climatic
conditions do not allow a safe production of French thyme. So far only the German
thyme is cultivated from seeds grown in nurseries or to a smaller extent directly sown
in fields (Rey, 1 9 9 3 ~always
) keeping in mind their cultural requirements (Rometsch,
1993). A race similar to French thyme but somewhat more rough, the cloned Vald8tain
thyme, could be recommended for warm and protected situations. The hybrids between
Vald8tain and German clones are more interesting because they are more robust.

VARICO, a n e w variety of thyme

In order to complement the demands from growers and consumers, a first variety of
hybrid thyme named VARICO was placed on the market in 1994. It is characterised
188 Charles Rey and Francisco Skez

Fzgure 6.4 VARICO thyme hybrid flowering IIJ spring

by the homogeneity in its phenotype, an erect habitus and great vigour (Figure 6.4). Its
greyish-blue foliage differentiates it clearly from varietieslpopulations of German
origin with their greyish-green colour. Its pale pink flowers bloom from May 20 until
June 10 in Arbaz (Switzerland, 9 2 0 m alt.). The productivity of dry matter teaches
~ 3 years of culture. Its good quality is reflected in the mean
more than 1 5 0 0 ~ 1 mafter
yield in essential oil (3.9 per cent vlw after 5 harvests) and its percentage of thymol of
over 50 per cent. Its resistance to frost is good (Rey, 1994a). The company DSP at
Delley (Switzerland) produces VARICO thyme seeds. Keeping in mind the selection
efforts involved, the price is higher than for other seeds in the market. The first cultures
of VARICO thyme established by plantlets and by direct sowing satisfy the growers
(Figure 6.5).

IN VITRO CULTURE OF THYMUS

The in vitro culture techniques provide a wide array of tools to the breeder that complement
the selection activities performed ex vitro out of the laboratory. By modifying the
conditions to which the plant is exposed the researcher is able to influence and even to
determine the metabolic pathways in the plant. These conditions include a multitude
of parameters such as temperature, quality and intensity of light, and composition of
the substrate or the atmosphere. Mulder-Krieger etal. (1988) reviewed the production
of essential oils and aromas in cell and tissue cultures of plants from about 70 species,
including bryophytes, conifers, monocotyledons and dicotyledons. Frequently significant
 Field czlltztve, in vitro cultwe and selectio~zof Thymus 189

Figure 6.5 Culture of VARICO thyme hybrid in Bassins (Switzerland) destinated to the production of
essential oil.

differences between in vitro and in vivo plant material are found; the desired compounds
may even be absent when grown in the laboratory.
Despite the potential applications of the in vit16o culture methods few researchers
have used them with Thymus. Furmanowa and Olszowska started their research with
thyme using T. vulgar& reviewed in 1992. L& (1989) took another approach using a
different culture medium following the modifications that Collet (1985) made to the
MS culture medium previously developed by Murashige and Skoog (1962). The CMS
medium suggested by Collet has also been applied by Sgez etal. (1984) working with
T . piperella, after tealising the beneficial effect of these changes compared with the original
formulation of MS salts. The experiments performed with these two species and the
results obtained will be described briefly.

In vitro Culture of T . vulgaris

T. vulgaris was cultured in vitro for the first time by Furmanowa and Olszowska (1992)
and references therein. They regenerated plants from buds in Nitsch and Nitsch ( N N )
culture medium, with varying concentrations of two auxins and three cytokinins, find-
+
ing optimal results with the use of either (a) 0.1 mgil Kin 0.1 mgil NAA. b) 0.1 mgil
Kin+0.3 mgil IBA or 0.5 mgil IBA. When using nodal segments, they tested different
concentrations of auxins (NAA, IBA) and cytokinins (Kin, BA, 2-iP). The best rooting
was achieved with 0.5 mgil IBA, and cytokinins were found to have little or no effect
on plantlet development at 0.05 mg/l. When higher concentrations were used, gradual
inhibition of rooting and shoot growth was observed.
170 Charles Rey and F~*anciscoSdez
L& (1989) tested eight treatments, i.e. two different compositions of mineral salts
(MS, CMS) and four combinations of growth regulators . Using stem cuttings bearing
axillary buds he concludes that the CMS medium without growth regulators performs
best when trying to get the best development, and the presence of growth regulators
disturbs the growth of axillary shoots, perhaps due to an interaction with these substances
from an endogenous origin. This is in agreement with Furmanowa and Olszowska
(1992), even though they use different culture media formulations. L@explains the better
performance of CMS medium than MS to be the reduction of concentration of the
ammonium ion ( N H ~in ) the CMS medium. These results by L@characterise mainly
the in vitro establishment of thyme. Under these conditions he describes an anomalous
development of axillary shoots in the presence of growth regulators. This was also
observed by SBez etal. (1994), but this behavior is only achieved at the beginning of the
experiments. When the plantlets are acclimated to the in vitro conditions of growing,
there is a more clear response to changes in the type and concentration of growth regulators.
The production of secondary metabolites in T. vz~lgariswas the object of Tamura
etal. (1993), who focused on the selection of the callus cells and management of cell
environments for the production of flavour metabolites. They recorded the relation
between the color of the callus and the presence of thymol and carvacrol. They found
that on an agar medium, green and yellow calli produced trace amounts of thymol,
while white calli produced ethanol and no rhymol. The addition of mevalonic acid enhanced
the amount of the volatiles produced up to two-fold in comparison to the reference
control, showing that enzyme activities of the monoterpene synthesis were latent in the
calli. Unfortunately the oil yield was very low, about 11500 to 111000 of the normal plant.

In witro culture o f T . piperella

The in vitro culture of T. piperella was started by SPez etal. (1994) with the aim of test-
ing the behaviour of a species quite different from the already studied T. vzlgaris.
Indeed T. piperella presents a more herbaceous habit, sometimes becoming lianoid, with
very long internodes. It is much less hairy, with glabrous leaves. Additionally, it is of
economic interest as a source of phenols and as a condiment. Furthermore, it is a species
with a small dispersal area and therefore it may not be collected intensively.
The procedure involves three major steps: (a) Acquisition of an i?z vitro established
population of individuals growing in the same conditions. (b) Determination of the
effects by different growth regulators on several characteristics of the plant material, to
help to select the culture media adapted to specific needs: multiplication, rooting.
(c) Determination of the effects that different concentrations of macronuttients, sucrose
and vitamins, have on the production of roots, to help to select the culture media ade-
quate for root production before putting the plants in ex vitru conditions. These steps
are described below.

Obtaining of plantlets growing i n witro

The initial step for obtaining an in vitru plant population was the collection of seeds from
natural populations (Figures 6.6 a,b). The bigger ones were selected and stored at a low
temperature until the activities in the laboratory started. Previous experiments had
shown two problems in these initial steps, one being the difficulties to remove completely
 Field culture, i n v i t r o cztlture and selection ofThymus 191

Thymus plant seeds Tween 20

(a) (b) (c)

sterile water bleach sterile water ethanol

(dl (el (9 (8)

1 5 min. 10 min. 15min. I seed 3 months

sterile water in vitr-o
(h) (i) (j)

SYSTEMATIC
TREATMENTS

base population
(1 i

Fig//" 6.6 Protocol for the establishment of an initial population of T. pipe~ellaplants in vitro.

the contaminants (bacteria, fungi) from the surfaces, and the other related to the different
ability to germinate andlor develop the initial plantlets.
The procedure to remove contaminants from the seed surface is described in Figure 6.6 c-h.
Once cleaned, the seeds are placed on the surface of the culture medium inside glass
tubes, 3 4 seeds per tube. Some of the tubes containing the seeds have to be removed
192 Charles Rey and Francisco Sa'ez
due to fungal growth (clearly visible after 2 days), bacterial growth (after 5-6 days) or
poor growth of the young plants, sometimes affected by vitrification (characterised by
an excess of water in their tissues).
After about two months the plants are 5-6cm long and present several internodes
(Figure 6.6 j). They are cut into portions with 2-3 internodes and transferred to larger
glass vessels (Figure 6.6 k) with translucent caps, the roots being removed. They pro-
duce axillary shoots that are periodically removed and transferred to new vessels, thus
producing a population of plantlets growing under the same conditions (Figure 6.6 1).
During this period of time, a change in the morphology of the leaves occurs, from
smaller, deeper green and tough, to wider, lighter green and herbaceous consistency
can be noticed.
The systematic treatments used portions of plants from this in vitro population
containing an apical bud and three nodes. They were transferred to new vessels with a
culture medium adequate to test the different combinations of growth regulators
(2 cytokinins and 2 auxins), sucrose, macronutrients and vitamins. Two groups of
treatments were performed. The first one tested five levels in the concentration of each
growth regulator in the products IAA x BA, IAA x Kin, NAA x BA and NAA x Kin
(100 combinations). The second one tested three concentrations of sucrose, four of
macronutrients and the presenceiabsence of vitamins (24 combinations). These two
experiments were run independently.

InJlztence of growth regulators

The effects of the addition of an auxin and a cytokinin to the growth media were
measured by (a) the number of shoots greater than 5 m m long, (b) the number of shoots
less than 5 mm long, (c) the quantity of roots produced per explant, (d) the number of
explants that showed abnormal growth, either by the presence of callogenic structures
in the base or by vitrification. The axillary shoots produced were classified into two
categories due to the higher ability of shoots greater than 5 m m to produce new well-
developed plantlets. Figure 6.9 represents the results (mean, standard error of the mean
and 95 per cent confidence intervals) obtained for the number of shoots > 5 mm,
comparing the different growth regulators and the concentrations used for each one.
Stronger activity of BA than Kin, when promoting the development of axillary shoots,
and when inhibiting the formation of roots was found. Similarly, NAA as an auxin
presents higher activity than IAA, expressed as stronger inhibition of shoot development
and promotion of root development. Furthermore, NAA produced calli at the base of
the explants more readily than IAA. Considering to all the variables studied, the most
suitable combinations of growth regulators were found to be 1.0mgll BA or 1.5 mgil
BA without auxin to promote shoot growth, and 0.5 mgil IAA without cytokinin to
promote root growth. Figures 6.7 and 6.8 show the effects of different combinations of
growth regulators.

Influence of macronutrients, sucrose and vitamins

The role of macronutrients, sucrose and vitamins added to the culture medium in
relation to the root development of the plantlets was tested in 24 experiments containing
different variations of them plus the addition of 0.5 mgll IAA (proved to enhance this
metabolism when testing the different growth regulators). The results are shown in
 Field culture, in vitro culture and selection of Thymus 173

Figwe 6.7 T. piperella growing in vztro with hlgh concentration of cytokinins.

Fzgzlre 6.8 T. pzperellu growing in vitro with high concentration of auxins.

Figure 6.10. The best combination seems to be a culture medium with 25 per cent
sucrose, and 50 per cent or 25 per cent macronutrients plus the addition of vitamins. I t
is advisable to reduce the concentration of sucrose to promote rhizogenesis, but in the
absence of vitamins, low levels of sucrose gave the lowest yields. Also a reduction to
194 Charles Rey and Francisco Sa'ez

cmED==
0

O 0 05 10 1 5 2 0 0 05 10 15 20
Cytok~ninBA Cytok~nln Kinet~n
Cytoklnul concentration (mgll)

O
-- --
0 02 05 15 30
Aux~nIAA
0 0.2 0.5 1 5
Aux~nNAA
30

Auxrn concentration (mgll)

Figz~ve 6.9 112 vitro culture of T. piperella. Production of axillary shoots greater than 5 mm long.
Comparison of results by cytokinin (up) and auxin (down) concentrations.

50 per cent or 25 pet cent of the macronutrients seems to promote rhizogenesis. There
is a very high variability in the results obtained, suggesting a certain influence from
endogenous growth regulators.

Obtaining different varieties of thyme suitable for field culture under different climatic
conditions is highly desirable, as previously stated. Renewed efforts have to be made in the
localisation of ecological/chemical variants in nature, as well as in the characterisation
of their properties under controlled environments, both ex vitro and in vitro. New
 Field culture, in vitro culture and selection of Thymus 175

Sucrose: 100% Sucrose: 50%

Macronutrients (% from full strength)
Sucrose: 25%

I
Figure 6. I0 In vztro culture of T. piperella. Root production in different concentrations of rnacronutri-
ents, sucrose, and vitamins, in CMS culture medium supplemented with 0.5 mgll of IAA.

techniques such as production of haploid and doubled-haploid plants from microspores

may help to obtain elite cultivars in the near future.

ACKNOWLEDGEMENTS

The collaboration of the following persons is sincerely appreciated: P. Acherrnann,

P. Bruchez, J.-P. Bouverat-Bernier, J. Burri, C.-A. Carron, F. Fournier, A. Fossati,
B. Galambosi, J. Gretillat, P. Imhof, L. Laflamrne, G . Marguerettaz-Gaetani,
B. Nendaz, I. Kapetanis, A. Schori, I. Slacanin and N. Verlet. Thanks are given also to
'Fundacibn Seneca', Murcia, Spain.

REFERENCES

Aflatuni A,, Pessala R., Hupila I., Sirnojoki P., Huhta H., Virri K., Kemppainen R., Jarvi A.
and Galambosi G. (1994) Yield of Thyvzw vulga~,zs,Melissa oj$ficinalis and Origanu~nvulgare
grown between 601 and 681 latitudes in Finland. Abstract of seminar n l . 240. Production of
herbs, spices and ?7zedzcinalplantsin the nordic countries. Mikkeli, Finland, August 2-3.
Anonymous (1992) ITEIPMAI. Technical dossier for balm (Melissa Officznalis, Lamiaceae).
ITEIPMAI, Chemillg, Angers.
Anonymous (2000) SRVA. Technical dossier for common thyme. In File for Medicinal and
Aromatic Plants. Working group coordinated by P. Amsler, SRVA, Lausanne.
Assouad, W. and Valdeyron, G. (1975) Remarques sur la biologie d u thym, Thynzw vulgaris L.
Bull. Soc. Bot. Fr., 122, 21-34.
Collet, G.F. (1985) Enracinement am6liorC lors de la production in vitro de rosiers. Rev. Suisse
Viticult. Arborzc. Ho~t.,17, 2 5 9-263.
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Furmanowa, M. and Olszowska, 0. (1992) Micropropagation of Thyme (Thynzus vulgaris L.). In
Y.P.S. Bajaj (ed.), Biotechnology in Agriculture and Forestry, 19, pp. 230-243.
Gammetter, M. (2000) Annual report Plantamont 1999.
Heeger E. F. (1 956) Handbuch des Arznei- 2nd GewirzpfLanzenbaues. Drogengewinnung. Deutscher
Bauernverlag, Berlin. pp. 775.
STEIPMAS (1983) Domestication de la production, conditionnement et definition du thym
(Thy??zusvulgaris L.). Bull. d'inform. serie Monographie.
Laflamme L., Tremblay N. and Martel C. (1994) Winter survival of medicinals plants in
Quebec (Canada). Compte-rendu d u seminaire no. 240. Production ofherbj, spices and ~nedicinal
plants in the nordic countries. Mikkeli, Finland, August 2-3.
Lawrence, B.M. (1992) Chemical components of Labiatae oils and their exploitation. In R.M.
Harley and T . Reynolds (eds), Advances in Labiate Science, Royal Botanic Gardens, Kew,
pp. 3 9 9 4 3 6 .
L@,C. L. (1989) Microbouturage in vitro du thym (Thymus vulgaris L.). Revue suisse Vitic. Arbo~,ic.
Hortic., 21, 355-358.
Mulder-Krieger, T., Verpoorte, R., Baerheim Svendsen, A. and Scheffer, J.J.C. (1988) Production
of essential oils and flavors in plant cell and tissue cultures. A review. Plant Cell, Tissue and
Organ Culture, 13, 85-154.
Murashige, T . and Skoog, F. (1962) A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol. Plant, 15, 473-497.
Rey, Ch. (1988) Comparaison de provenances de thym vulgaire. Internal report RAC.
Rey, Ch. (1989) Le thym vulgaire (Thymus vulgaris L.) d u Val d'Aoste: une particularit6
botanique de haut inter@t.Rev. Vald6taine 2Hist. Naturelle, 4 3 , 79-97.
Rey, Ch. (1990a) La culture d u thym en Suisse. Revue horticolesuisse, 63, 20-22.
Rey, Ch. (1990b) Thymus vulgaris L. du Val d'Aoste (Italie): un ecotype intgressant pour les
zones marginales. Revue suisse Vitic., Arboric., Hortic., 22, 3 13-324.
Rey, Ch. (1991) Incidence de la date et de hauteur de coupe en premii.re annee de culture sur la
productivitC de la sauge officinale et d u thym vulgaire. Revue suisse Vitic. Arboric. Hortic. 23,
137-143.
Rey, Ch. (1993a) Hybrides de thym prometteurs pour la montagne. Revue suisse Vitzc. Arbol-ic.
Hortic., 25, 269-275.
Rey, Ch. (1993b) Selection of tyme (Thynzu vulgaris L.). Acta Hort., 344,404-410.
Rey, Ch. ( 1 9 9 3 ~Semis
) direct au champ d u thym (Thymus vulgaris L.). Revue sztisse Vitic. Arboric.
Hortic., 25, 401-403.
Rey, Ch. (1994a) Une variCt6 de thym vulgaire 'Varico'. Revue suisse Vitic. Arboric. Hortic., 26,
249-250.
Rey, Ch. (1994b) La selection du thym (Thymus vulgaris L.). Actes du 3e Colloque Mediplant sur
le thi.me 'Ressources et potentiels de la Flore medicinale des AlpesA', 20 october 1994,
Domaine de Bruson (RAC) Bruson-Bagnes (ValaisiSuisse).
Rometsch, S. (1993) Ecology and cultivation assessment of Thyme (Thymw vulgaris L.) in the
Canton Valais, Switzerland. Acta Hort., 344, 41 1 4 1 5 .
Sbez, F., Sknchez, P. and Piqueras, A. (1994) Micropropagation of Thymw piperella. Plant Cell,
Tissue and Organ Culture, 39, 269-272.
Simojoki P., Hupila I., Pessala R., Galambosi B. and Aflatuni A. (1994) Yield potential of
thyme, lemon balm and anyse hyssop grown in different latitudes of Finland. Abstract of
seminaire no. 240. Production of herbs, spices and medicinal plants in the nordic countries. Mikkeli,
Finland, August 2-3.
Tamura, H., Takebayashi, T. and Sugisawa, H. (1993) Thynzus vulgaris L. (thyme): In vitro
culture and the production of secondary metabolites. In Y.P.S. Bajaj. (ed.), Biotechnology in
Agriculture and Forestry, 2 1 , pp. 4 13-42 5.
7 Harvesting and post-harvest handling
in the genus Thymus
Petrus R. Venskutonis

INTRODUCTION

There are many Thymus species, however only few of them are of commercial significance
(Reineccius, 1994; Clarke, 1994), namely T . vulgaris L., T . zygis L. ssp. gracilis Boiss.
(red thyme), T. satareioides Cosson, T . serpyllum L. (wild thyme), T. capitatas Hoffmanns.
and Link (syn. Thymbra capitata (L.) Cav., Spanish "origanum"). The first two are the
most widely used Thymw species and surveyed literature sources deal mainly with these
two herbs. T. vulgaris is the only species, which is cultivated commercially in reason-
able amounts. Other Thymus species are collected in wild-growing sites mainly as sources
of dried medicinal herbs.
In order to obtain the product of the best quality, harvesting as well as growing and
all other processing steps of thyme should be optimised considering several important
factors. These factors, which in general are very common to many aromatic and medi-
cinal plants, shall be briefly discussed in this chapter. Two flow diagrams of manual
and mechanised herb harvesting and processing are presented in Figure 7.1 (Heindl and
Miiller, 1997). The cutting and processing steps shown in the diagram represent most
traditional procedures, which have been generally used in the preparation of dried
aromatic and medicinal herbs.

Cutting Loading and transporting Drying Sorting, cleaning

Harvesting Sorting Comminution Sifting Drying

Ftgure 7. I Flow diagrams of harvesting and processing of herbs (Hemdl and Miiller, 1997).
198 Petras R. Venskutonis
HARVESTING METHODS A N D THEIR INFLUENCE O N THE
QUALITY OF THE PRODUCT

Harvesting date, time of day a n d weather conditions

T. vulgaris is indigenous to the Mediterranean region, however it is also widely cultivated
in different parts of the world including North America, Europe and North Africa.
In general, thyme is most aromatic during the period of blooming (or at the beginning
of full bloom); therefore this period is considered as the best time for harvesting. How-
ever, the period of vegetation and blooming can be different in various geographical
zones depending on their climatic conditions. Spain is one of the major producers of
thyme in the Mediterranean region, and harvest takes place during the blooming
period from February to August, depending on the species (Tainter and Grenis, 1993).
Collection of plant material for Moroccan thyme oil (T. saturezoides) takes place in the
beginning of April with plants (flowering tops) growing at l 0 0 0 m above sea level,
the harvest ends in August with plants growing up to 2000 m. In France, thyme can be
harvested twice a year, once in May and then again in September.
In the central regions of Russia thyme is harvested during the second year of plant
vegetation (Poludennij and Zhuravlev, 1989). Usually, the first cutting is performed in
June during flowering, the second one in September-October, i.e. 1.5-2 months before
the end ofvegetation. Aerial parts are cut at 10-15 cm height from ground. T. serpyllunz
is harvested starting from the first year of vegetation in June-July at the beginning of
full blooming (Kudinov etal., 1986; Mashanov and Pokrovskij, 1991). It has been
recommended to harvest T. serpyllum during the flowering period. The best time is con-
sidered to be the middle of August, because after cutting there will be enough time for
new stems to grow and this helps plants to prepare for the winter (Jaskonis etal., 1981).
The quality of the herb depends on the site and climatic conditions of growing.
Accumulation of essential oil (EO) in herbs directly or indirectly depends on light. Li
etal. (1996) studied the essential oil production in thyme and sage grown at 15 per
cent, 27 per cent, 45 per cent and 100 per cent of full sunlight. Their results showed
that the highest yield of essential oils and percentage of thymol and mytcene in thyme
occurred in full sunlight.
Weather conditions during the day of harvest are also important. In general, sunny
days should be preferred, after morning dew has disappeared. The plants harvested after
rain or with dew moisture are difficult to dry; they deteriorate much faster, their colour
and useful properties become inferior (Kauniene and Kaunas, 1991).

Harvesting techniques a n d machinery

Most wild growing medicinal plants ate collected by hand. The main objective of such
harvesting is to collect the most valuable anatomical parts of the plants. In case of
thyme (T. serpyllunz, T. pulegioides, T. vulgaris and other species) these are the leaves and
flowering parts. Woody stems, which are of minor value, must be avoided as far as pos-
sible. For instance, recently GuillCn and Manzanos (1998) studied the composition of
a Spanish T. vz~lgarzsand found that the yield obtained by the extraction with dichloro-
methane from leaves and flowers was much higher than that obtained from stems; the
chemical composition of the extracts obtained from different anatomical parts was
also different. It is also important to avoid damaging plant growing sites to ensure
 Harvesting andpost-harvest handlzng in the genw Thymus 199
renewable crops during the following years of harvesting. Simple means are suggested
in the numerous manuals for medicinal herb gatherers, e.g. to use scissors or knives to
cut the herb instead of tearing it out with roots (Borngen, 1979). T . vulguris as a com-
mercially cultivated herb can be harvested more effectively by common harvesting
machinery, which cuts the plant at 10-15 cm height from the ground (Mashanov and
Pokrovskij, 1991).

Effect of harvesting time o n t h e essential oil content

and composition
The essential oil yield and chemical composition are the most important characteristics
of aromatic herbs. Senatore (1996) investigated the oil of a wild Italian thyme, Thynzus
pulegioides L., at different growth times (Table 7.1). From April to September the essen-
tial oils consisted mainly of a-terpinene, p-cymene, thymol, and carvacrol, which varied
from 57.3 to 62.5 per cent of the total oil content. From their results it was concluded
that the best time to harvest this species of thyme, for both essential oil yield and
phenol content, is during or immediately after full bloom.
Cabo etal. (1987) collected the aerial parts of Thy??zwhyenzalis Lange throughout its
complete vegetative cycle (April 1981 to March 1982) and determined the content of
essential oil and its composition. The results of this study are summatised in Table 7.2,
showing that the oil yield varied from 0.15 per cent (December 23) to 0.58 per cent
(July 4). The percentages of the main constituents were quite stable during the period
of investigation with few exceptions. For instance, the content of monoterpene hydro-
carbons (MH) was significantly lower during the period of September-November and
February-March. Thymol and carvacrol were determined in reasonable amounts only
during October-December. However, it should be noted that recently SBez (1998)
reported the results of a comprehensive study of the variability in essential oils from
populations of T . hyemulis, which supported the concept that the linalool chemotype is
intrinsic to this species, while borne01 and 1,s-cineole types are not. The latter com-
pound was one of the major constituents in T . hyenzulis investigated by Cabo etul. (1987).
Karawya and Hifnawy (1974) examined the oil of T , vulgdrzs grown in Egypt and
collected at different stages of growth, before flowering, during flowering, and at fruiting
stages. They found the highest thymol and carvacrol concentrations during the beginning
of the flowering stage.
Arrebola etul. (1994) investigated Thynzw serpylloides Bory ssp. serpylloides for 3 years.
They found that the oil content in stems obtained by steam distillation was lower than
0.5 per cent vlw. Independent of its sexual characteristics, the highest oil yield had
been obtained from plants collected during the full flowering period (average measures
of the 3 years: 1.56 per cent vlw from hermaphrodite, and 1.05 per cent v/w from
females). The authors also observed that the highest content in carvacrol was found in
a sample of hermaphrodites collected during full flowering in 1991, while the lower
content was found during fruiting stage. In previous years, the amount of carvacrol
during the full flowering period was lower (1989) or quite similar (1990) to the fruit-
ing stage. The annual variation of carvacrol may be due to variations in the climate
experienced during the 3 years of harvesting.
The essential oil composition of T . vzdguris grown in Lithuania has been investigated
at different vegetation phases (Venskutonis, unpublished data). The results on the total
oil content and some of its constituents in leaves (L) and flowers (F) are tabulated
Table 7.1 Essential oil yielda and compositionb of Italian thyme (Thynzuspzllegtoides L.) at different dates of harvest

Characteristic Date qf harvest

Apr 18' May 2' May 12' May 1 2 ~May 24' May 3 1' May 3 1 June 10' June 21' July 7' July 20' A?% 2' Aug 19' Sept 13'

Yield of oil
MH
OCM
p-Cymene + y-terpinene
Thymol + carvacrol
TPC
SH
OCS
Others
Undetermined

Notes
a g/lOO g of fresh material.
b G C peak areas percentages.
c Leaves.
d Flowers
MH, Monoterpene hydrocarbons.
OCM, Oxygen-contain~ngmonoterpenes
TPC, Total phenol content.
SH, Sesquirerpene hydrocarbons.
OCS, Oxygen-containing sesquiterpenes.
Source: Senarote, 1996.
Table 7.2 Thynzus hya~zulitLange - essential oil yield and its composition (%) at different dates of harvest

Date of harvest

Charucterzstzcs ~ p 22
? may26 jllly 4 A Z ,3~ Sept 4 Oct I0 Nov 23 Dee 23 Jan 29 Feb 27 Md~ch30

Yield of 011(% vlw)

MH:':
Myrcene
1,8-Cineole
Camphor
Alcohols
Acetates
Thymol + carvacrol

Notes
" MH, monoterpene hydrocarbons (includ~ngmyrcene)
rr content below 1%.
Source: Cabo etal., 1987.
202 Petras R. Venskzttonis

Table 7.3 Composition (%) of the essential oil from thyme (Thynzus vzilgarzs) at different dates of harvest

Conzpound L1 L2 LF3 LF4 LFj LF6 LF7

May 2 j June 6 June 16 June28 Jzuly 7 July 1 9 Az~gzlst7

a-Thujene 0.59 0.62 0.67 0.72

a-Pinene 0.67 0.50 0.55 0.50
Camphene 0.52 0.26 0.25 0.22
Oct-l-en-3-01 0.61 0.56 0.51 0.7 1
Myrcene 1.17 1.07 1.19 1.14
a-Terpinene 1.37 1.00 1.23 1.03
p-Cymene 14.43 10.21 11.19 8.27
Limonene 0.29 0.25 0.28 0.24
,6-Phellandrene 0.76 0.45 0.48 0.50
1,s-Cineole 0.29 0.25 0.28 0.24
7-Terpinene 13.40 6.52 7.50 7.58
tr-Sabinene hydrate 0.27 0.64 0.60 0.7 1
Linalool 1.60 2.04 1.91 2.23
Camphor 0.25 0.19 0.08 0.12
Borneo1 1.30 0.78 0.65 0.63
Terp~nen-4-01 0.83 0.67 0.67 0.62
a-Terpineol 0.27 0.17 0.22 0.23
Thymol methyl ether 2.61 0.59 0.34 1.33
Carvacrol methyl ether 1.58 0.44 0.23 0.89
Thymol 49.12 65.98 63.11 65.20
Carvacrol 1.66 3.35 3.33 2.95
,6-Caryophyllene 1.87 2.74 2.44 2.41
7-Cadinene 0.40 0.31 0.30 0.37
bcadinene 0.22 0.40 0.28 0.33
Caryophyllene oxide 0.44 0.41 0.37 0.32
T-Cad~nol 0.78 0.28 0.38 0.48
Essential oil (5%) 1.75 1.40 2.90 2.86

Notes
L, Leaves.
F, Flowers.

in Table 7.3. The highest amount of essential oil was distilled from the flowering parts
harvested at the later phases. The percentage ofp-cymene was highest in May, while the
content of the major phenolic constituent thymol at the same phase was the lowest.
Further it increased and was quite stable during the period of vegetation. It is interest-
ing to note that the percentage of a-terpinene in June - beginning of July was rather
low, however it considerably increased in the end of June and beginning of August,
when some reduction in the content of thymol was determined. Mohamed (1997) also
reported the effect of time of harvest on the composition of essential oil from T. vulgaris.
MoldZo-Martins etal. (1779) investigated seasonal variations in yield and composition
of T. zygzs ssp. sylvestrzs (Hoffmanns. et Link) Brot. essential oil. The authors determined
that the yield in essential oil peaked at the flowering stage (0.9-1.4 per cent) and was
lowest during the dormancy period (about 0.15 per cent). The composition also showed
different patterns at different phases of the vegetative cycle. At the flowering stage, the
essential oil was rich in thymol and geraniol while p-cymene was highest when thymol
was at a minimum (post-flowering period). Concerning the use of the essential oil as
a food ingredient, it is suggested that the most interesting stage is the post-flowering
 Harvesting andpost-halvest handling in the genus Thymus 203
period, the essential oil at this time being rich in thymol (about 21 per cent), geranyl
acetate (about 17 per cent) and geraniol (about 1 3 per cent). Baser etal. (1999) also
reported the variations in chemical composition of the essential oils of T. pectinatus
Fisch. et Mey. var. pectinatus at different stages of vegetation.

PROCESSING OF FRESH AND DRIED PRODUCTS

Only a small part of harvested thyme can be consumed as fresh plants. Processing tech-
nologies in general and their parameters in particular, which are andlor could be, applied
to Thylnus genus are similar to the processing technologies commonly applied for many
other labiates. These technologies and processing parameters have been comprehensively
described in several internationally recognised manuals, handbooks, edited books and
monographs (Tainters and Grenis, 1993; Heath and5 Reineccius, 1986; ~ a r r e l l 1985; ~,
~ e a t h 1981;
~ , Reineccius6, 1994; Underriner and Hume, 1994; Richard7, 1992; ~ s h u r s t ' ,
1991; ~ e r h a r d t ' , 1994). The content of this chapter is based on the materials provided
in the above mentioned literature sources. It should be mentioned that specific inform-
ations on the processing of thyme is rather scanty, therefore, the description of processing
treatments provided in this chapter is mostly of a general character.

Processing of fresh products

Shelf life of fresh herbs is usually very short and therefore, traditionally, herbs have been
used as dehydrated products. However, processes to prolong shelf life of herbs and
spices have been developed and used. Storage of freshly harvested herbs at a temperature
close to 0 OC is the simplest method to prolong shelf life of the fresh herbs. Such storage
can delay deterioration only a few days. Some processes for producing frozen herbs,
which retain their flavour and appearance for a considerably longer time, have been
recently developed and tested. For instance, LaBell (1991) describes a process during
which herbs are cleaned, chopped and coated lightly with canola oil within a few hours
of harvesting. A small amount of acid is also added to the herbs to inhibit browning.
The herbs are then stored frozen and this way they are stable for 1 year. Such processing
enables more of the volatile top notes to be retained than by drying.
Also chemical agents were used to prolong shelf life of the fresh herbs. Mohammed and
Wickham (1995) used this method for the plants of shado benni (Eryngiunzfoetidu~nL.),
which were harvested with intact roots, dipped in Gibberellic Acid (GA3) and stored
in perforated and non-perforated low density polyethylene (LDPE) bags up to 22 days
at 20-22 OC and 28-30 O C . It was shown that GA3 effectively retarded plant senescence
up to 22 days at both temperatures when stored in non-perforated LDPE bags. Despite
the external maintenance of marketable quality, flavour life was 17 days as development
of off-flavours and reduction in pungency occurred after this period. Thus, the combin-
ation of polyethylene packaging, GA3 dip treatment and reduced temperature storage
extended the shelf life of the plants in a fresh, turgid and decay-free condition for an
excess of 2 weeks. Most likely, such procedure could be applied to other plants including
Thynzus species.
The processing method of fresh vegetable products and particularly fresh herbs and
spices invented by Hsieh and Albrecht (1988) is summarised in Figure 7.2. The flow
diagram shows that modern technologies, such as fluidised bed drying (the process
204 Petras R. VensRutonis

visual rnspection and cleaning

Figzure 7.2 Fresh vegetable products creating flow chart (Hsieh and Albrecht, 1991)

when the solid particles are suspended in a rising stream of air) and using humectants
(substances having distinct hygroscopic properties and retarding moisture changes)
provide several different treatments for preserving the quality of fresh herbs.

Processing of dried products

Drying is undoubtedly the most ancient and still most widely used method of the fresh
herb processing. At the time of harvest, most herbs and spices contain 60-85 per cent
of water. In order to obtain stable products, which will withstand long periods of
storage without deterioration this must be reduced to 8-10 per cent. Drying is the
most critical process due to the volatility and susceptibility to chemical changes of the
contained volatile oil (Heath, 1982).

Cleaning
In 1975, the Food and Drug Administration (FDA) initiated a three-year study, including
more than 1000 samples, to develop data on insect, bird, rodent and other animal
contamination levels in selected retail market, ground and unground spices and mould
in ground paprika (Gecan etal., 1986). The sampling and analytical details on nine
spice products (including thyme) from that program were presented in the report of
that study. Frequency distribution of insect fragment counts in ground and unground
thyme are presented in Table 7.4, while a statistical summary oEdefects found in thyme
 Harvesting and post-harvest handling i n the genus Thymus 205

Table 7.4 Frequency distribution of insect fragment counts; 1267 of 25 g samples of unground thyme
and 1332 of 10g samples of ground thyme

Insect Rodent Feather

fragments Ground Unground hair Ground Unground barhule Ground Unground

Mite Thrzps Aphid

Ground Unground Ground Unground Ground Unground

0 1293 588 0 1284 582 0 1291 694

1 26 196 1 33 164 1 21 130
2 6 115 2 7 107 2 8 102
3 2 74 3 2 74 3 3 64
4 2 52 4 71 4 2 42
5 1 24 5 1 32 5 3 30
6 23 6 1 27 6 1 21
7 9 7 1 22 7 19
8 14 8 33 8 3 26
9 7 9 19 9 11
10 2 10 2 19 10 20
11-20 34 11-20 1 70 11-20 71
2 1-30 15 21-30 24 21-30 24
31 4 0 6 31 4 0 11 3 1-40 5
41-50 4 41-50 2 41-50 4
5 1-100 1 22 5 1-100 10 5 1-1 00 4
101-200 1 18
201-300 20
301400 3
401-500 7
501-999 34

Source: Gecan etal , 1986.

is presented in Table 7.5. The investigations show that thyme was a heavily contaminated
herb, particularly with insect fragments, mites, thrips, and aphids.
Count means of insect fragments varied from 7.8 for l o g of ground allspice to 287.7
for l o g of ground thyme; samples containing insect fragments ranged from 70.8 per
cent for ground allspice to 98.6 per cent for ground thyme. Mite counts ranged from
0-2 for 25 g of ground paprika to 0-999 for 25 g of unground thyme; count means
varied from 0.0 for 25 g of ground paprika to 35.4 for 25 g of unground thyme; samples
containing mites ranged from 2.8 per cent for ground thyme to 53.6 per cent for
Table 7.5 Statistical summary of defects found in thyme

Dejieiem Ground thjime* Unground thyme2**

Counts Sunzples Counts Samnples with

zvith dejiects dejiects (%)
Mean Range (%) Mean Range

Insect fragments 287.7 0-3625 99.6 100.7 0-2257 96.6

Rodent hairs 0.2 0-3 2 12.6 0.2 0-28 12.2
Feather b u r b ~ ~ l e s 0.2 0-12 12.8 0.5 0-50 21.5
Mites 0.2 0-179 2.9 35.4 0-999 53.6
Thrips 0.1 0-12 3.6 3.8 0-99 54.1
Aphids 0.1 0-8 3.1 3.1 0-83 45.2

Notes
* In total 1332 of 10 g samples were examined.
";" In total 1267 of 25 g samples were examined

Source: Gecan et nl., 1986.

unground thyme. Thrip counts ranged from 0-1 for 10 g of ground allspice and 2 5 g of
ground paprika to 0-99 for 25 g of unground thyme; counts means varied from 0.0 for
1 0 g of ground allspice and 2 5 g of ground paprika to 3.8 for 2 5 g of unground thyme;
samples containing thrips ranged from 0.2 per cent for ground paprika to 54.1 per cent
for unground thyme. Samples containing aphids ranged from 0.0 per cent for ground
paprika to 4 5 . 2 per cent for unground thyme.
Tainter and Grenis (1993) have described the general principles of cleaning methods
and equipment. The principles of all cleaning equipment are based on physical
difference (i.e. shape, density) between the spice andlor herb and the foreign material
being removed. The equipment can consist of magnets, sifters, air tables, destoners, air
separators, indent separators and spiral separators. The choice of cleaning methods and
equipment depends on the physical characteristics of the herb, the cost of the machinery
and the process effectiveness of the removal of foreign objects, and the loss of the main
material during cleaning. Anyway, it is impossible to perform a cleaning operation at
reasonable production rates that result in a pile of foreign material completely free of
thyme and a pile of thyme completely free of foreign material. Therefore in optimising
the process it is necessary to define the limits of foreign material in the cleaned products.
Some of these characteristics are usually provided in the regulations for a particular
spice or herb.
Magnets are widely used for eliminating ferromagnetic particles from herbs and spices.
Modern quality assurance systems, e.g. HACCP (Hazard Analysis Critical Control
Points), require that every herb cleaning system should include magnets or other metal
detecting devices. The main purpose of removing metals from the products is to protect
the end-user from physical hazards. Remaining pieces of metals can also damage other
processing equipment, e.g. milling machinery.
Dehydrated thyme can already be considered as a prepared product for utilisation.
However, when leaves andlor blossoms are used as a spice, a separation process is necessary
to remove stems, especially woody parts, which usually are of inferior quality as compared
to green plant parts. Several techniques can be used for that purpose. The simplest
method is to dry the product in such a way that the flowering parts are sufficiently
 Hayvesting and post-hawest h a n d i n g i n the g e n w Thymus 207
dried to detach them, whereas the stems remain humid. The other technique deals with
passing dehydrated plants through modified threshers.
The most basic cleaning operation is the utilisation of sifters. By running the herb
over a set of screens (Table 7.6),it is possible to remove larger and smaller particles from
the product that is being cleaned. Although the principle of sifting is quite simple, it is
rather difficult in operation, because dried herbs are random pieces of leaves. Therefore,
sifters are generally not used for cleaning, but for sizing.
An air table or a gravity separator is the most versatile piece of cleaning equipment
for herbs and spices. Actually, an air table is a wire mesh screen with a stream of air blow-
ing up through it. The lighter pieces on the screen are suspended higher than the
heavier ones. The air stream blows the very lightest pieces out of the system. During
operation the screen is tilted and all thyme particles move to the bottom end of the
screen. Rotational vibration is imparted to the screen, which is adjusted so as to just
touch the heavier particles and tap them, pushing up the screen, while the light filth
migrates to the bottom of the screen. In practice, the tilt of the screen, the rotational
vibration of it, and the airflow through the screen are adjusted so that the cleaned
thyme migrates to the middle of the screen, the heavy filth to the top of the screen.
The main disadvantage of an air table is that it may or may not be able to separate
particles of different sizes and different densities if the air stream floats a large surface area
particle of relatively heavier weight at the same height as a small surface area particle of
lighter weight. However, it can efficiently separate particles of the same density and
different size and the particles of the same size and of different densities.
There are some other types of cleaning equipment, e.g. indent separators which try
to make use of the difference in shape between the spice and the foreign material, spiral
separators, which work well separating round seeds from nonround foreign material.
However, physical characteristics of dried thyme are not convenient for such techniques.
Very fine dusty particles can be removed from dried raw herb materials by using an
apparatus
- - called a cyclone.
Harvesting of thyme should be performed in a way to minimise the contamination
with stones and rocks. However, depending on the excellence of the whole process some

Table 7.6 Compar~sonof various screen size measurement sysrems'g

USS Tyler Mill Stainless-steel USS Tyler Mill Stainless-steel

scyeen sc~~een .screen screen screen screen screen J ween

Note
* This information shows the closest match of screen sizes for various measurement systems. The actual aperture for
each is not necessarily identical and some tolerance is needed to build into specifications.
r Grenis, 1991.
Source: T a ~ n t e and
208 Petras R. Venskutonzs

amount of stones will remain in the dried product. Destoners work on the same principle
as the air tables but are generally much smaller in size. Where an air table is able to
separate the product stream into as many divisions as is desired, a destoner is generally
set up to remove only heavier stones and rock from thyme. Once again, by varying the
airflow, the inclination, vibration and the type of screen, it is possible to make the
stones "walk" up the screen and thus affect a separation from the lighter leaves of
thyme (Tainter and Grenis, 1993).
Dried thyme can be imported from various countries or sources, which sometimes
do not ensure good sanitary processing conditions. Therefore, its reconditioning is used
to remove contaminants and bring the product into conformity with specifications.
Reconditioning involves the same cleaning steps, which are briefly outlined above.
Some legislation institutions, e.g. FDA in the US, want to know if the spice is planned
to be reconditioned prior to performing the work. They may want to supervise the
operation to ensure adequate removal of the contaminant. Under American Spice Trade
Association (ASTA) procedures, supervision is not necessary, but the lot must be resampled
and tested by an independent laboratory.

C o m m i n u t i o n a n d particle size selection

Thyme can be used as a whole, leaves and flowering parts, and as a ground product. The use
of whole herbs in food processing is limited as they are not ready sources of flavour but
they do improve the appearance of certain products. With few exceptions, herbs and spices
are normally milled to powder before use. Size reduction may also be an essential
primary stage for extraction or distillation, for incorporation into food product mixes or
for use as they are at table or in cooking.
There are many different ways to cut, grind, break or otherwise reduce the size, how-
ever the basics of herb comminution are very simple. There is a variety of equipment
used to grind thyme, and they are generally designed to crush, or to shatter the plant
particles. To minimise negative grinding effects such equipment ranges from high-speed
disintegrators, hammer mills, pin mills, plate mills, roller mills, ball mills to slow
speed, vertical and horizontal buhrstone mills in a whole range of sizes and capacities.
The machinery can be roughly classified into the following groups:

(a) course cutters, crushers or breakers designed to give an intermediate product further
reduction from the large plant parts received in bulk;
(b) slow-speed attrition mills, which reduce the product to a fine powder;
(c) high-speed impact mills, whereby a wide range of sizes may be produced, and
throughput is rapid;
(d) micromills for extremely fine powders.

There are two main problems encountered in the grinding procedure: (i) breaking of herb
oil bearing particles and (ii) formation of heat. In natural herbs the aromatic components
are retained within a protective cell wall. This gives to the whole herb a long shelf life
so long as certain basic storage conditions are observed. By grinding all the oil structures
containing the volatile oil, the oil becomes available for reaction (e.g. oxidation) or
evaporation. Grinding also generates some heat, which will tend to vaporise the volatile oil
leading to a reduction in flavour strength. The strong odour of thyme, which penetrates
environment during grinding, indicates some degree of flavour loss. Therefore it is
 Harvesting and post-harvest handling in the germs Thymus 209
necessary to keep the temperatures during the grinding process as low as possible to
minimise the loss of volatile oil.
Various mills can be configured in various manners by changing internal screens,
speed and internal clearances to control the heat build-up. As a rule, finer particle sizes
develop higher temperatures during grinding. To limit the formation of heat the product
can be prechilled before feeding. Usually the user defines particle size. There are
sophisticated grinding methods that avoid heat release during grinding. For instance,
Cohodas in 1969 patented a process for cryogenic milling in which the spice stock and
a gaseous refrigerant (e.g. nitrogen) are fed into the milling head at a constant rate.
Such a technique considerably minimises losses of volatiles and discoloration of herb. By
freezing the herb and solidifying the volatile oils, these herbs grind and sift a lot easier.
Cryogenic grinding will also minimise oxidative deterioration of the flavours due to the
nitrogen blanket during grinding. Therefore, a spice ground cryogenically may have
a different flavour profile, usually retaining the top notes and giving a fuller flavoured
product.
Comminution operations often include sieving and sifting procedures. The mills may
have internal screens that in part dictate the final particle size, or the sifting operation
may be a separate operation where the oversized particles are returned to the mill for
further processing. In either case, the set-up of the mill or sifters determine the particle
size of the finished spice. Since nearly all spice and herb specifications contain a granu-
lation parameter, e.g. granulation of ground thyme should be 95 per cent minimum
through a United States Standard (USS) #30 (ASTA), it is important to look at the
particle size control of the ground herb. The manufacturers may also adopt their own
empirical classification.

POSTPROCESSING TREATMENTS

Control or reduction of microbiological populations found in herbs and spices as they

are harvested is an important task demanding some postprocessing treatments of the
product. Kneifel and Berger (1994) screened a total of 160 samples of 55 different
spices and herbs originating from six different suppliers and retailed at outlets in
Vienna for their microbial quality. Arithmetical mean values and a range of viable
counts of thyme are presented in Table 7.7. In the other source the contamination of
rubbed thyme herb is estimated as 3.5 x lo4 countslg (Heath, 1982). High microbio-
logical loads can lead to a significantly reduced shelf life and may produce problems if
there is not a significant heat processing step in the finished food. The process of steril-
isation adds to the cost of the herb or spice but it is well worth it to the ultimate user
(Farrell, 1985).
Pathogens (microorganisms which cause human diseases) can be a problem, espe-
cially for herbs and spices processed in countries with lower sanitary standards compared
to those countries that may have well-defined levels. As the presence of microorganisms
in herbs can cause food spoilage and illness, a case can be made for sterilisation. How-
ever, herbs and spices generally constitute only 0.1-2.0 per cent of most food products
and are most often used in cooked form. As a result, the risk of spoilage and illness is
greatly reduced.
A number of different sterilising procedures have been applied to spices and herbs in
an attempt to reduce high microbial levels. Both physical and chemical methods have
2 10 Petras R. Venskutonis

Table 7.7 Microbial contam~nationof random samples of thyme

Mzcroorganisnz~ Mean values Viable count range

log CFUig
--

Total aeroblc mesophll~c 5 1 x lo6 4 1-7 6

bacteria
Enterobacteria 4.5 x 10'
Pseudomonades and 3.0 x 10' 3.4-6.4
Aeromonades
Bac~lli 6.6 x 10" 4.1-6.4
Lactobacllll <lo
Enterococci 8.8 X 10'
Viable counts 2.8-5.0

Source: Kne~feland Berger, 1994

been used with varying degrees of success. These include steam and heat sterilisation,
ultra-violet (UV) irradiation, ionising irradiation, filtration, infrared irradiation, and
fumigation. Mention may also be made of methods such as drying, freezing and salting
which are generally used in a supplementary role. A common industry practice is to refer
to these treatments as sterilisation or "bacteria treatment". Neither of these terms is
accurate. The treated spice is not commercially sterile nor has it been treated with
bacteria (ICMSF, 1980; see Table 7.8). All microbial decontamination methods can be
classified into physical and chemical treatments. The principles of these methods ate
comprehensively described elsewhere (Gerhardt, 1994; Tainter and Grenis, 1993).

Ethylene oxide ( E T O ) or methyl bromide treatment

Many commercial food processors fumigate herbs and spices with methyl bromide to
eliminate insects or with ETO or its mixture with carbon dioxide (90 : 10) to eliminate
bacteria and mould. Both methyl bromide and ETO are extremely toxic, and methyl
bromide is potentially capable of depleting the atmospheric ozone layer (Thayer etal.,
1996). The US Environmental Protection Agency (EPA) (1982) places a maximum
tolerance of 50ppm for ETO in or on ground spices; after 25 years of use in Germany
ETO was recently banned as well as in the other European Union (EU) member states.
Propylene oxide is also approved as a microbiological treatment process for spices, but
it is not nearly as effective as ETO because its penetrating ability is weaker.
Blends of spices can be treated with ETO as long as no salt is present. Salt will
react with ETO to form chlorohydrins that are toxic. Gustafsson (1981) examined the
residue levels of ethylene chlorhydrin (2-chloroethanol) in various products and found
that 5 samples of thyme contained 105, 230, 390,450 and 1290mglkg of chlorhydrin.

Irradiation

Most of the chemical microbial decontamination treatments are insufficient or have

a serious disadvantage (Eiss, 1984). Irradiation for the sterilisation of spices has been
studied for several years and the treatment has been shown to be a very effective tool of
reducing microbial populations. Alongside traditional methods of processing and pre-
serving food, the technology of food irradiation is gaining more and more attention
 Harvesting and post-harvest handling in the genw Thymus 211
around the world. Herbs and spices are being irradiated in many countries including
Argentina, Brazil, Denmark, Finland, France, Hungary, India, Indonesia, Israel, Norway,
the United States, and Yugoslavia.
The Joint Expert Committee on Food Irradiation (JECFI) in 1980 concluded that "the
irradiation of any food commodity" up to an overall average dose of lOkGy "presents
no toxicological hazard" and requires no further testing. Dried herbs and spices are
allowed the highest radiation treatment level (up to 30 kGy) because (i) they are con-
sidered as a minor part of our diets; (ii) they are safer to irradiate due to the low mois-
ture content; and (iii) they are irradiated for the purpose of controlling both insects and
microorganisms, which necessitates high doses of radiation.
The International Atomic Energy Agency (IAEA) and the International Consultative
Group on Food Irradiation (ICGFI) concerning herb and spice irradiation issued the
following important documents:

IAEAITECDOC-688 (Technical Document 688): Irradiation of Spices, Herbs and

Other Vegetable Seasonings - A Compilation of Technical Data for its Authorisation
and Control;
ICGFI (International Consultative Group in Food Irradiation) Document No. 4:
Code of Good Irradiation Practice for the Control of Pathogens and Other Micro-
flora in Spices, Herbs and Other Vegetable Seasonings.

Ozone and other methods of treatment

Ozone is an extremely reactive substance and a very effective antimicrobial agent.

Decontamination can be performed in storage tanks or in the milling machinery during
the comminution procedure. Ozone treatment can reduce microbial counts down to
2-3 per cent of the initial load (Gerhardt, 1994). However, the main disadvantage of
ozone application deals with the safety issues due to the possibility of explosion.
UV-irradiation, microwaving, treatment with high frequency electric currency, high
pressure treatment are rarely used methods for herb decontamination, some of them are
in the stage of development, laboratory and pilot-plant testing. Some other methods,
such as a focused microwave system (Anonymous, 1991a) and pulsed electric fields
(PEF) (Keith etal., 1997) were also tested for disinfestation of herbs and spices.
Steam sterilisation is usually applied to the whole herb or spice before grinding and
decontamination can be performed by pressurised, atmospheric and sub-atmospheric
steam (Anonymous, 1999). Although described in the literature, steam sterilisation
methods and apparatus only in some cases were tested on thyme, it is reasonable to
briefly overview these methods, so far as they can be applicable for the sterilisation of
Thymus herbs as well.
Hosokawa Micron Europe BV developed a process for sterilisation of herbs and spices,
based on rapid heating to the required temperature with saturated steam, holding for
the required period, and rapid vacuum coolingidrying (Spook, 1993; Anonymous,
1993). Dudek (1996) patented the process involving exposure to elevated pressures and
temperature for a predetermined time in a series of chambers; Leife (1992) described
a method for sterilisation of spices and herbs with rapid pulses of steam. Herbs and
spices can be pasteurised in Torbed equipment (from Torftech, UK) described by
Dodson (1996). Particles are forced into a toroidal motion, exposing all surfaces to
heat and breaking down the insulating boundary layer in seconds. A natural steam
pasteurisation process for herbs and spices was developed by McCormick as a superior
alternative to UV or Infra-Red (IR) radiation, microwave, cryogenic or ultrasonic
methods of microbial control (Anonymous, 1991b). Mercati (1992) proposed a multi-
step processing method, which is characterised by a stage in which the herbs stay in hot
air chambers at an adjustable temperature and for a period of time ranging from 1 to
60 h sufficient to cause the death of the bacteria.

DRYING TEMPERATURES, CONDITIONS AND DURATION

OF STORAGE

Dehydration (drying) is undoubtedly the most ancient process for the preservation of
spices and aromatic herbs. As medicinal and aromatic plants are usually harvested at
80 per cent moisture content and stored at 11 per cent, drying of this crop requires high
energy equivalent to 1-2 1 of fuel oil per kg of crude drug. Depending on the herb or
spice the ratio of the weight of fresh raw material with the weight of dry product can be
from 1:1 to 10:1; for thyme this ratio can be from 3: 1 to 5:l (Gerhardt, 1994). The
process of drying for many medicinal and aromatic plants is the crucial one in deter-
mining the quality of the dried material and products produced from it. Heindl and
Miiller (1997) comprehensively described drying of medicinal plants and spices.
Traditionally herbs have been cut and dried in the sun or in the shade. Dried herbs
then were sold to a processor who beats the leaves from the stalk by hand or machine
and then sieves them to remove stalks and stones. The process of removing the leaves
from the stalk is called "rubbing" and the sieving called "sifting".

Methods of drying
In general, all drying methods can be divided into thermal and non-thermal drying.
Thermal methods can be classified into (1) natural direct drying (air drying with the
aid of sun energy), (2) solar indirect drying, and (3) artificial drying (with the aid of
heat, cold or IR). Non-thermal drying can be performed by using (1) moisture-
absorbing materials, (2) drying agents and (3) electrolytes (Gerhardt, 1994). There are
different modifications of these methods, which are used depending on the economical
and quality requirements.
Natural drying is the simplest way to prepare herbs for storage and further processing.
So far as the method is cheap and does not require costly equipment it is still widely
used in many countries. There are several methods of drying of raw material such as
sun-drying (SD), drying in the shade, solar drying, hot air drying, practised in com-
mercial processing in the different countries.
The method of sun-drying is the most usual as well as the one most widely
employed. In most cases the material is first comminuted and spread on the ground on
mats and exposed to direct sunlight. This method is simple and quite cheap; however,
it possesses some disadvantages: (a) the possibility for the introduction of contaminants
in many different ways (rodents, insects, and their resultant contamination), (b) the loss
of volatile matter, (c) the degradation of heat or light-sensitive constituents, (d) dif-
ficulties in controlling the process, (e) usually long time of drying. Natural drying of
the whole T. vulgaris herb is particularly problematic because the shrub consists of
comparatively fast drying leaves and slowly drying rather hard stems. Therefore the
 Harvesting and post-harvest handling in the genus Thymus 213
process can take up to 120 h causing severe loss of the volatile oil (Raghavan etal.,
1775).
A more sophisticated method of solar drying is also employed, but this is only used
in cases of small volume high value products. This method is excellent for leafy material
as it maintains the rich green colour making the product look attractive. It is, however,
a method, which involves much initial outlay and will be uninteresting for most producers.
Different types of solar dryers have been developed. They have been successfully tested
on different Labiatae aromatic plants (mainly mint and sage) and can be used for drying
Tbymw spp. In 1771 a solar drying device, based on the use of solar-energy, started to
operate in Krka - Drug factory, p.0. (Novo mesto, Yugoslavia, Program of Green
Drugs). In 1793 Miiller etal. developed a solar-heated dryer in modular design at the
Institute for Agricultural Engineering in the Tropics and Subtropics of Hohenheim
University (Figure 7.3). The roof of a standard plastic film covered greenhouse was used
as a solar air heater area. A batch dryer was installed inside the greenhouse. A comparat-
ive economic evaluation showed that the plastic-house type solar dryer is economically
more efficient than the conventional drying system, as soon as supplementary heating
is used.
Hot air drying allows for more rigid control of the process, it is rapid and clean,
however requires high capital and operational costs and can cause overheating. This
method is useful if the end product is of high value and the quantity to be handled is of
reasonable magnitude (Wijesekera, 1973). Some companies provide high output, auto-
matically controlled dehydration lines specially designed for leafy plant material. The
scheme of one such line produced by Heindl GmbH, Maschinen- und Anlagenbau,
Germany, is provided in Figure 7.4.
Although traditional hot air drying is a simple method in herb processing, however,
its main parameters can be varied and should be tailored to every particular herb
mainly to minimise flavour loss and to perform the process at reasonable time and
energy costs. Raghavan etal. (1775) compared cross flow and through flow drying
methods on Indian thyme at 40, 50 and 60 OC and found that through flow drying at

Recirculation
flap

Fzguve 7.3 A solar heated dryer in modular design (Miiller etal., 1993).
2 14 Petras R. Venskzltonis

Figwe 7.4 Dehydration line for leaf vegetables and officinal herbs (Heindl G m b H , Maschinen-und
Anlagenbau). (1) feed hopper, (2) leaf cutter, (3) conveyor belt, (4) conveyor belt, ( 5 ) stalk sep-
arator, (6) dosing hopper, (7) paddle washer (optional), (8) fan washer, ( 9 )vibratory conveyor
for removal of surface water (optional), (10) oscillating conveyor belt, (1 1) five-band-dryer,
(12) milling and air separating unit.

4 0 C gave the best results. Kakis (1986) invented the method by which foodstuff is
firstly pressed to remove a substantial amount of its less tightly bound water, then
contacted with an absorbent to remove a substantial amount of its more tightly bound
water and provide a low moisture foodstuff. Subjecting this low moisture foodstuff to
ambient temperature air drying in a low moisture atmosphere provides a dry-to-the-
touch dehydrated foodstuff which retains its flavourful and aromatic volatiles and is
resistant to spoilage. Ground fresh thyme (63.2g) was subjected to the pressing at
8000-10000 psi and absorption steps by using 5 g of Syloid 244. After the initial
pressing step the sample lost 32 per cent and after the absorption step an additional 25
per cent of its original weight. After air drying at 26 OC for 1 8 hours, 1 8 g of dry mate-
rial were obtained. Thus, the total weight loss for the three-step process was 72 per cent
of the original.
Freeze drying is based on evaporation of water directly from ice under a high vacuum.
Herbs are rapidly frozen to less than -18 OC temperature to form microcrystalline ice
structure, which does not damage plant cell structure. The products obtained by this
method are usually of a better appearance (colour) and aroma quality. High cost is the
main disadvantage of freeze drying limiting wider use on a commercial scale. The effect of
freeze drying on the chemical composition of thyme will be discussed in the Chapter 8.

Drying conditions
It is well established that the higher the drying temperature is, the shorter the time of
the process is; however, bigger losses of volatile oil occur. Drying temperature and
conditions should be tailored for every aromatic plant to achieve optimal quality char-
acteristics.
Poludennij and Zhuravlev (1989) recommend drying harvested thyme in the shade
in ventilated premises. When oven-drying is used, the temperature should not exceed
4 3 4 5 OC. T. serpyllum is also dried in the shade or in special oven-dryers at 40 OC. After
drying, leaves and flowers are ground and sifted. Wooden stems and branches are
removed (Kudinov etal., 1986; Mashanov and Pokrovskij, 1991).
So far traditional drying conditions require long drying time. There are some inter-
esting reports about using different techniques intended to improve the process of herb
processing. Sometimes very simple alterations can be implemented to improve natural
drying conditions, e.g. Aliev and Kuliev (1989) suggest to hang the herbs up to dry in
0.5-4m plaits to reduce product loss, and facilitate subsequent storage and transport.
Some authors describe more sophisticated new combination techniques, e.g. including
 Harvesting and post-harve~t handling in the genw Thymus 215
such procedures as blanching, treatment with osmotic agents and surfactants. So far as
the application of these techniques can be considered in the processing of thyme they
will be briefly described.
Mastrocola etal. (1988) applied four different sets of drying conditions to retail samples
of basil and found the importance of blanching in retaining the colour. Rocha etal.
(1993) used steam blanching and surfactant pretreatment to increase the drying rate of
basil and found that drying rates were increased by a factor of 10 and 14 for steam
blanching and surfactant pretreatments, respectively. Blanching leaves of Indian
spearmint (Mentha spicata L.) prior to drying yielded products, which were unattractive
with respect to colour and appearance and were also bland and odourless due to loss of
volatile oil during drying whereas shade-drying leaves resulted in a product with a
good green colour and small loss of volatile oil (Raghavan etal., 1994). Blanching of
Eryngium foetidurn L. herb in hot water at 96OC using a quick dip step followed by
drying in the indirect drier reduced the loss of green colour normally observed on direct
drying without pretreatment (Sankat and Vashti-Maharaj, 1994).
Investigations with parsley leaves showed that compared with convection drying, when
leaves where dried in a microwave oven the loss of the aroma fractions was approximately
five times less, sensory scores were considerably higher, and drying times were three
times shorter (Zarebski and Mroczkowski, 1995). Aung and Fulger (1993) describe
a method for drying fresh herbs whilst preserving their colour, aroma, flavour and overall
appearance involving treatment with an osmotic agent which, on completion of the drying
process, forms a solid amorphous mass. This mass coats and infuses the treated herb.
Effect of SD, solar cabinet drying (SCD) and tray drying (TD) on the colour of
dehydrated fenugreek (Trigonella foenzlm-graecum) and mustard (Brassica campestris var.
sarson) leaves was investigated and significant differences were observed. It was recom-
mended that SCD could be used in tropical regions (Ramana etal., 1988).
A process for preservation of herbs or other aromatic plants is based on mixing the
edible part of the herb plant with a substance which reduces water activity; freezing the
mixture; partial freeze drying to yield a mixture of dried and frozen or non-dried herb
material; and homogeneous mixing to achieve equilibration of moisture content
between the dried and partially dried material (Darbonne, 1996). Various spices were
dried by a dehumidifying dryer, in which trays of products are superimposed and air is
circulated by a fan. After passing through the product, the humid air is cooled in order
to remove the water vapour, and then heated before re-entering the products (Rattanapant
and Phongpipatpong, 1990).
A process for osmotic partial drying of edible plant material (e.g. vegetables, herbs)
has been described. The plant material is treated with an aqueous solution (refracto-
metric dry matter (DM) content 25-40 per cent) containing one ingredient selected
from the group gum arabic, carboxy methyl cellulose (CMC), modified starch and ethyl
maltol, together with a polyol (sorbitol, mannitol, xylitol or glycerol) and/or a sugar
(sucrose, glucose, lactose, or maltodextrin with dextrose (DE) less than 30). The solution
should have p H in the range 4.3-6.5. Contact time between the plant material and the
solution is 15-50min, at 10-50C. The plant material is dried to a residual moisture
content of 10-15 per cent, corresponding water activity (aw) of 0.3-0.6. Optionally,
the plant material may be blanched before the osmotic drying process (Darbonne and
Bain, 1991).
Bousser (1990) describes a method for improvement of natural drying of biological
materials (including fruit, vegetables, herbs and medicinal plants) which is based on
addition of glucose to the raw material, then comminution of the mixture to a homo-
geneous mass which is then dried. Optionally, sorbitol andlor citric acid andlor salt
may be added to the glucose; a typical formulation is 95 per cent glucose, 2.5 per cent
sorbitol, two per cent NaCl and 0.5 per cent citric acid. Metabisulphite may optionally be
added. The glucose-based mixture is added to the raw material at a level approximately
three times higher than the quantity of water in the product to be dried. The mixture is
dried in a thin layer at 25-30 OC with gentle ventilation. The products may also contain
other ingredients, e.g. dried milk, chocolate, starch or breadcrumbs.
A drying system, utilising conductive and convective heating to dehydrate a variety
of foods (including vegetables, herbs, fruits, cheese, meat and dairy products) with
low-temperature air, is described. Feed material enters the top of the dryer and is uniformly
distributed across a moving bed of balls made of stainless steel, ceramic, aluminium or
food-grade plastics. Heated air enters at the base of the dryer. Products can be dried to
3-7 per cent moisture, and have flavour and colour characteristics comparable to freeze-
drying (Swientek, 1988).
A method and apparatus for treating (washinglsteepingldryinglcooling) freshly
harvested vegetable products, particularly herbs and spices, are described (Hsieh and
Albrecht, 1988).
Herbs are prepared by stabilising them in the freshly harvested state (or, if frozen
products are used, during or immediately upon thawing), and drying them in the
presence of a suitable carrier under mild conditions. As carriers, salts (electrolytes), pro-
teins, carbohydrates, or mixtures can be used. The stabilisation step consists of either
heating to 50-150C or mixing with an electrolyte or both (Bezner etal., 1987).

PACKAGING A N D STORAGE

Dried thyme is a long shelf life product. As a rule dried herbs are considered stable
until the development of some noticeable off-flavour (unusual taste andlor odour). The
period of minimal stability is a time during which the herb is fully suitable for use and its
essential specific characteristics such as aroma, pungency, colour, etc. are maintained.
The quality and minimal period of stability can be assessed by the sensory evaluation of
colour, odour and taste and by the determination of essential oil (Gerhardt, 1994).
The stability of thyme depends on the following aspects:

moisture content;
comminution method (finer grinding means lower stability);
quantity and package size (bigger package brings higher stability);
packaging material (lower permeability to water and air results in higher stability);
penetration of air into the package;
effect of light and humidity (higher humidity and light access causes lower stability);
storage temperature (lower temperature means higher stability).

In general storage below -18 OC is a guarantee for unlimited stotage time; when the
product is stored at 5-7 OC dried herb can be stored more than 12 months, whereas at
room temperature stability considerably decreases.
After all processing steps have been completed thyme herbs are packaged. It is an
important procedure, because during comminution the structure of the cells is usually
 Harvesting and post-harvest handling in the genus Thymus 217
more or less damaged and volatile constituents can be easily released. There are two
main tasks for packaging (Niebergall etal., 1978): (a) to protect against exterior effects,
(b) to increase the stability against negative internal changes (enzymatic, non-enzymatic,
chemical reactions, etc.).
Optimal packaging materials are glass and metals: both are completely impermeable
and provide the best protection of aroma. Due to economical and some other reasons
different new materials, mostly synthetic plastics, have increasingly substituted those
traditional packaging materials. However, among them there is no ideal material for
the packaging of herbs. For instance, polyethylene efficiently protects against water and
gives resistance, however it is permeable to fat and aroma compounds and is difficult
for machine treatment; polyamide is impermeable to gas, whereas its waterproof
qualities are less efficient (Gerhardt, 1994). Modified atmosphere packaging can be used
to protect packaged herbs against oxidation. The oxygen can be removed by vacuuming
or by replacing air with inert gas. Oxygen absorbents can also be used for this purpose.
The quality of thyme decreases during storage. The changes depend on several factors,
drying method and parameters, moisture content, cleaning procedures, grinding tech-
nique (e.g. perfection of milling equipment minimising heat build-up during grinding)
and particle size, sterilisation treatment, storage conditions, packaging type, etc. In
general, dried thyme should be stored in cool, dry conditions away from light. Ideally,
it should be in airtight packaging to reduce oxidation.
Finely milled thyme is often advantageous in use, however, it does tend to lose vola-
tiles more rapidly than medium or coarsely ground material and must be stored in
well-closed containers. Storage in multi-layered paper sacks having an impervious lining
is also satisfactory but not so good once the sack has been opened (Heath, 1981).
The content of essential oil in herbs and spices reduces during storage. The loss of
volatiles depends on various factors, e.g. botanical plant characteristics (structure and
distribution of oil-bearing particles), essential oil composition, processing (mainly
drying and grinding procedures), packaging and storage conditions. For instance, in
one of the early studies, thyme herb, which was packaged in paper bags, was stored in
dark premises at ambient temperature and after 6 years of storage it was found that the
loss of essential oil constituted 71.4 per cent, whereas essential oil reduction in sage
after 7 years of storage was only 20.6 per cent (Stamm and Willner, 1934). Some
changes in volatile oil composition also take place during storage.
Fehr and Stenzhorn (1979) studied the change of essential oils in relation with the
storage time (up to 38 months). The essential oil content in thyme decreased at a rate of
0.002-0.022 mllmonth. The authors determined significant differences in the compos-
ition of essential oils (Table 7.8) and proposed mathematical description of the long-
time storage stability of dried thyme.
The concentrations of some quantitatively important thyme flavour compounds after
a storage period of 0, 1, 5 and 1 0 months were also studied by Venskutonis etal. (1996)
and are presented in Table 7.9. It was found that the changes during storage are highly
significant, but the differences vary between compounds and it is possible to divide the
compounds into groups of relatively small changes and relatively large changes. The
compounds belonging to the two groups are as follows: (a) small differences: a-pinene,
p-cymene, linalool, borneol, thymol, carvacrol, and P-caryophyllene; (b) large differences:
a-thujene, myrcene, a-terpinene, y-terpinene, trans-sabinene hydrate and caryophyllene
oxide. Some of the compounds did not show systematic changes and generally were not
reduced during the storage period, that is the case with thymol, while others like
2 18 Petras R. Venskzltonis

Table 7.8 Contamination of some untreated Labiatae herbs with

bacteria and moulds (ICMSF)

Spice Cumuhtive percentage incidence of

Aerobic plate countig Mould countig

Thyme 85 53 0 87 6 0
Basil 86 38 0 6 0 0
Marjoram 78 33 5 29 0 0
Oregano 32 9 0 9 0 0
Sage 47 6 0 50 0 0
Savory 10 0 0 0 0 0

Table 7.9 Composition of thyme essential oils (%) depending on storage time

Compound 1175 2/76 3176 4176 5177 6/77 7178 8178 9178 Range Mean
- -

a-Pinene 1.1 1.4 1.4 1.7 1.5 1.3 1.9 1.3 1.9 1.1-1.9 1.5
Carnphene 0.5 0.6 0.6 0.7 0.6 0.6 0.7 0.5 1.0 0.5-1.0 0.6
Myrcene 0.7 0.9 0.8 0.9 1.3 0.8 1.3 0.8 1.0 0.7-1.3 0.9
a-Terpinene 0.8 1.0 1.0 1.0 1.3 0.8 1.4 0.9 1.0 0.8-1.4 1.0
Limonene 0.3 0.4 0.4 0.5 0.5 0.4 0.4 0.4 0.4 0.3-0.5 0.4
1,8-Cineole 0.6 0.6 0.7 0.8 0.8 0.6 0.7 0.6 0.8 0.6-0.8 0.7
y-Terpinene 3.2 4.1 3.9 3.8 6.9 3.2 7.6 3.2 6.0 3.2-7.6 4.7
p-Cymene 30.6 31.4 34.1 42.8 29.3 34.8 27.3 33.2 34.6 27.3-42.8 33.1
Linalool 2.2 2.3 2.6 2.5 2.9 2.7 3.0 2.7 3.0 2.2-3.0 2.7
Terpinen-4-01 2.0 2.2 2.2 2.5 2.3 2.3 1.4 1.5 1.2 1.2-2.5 2.0
0-Caryophyllene 0.2 0.3 0.3 0.3 0.2 0.3 2.1 1.1 2.2 0.2-2.2 0.8
Borneo1 1.1 1.2 1.2 1.2 1.3 1.2 1.5 1.1 1.8 1.1-1.8 1.3
Thyrnol 43.6 42.7 39.1 30.0 39.5 39.3 39.6 41.1 33.2 30.0-43.6 38.7
Carvacrol 4.3 4.1 4.0 3.5 4.1 4.3 3.6 5.4 3.5 3.5-5.4 4.1
Hydrocarbons 37.4 40.1 42.5 51.7 41.5 42.2 42.7 41.4 48.1 37.5-51.7 43.1
Oxygenated 53.8 53.1 49.8 40.5 50.9 50.4 49.8 52.4 43.5 40.5-53.8 49.4
compounds

Source: Fehr and Stenzhorn, 1979.

a-thujene, myrcene and sabinene hydrate are reduced by 21-40 per cent after 10
months of storage.
Besides the most important compounds quantitatively caryophyllene oxide is also
included in Table 7.10 as the content of this compound increased during storage. This
could indicate some oxidation during storage. It is interesting to note that the reduction
of the content of ,!?-caryophyllenebetween 1 and 10 months of storage was by 69mg/kg,
whereas the increase of the concentration of its oxide during the same period was by
56mg/kg, i.e. nearly equal. However, this tendency was not found during the first
month of storage, when according to the statistical assessment there were no significant
differences in the content of 0-caryophyllene. Tress1 etal. (1978) found 12-fold increase
in the concentration of caryophyllene oxide in dried quince after a storage period of
 Harvesting andpost-harvest handling in the genzs Thymus 219

Table 7.10 Changes of the average content of volatile compounds in

thyme during storage (mglkg)

Constitz~ent Tzme of storage, months

0 1 5 10

a-Thujene 255" 179" 54d

a-Pinene 257" 222C 92;
241 25Pb
Myrcene 299" 26ab 166' 1
a-Terp~nene 220" 198" 15 1' 10Gd
p-Cymene 3863" ?1694~ 3360' 3538bc
r-Terpinene 1296" 1173" 80jb 410'
tr-Sabinene hydrate 260" 233" 97' 8lc
Linalool 345" 355" 323b 324b
Borneo1 189" 160' 169bc 183~~
Thymol 6912~ 8153" 6441b 6426b
Carvacrol 827b 1027" 880b 979"
,!!-Caryophyllene 283" 311" 303" 242b
Caryophyllene oxide 34' 58" 6gb 114"

Note
a-d Values with the same letter within same row are not significantly different.
Source: Venskutonis eta(., 1996.

3 years, whereas the content of all identified non-oxygenated terpenes severely reduced
during the same period.
The changes in the amount of the aroma compounds during storage may be explained
by oxidation and other chemical changes, as indicated by the increase of caryophyllene
oxide. However, as the polyethylene bags were not aroma tight some losses may be due
to evaporation of volatile compounds (most of non-oxygenated monoterpenes) through
the PE bags. In this view fairly stable content of a-pinene during storage seems rather
contradictory.
Bend1 etal. (1988) studied the effect of freeze-drying of sage and thyme on the stability-
properties following storage in comparison to drying at increased temperature. The
authors determined that by using freeze-drying there could be found greater amounts
of the characteristic flavour components particularly immediately after preparation but
also after 8 0 days of storage (Table 7.11). These studies indicate that by freeze-drying
sage and thyme there could be obtained a storable product with high spice and flavour
quality. Some special measures to improve storability were applied. For instance, stor-
ing fresh leaves of Indian spearmint (Mentha spicata L.) for 12 h after spraying with
water increased volatile oil content by about six per cent (Raghavan etal., 1994).
Anokhina etal. (1990) performed tests on the effect of freeze-drying on dill and parsley
from a retail perspective and reported that storage in laminated polyethylene containers
is recommended in order to minimise vitamin C loss.
Koller (1988) gives examples of inadequate treatment changing the characteristic
aroma of herbs and spices, so that they no longer fulfil their function. Processes such as
drying (temperature, method, pH) and storage conditions (air access, light, tempera-
ture, pH, packaging material), as affecting colour and aroma of thyme, sage, cloves and
marjoram were investigated. Storage temperature has been found to be most decisive
on the changes of headspace volatiles and consequently aroma.
220 Petras R. Venskutonis

Table 7.1 1 Effect of storage on the content of the essential oil, thymol and
carvacrol in thyme

Storage, Anzozlnt in ground herb

days
Essential oil (pllg) Thyrnol (mglg) Caruacrol (mglg)
FD OD FD OD FD OD

Notes
FD, freeze dried.
OD, oven dried.
Source: Bendl et dl., 1988

Other constituents of herbs also can change during storage. Bakowski and Michalik
(1988) investigated suitability of some plants for drying and observed high losses of
vitamin C during dehydration and storage (after 6 months by 90 per cent). After dehy-
dration the carotene content decreased by 10 per cent, after 6 months storage, by 20 per
cent. Chlorophyll content in leaves also decreased during dehydration and storage period
but did not change the colour. The dehydrated leaves had high contents of calcium,
phosphorus, potassium, magnesium and iron.

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8 Thyme - processing of raw plant
material
Petras R. Venskutonis

INTRODUCTION

Traditionally dried ground spices and herbs, although being widely used products, possess
several serious disadvantages. The most common disadvantages of dried thyme are:

variable flavour strength and profile;

unhygienic;
often contaminated by filth;
easy adulteration with less valuable materials;
presence of lipase enzymes;
flavour loss and degradation on storage;
undesirable appearance characteristics in end products;
poor flavour distribution (particularly in thin liquid products such as sauces);
discolouration due to tannins;
unacceptable hay-like aroma;
dusty and unpleasant to handle in bulk.

Therefore, manufacturers are increasingly recognising the advantages of seasoning

based on herb extractives. In general, the methods of extraction depend on the desired
properties of a final product, characteristics of plant material, economical and technical
issues. The most important extraction products that are obtained from thyme are essential
oils, herbs, oleoresins and extracts. Therefore these three processes will be discussed more
thoroughly in this chapter.

ESSENTIAL OIL: PRODUCTION A N D CHARACTERISTICS

The definitions of the most applicable terms to thyme products can be seen as follows
(Lawrence, 1995, modified):
Essential oil: The isolated volatile aromatic portion of a plant, produced within
distinctive secretory structures. The essential oils generally constitute the odorous
principles of the plants. They are either distilled or expressed. In exceptional cases,
they may be formed during processing when the plant tissue is brought into contact
with water.
 Thyme -processing of raw plant material 22 5
Extract:A concentrated product obtained by treating a natural raw material with a
solvent. True extracts do not contain significant amounts of the solvent. Depending on
the polarity of the solvent extracts consist of polar or less polar compounds.
Oleoresin: Liquid preparations extracted from herbs or spices with solvents which can
extract oil and resinous matter from the botanical drugs yielding the oleoresin as evaporation
residue. Oleoresins are often used in food and pharmaceutical industries as a replacement
of ground spices and spice tinctures. Prepared oleoresins may also contain fixed oils.
Natural oleoresins are exudations from tree-trunks, barks, etc.
It can be noticed that the differences in the definitions between extract and oleoresin
are not very strict. The content of volatile oil can be considered as the most important
characteristic in distinguishing these two products. Oleoresins usually contain significant
amounts of volatile oil whereas its content in the extract is much smaller or absent.
Artificial combinations of the essential oil and an extract of the same plant are also
called oleoresins.

Principles of essential oil isolation

Essential oils are accumulated in different types of secretory structures of the plants,
and they can be categorised into superficial and subcutaneous oils, and Labiate's oils
belong to the first group. The main methods to obtain essential oils from plant material
are water distillation, steam and water distillation, steam distillation, maceration
distillation, empyreumatic (or destructive) distillation, and expression. With the excep-
tion of the latter process, all others need heat to release the oil.
Water distillation is the simplest method to obtain volatile oils; therefore this method
is usually performed in rural areas where no access to a steam boiler is possible. The
plant material is loaded into a still fitted with a slow-speed paddle stirrer (to avoid
agglomeration) and is always in direct contact with water. The water can be boiled by
direct fire and by submerged stem coils. It is very important to maintain a sufficient
level of water in the still to avoid overheating andlor charring of plant material and
consequently undesired essential oil off-flavours. During the process of boiling, volatile
oil evaporates together with water and the vapour afterwards is condensed. Due to its
highly hydrophobic property the essential oil can be easily separated from the water in
the so-called florentine flask. Thyme essential oil, being lighter than water, separates
on its surface. The scheme of the apparatus for water distillation is shown in Figure 8.1
(Heath and Reineccius, 1986).
Steam distillation is performed with the aid of steam, which is generated outside the
still, in steam generators generally referred to as boilers. Plant material is loaded into
a suitable still on a perforated grid through which steam may be injected from the base.
The process of steam distillation is more effective and the most widely accepted process
for the production of essential oils on a large scale. Steam distillation units can be sta-
tionary or mobile. In case of mobile distillation the process is performed in the harvesting
fields, and therefore such time and labour consuming operations as loading of herbs
into a cart, transporting to the still and unloading can be avoided. This may result in
reducing the time for the whole process, from chopping the wilted plant material to
disposing of the spent material, from 6-8 h to less than 3 h, the size of the labour force
could be reduced by at least 50 per cent. The mobile steam distillation process is used
for the production of oils from such important commercial plants as mints, clary sage
and dill.
226 Petras R. Venskzltonis

Figure 8.1 Diagrammatic representation of a water distiilation unit (Heath and Reineccius, 1986).

The only difference between water distillation and steam and water distillation is
that during the latter process the plant material is separated from the water. It can be
loaded onto a frame within the still body, fixed above a layer of water. T o increase the
effectiveness of water and steam and water distillation, cohobation is commonly used,
consisting in the return of distilled water to the still after the oil has been separated
from it so that it can be re-boiled. This procedure is very important for thyme, because
its oil is rich in phenols, which to some extent may dissolve in distilled water. Cohobation
on the one hand minimises the loss of oxygenated compounds, on the other hand how-
ever, it increases the risk of hydrolysis and degradation of constantly re-vaporised and
condensed oxygenated compounds. Therefore, it is not recommended unless the tem-
perature to which the oxygenated compounds dissolved in the distillate exposed is
maintained not higher than 100 OC.
Continuous distillation possesses many advantages in comparison with conventional
distillation procedures. Short time and high output, reduced energy and water needs,
reduced disposal costs for spent material, reduced labour costs, possibility of automation,
improved process reproducibility and consequently quality of oil are the most important
ones. However, such a process can be efficiently used only when large quantities of essential
oils are required.
The principles of essential oil distillation are common to many oil-bearing plants,
however, to obtain the highest yield and the best quality product the process has to be
tailored for every particular herb depending on its characteristics. The quality of an
essential oil is adversely affected by heat, light, air and moisture and since these are
inherent parameters of distillation it is small wonder that many commercial oils differ
markedly in sensory character (Heath, 1982). The quality of the oil is also affected by
the method of distillation. Water-distilled oils are commonly darker in colour and have
stronger still notes than oils produced by other methods (Lawrence, 1995). Distillation
can cause chemical changes of natural constituents, e.g. formation ofp-cymene from
y-terpinene, both compounds being important for thyme (Moyler, 1991).
 Thynze -processing of raw plant material 227
Characterisation of different thyme oils
In commerce, the designation "thyme oil" is occasionally applied indiscriminately, and
erroneously, to oils distilled from plants belonging to species other than Thymus
vulgaris L. or Thymus zygzs L. In Fenaroli's Handbook of Flavour Ingredients (Burdock,
1994) the essential oil obtained from T. vulgaris and T . zygis is described as a brownish-red
liquid exhibiting a strong, aromatic odour and a warm, somewhat sharp flavour (red
thyme oil). White thyme oil is a pale-yellow liquid obtained by rectification of the
distilled red thyme oil, exhibiting similar but milder odour and flavour characteristics.
The main constituents of thyme oil are thymol and carvacrol (up to 70 per cent). Other
chemotypes of T. vulgaris are limited to specific areas and yield, e.g. oils that contain
geraniol, linalool, a-terpineol, and 1,8-cineole; these oils are of minor importance. Some
chemotypes of T. zygis produce an essential oil with other dominant constituents (linalool,
carvacrol, geraniollgeranyl acetate, 1,8-cineolellinalool linaloollthymol, 1,8-cineolei
linaloollthymol). For instance, Sfiez (1995) who comprehensively reviewed recent inves-
tigations on T . zygis in one of the samples grown in south-eastern Spain (ssp. graczlis)
determined 91.40 per cent of linalool, and only 0.3 1 per cent of thymol.
The essential oil of Thymzls capitatus (today: Thymbra capitata) is a clear, pinkish to red-
dish-brown oily liquid with odour reminiscent of origanum (Spanish origanum). Usually
the oil from Thymzls capitatus is richer in carvacrol than the oil from T . vulgaris and T . zygis.

EXTRACTION: METHODS A N D EXTRACT CHARACTERISTICS

Numerous companies all over the world produce different extracted thyme products. The
examples of such products are provided in Table 8.1. Usually standardised thyme oleoresins

Table 8.1 Examples of standardised thyme products

--

Product Prodt~rer Chararterzstzrs

Standardised oleoresin Bush Boake Allen Limited, Volatile oil content (%, vlw)
Thyme FD0718 London, England 54-60
Standardised oleoresins Lionel Hitchen Essential Oil Volatile oil content (%, vlw) 50
Thyme HX2089 Company Limited, Dispersion race kgs = 100 kg of
Barton Stacey, Hants, UK spice 1
Dispersed spices - salt Thyme Bush Boake Allen Limited, Volatile oil content 0.3-0.4%
London, England (vlw)
Dispersed spices - dextrose Thyme Bush Boake Allen Limited, Volatile oil content 0.3-0.4%
London, England (vlw)
Dispersed spices - rusk Volatile oil content 0.6-0.8%
Thyme FD5781 (vlw)
Standardised emulsion oleores~ns Felton Worldwide SARL, Strength compared to ground
Thyme HF107 Versailles, France spice 4 X
Standardised emulsion oleoresins Bush Boake Allen Limited, Strength compared to ground
Thyme FD6136 London, England spice 5 X
Encapsulated standardised oleoresins Bush Boake Allen, "Saronseal Strength compared to ground
Thyme FD4040 Encapsulated spices" spice 10 X
 Y GROUND THYME HERB

1 Distillation
I 1 Solvent extraction 1

L_1 Essential oil

a Oleoresin

oleoresin

Dispersed in Dispersed in
neutral carner acid-stabilised
starch solution

Blended w ~ t h
other flavouring
material

-
Commercial Double-
Dispersed Liquid encapsulated
dispersed Spice
standardsed
standardised In high melting emulsion
oleoresin oleoresin
oleoresin point fat
- -

Flgzre 8.2 Flow chart of a typ~calstandardised oleoresin range of thyme (Moyler, 1991)

are produced by adding to the extract some distilled essential oil. Such oleoresins can be
hrther used in preparation of different thyme products, which are shown in Figure 8.2.

Extraction methods

Extraction solvents

General principles of the extraction and equipment used for this purpose are compre-
hensively described in various handbooks and manuals on herb and spice processing
(Heath, 1781; Moyler, 1991; Peyron and Richard, 1972; Richard and Loo, 1772;
Reineccius, 1774; Lawrence, 1975). There are several solvents, which are legally
approved for the extraction of aromatic materials. These are tabulated in Table 8.2.
 Thyme -processing of raw plant material 229

Table 8.2 Extraction solvents (IOFI, Europe)

Butane Ethyl acetate

Propane Diethyl ether
Isobutane Dibutyl ether
Toluene Dichloromethane
Cyclohexane Trichloroethylene
Petrol ether Dichlorofluoromethane
Methanol Dichlorodifluoromethane
Butanol- 1 Trichlorofluoromethane
Acetone Dichlorotetrafluoromethane
Ethyl methyl ketone Carbon dioxide

The quality of the extracts and often their composition depends on the solvent
nature, particularly its polarity: the polar solvents better extract polar constituents.
Therefore, possessing the knowledge of the constituents of a plant, it is possible to
predict which components will be extracted under a given set of extraction conditions.
For instance, the main constituents in the essential oil of T. vulgaris are both polar
(thymol, carvacrol) and non-polar (p-cymene, y-terpinene) compounds. Another important
characteristic of the solvent influencing the profile of an extract is its boiling tempera-
ture. So far as the solvent has to be removed from the extract some natural volatiles can
be lost. To avoid losses of volatiles, the choice of the solvent has to be a compromise
between extractive potency and boiling temperature.
Solvent viscosity and its latent heat of evaporation are also important characteristics.
The former affects solvent penetrability into the extracted material, the latter is directly
related to the extraction energy costs. The removal of solvent from the extract is of
ultimate importance due to the following reasons: (a) the residues of most of the
solvents are strictly limited by laws; in the US maximal permitted residue of acetone is
30 ppm, methanol 50 ppm, isopropanol 50 ppm, hexane 25 ppm, and chlorinated
solvents 30ppm (Reineccius, 1994); (b) the residue of the solvent affects the quality of
the extract and has to be minimal; (c) removal of the solvent can cause the loss of more
volatile constituents, which are usually extracted together with less volatile substances.

Extraction at atmospheric pressure

The most widely used extraction process at atmospheric pressure involves the following
unit operations:

preparation of the raw material;

exposure of the material to the solvent;
separation of miscella from the extracted material;
removal of the solvent.

Comminution is the main and most important preparation procedure for herb extraction.
It is necessary to obtain the optimal particle size, sufficient to enable the solvent to
penetrate the mass completely, but not too fine to reduce the rate of penetration. Expos-
ure of the material to the solvent involves three phases: (i) the addition of the solvent
and its penetration into the dry mass; (ii) the achievement of equilibrium; and (iii) the
replacement of the solute with new solvent (Reineccius, 1994). The process can be
carried out on a batch basis (e.g. when small quantities of different materials are handled)
or continuously, when large amounts of unique raw materials are processed. Separation
of miscella from extracted material is a process during which the ground material acts
as its own filter and consequently the clear miscella passes directly to a still or evaporator.
To meet all extract concentration requirements the miscella is usually processed in
two stages: (1) the removal of the main part of the solvent (approximately 95 per cent
of the solvent can be removed in a standard falling-film, raising-film or other type of
evaporator); (2) removal of the rest of the solvent, e.g. by using vacuum treatment.
Besides the conventional extraction procedures some authors proposed to use an
optional measure. For instance, Honerlagen and Steiner (1990) in their patent proposed
to add a drying agent before or after separation of the extract from the exhausted solids, to
eliminate water from the extract. The solvent is then distilled off, to leave the extracted
lipophilic material. Recently some interesting experiments were carried out on the use
of microwaves in the development of extraction processes. The so-called microwave
transparent solvents, which allow all the energy to be absorbed by the plant material
have been used in such experiments with mint, cedar leaves and garlic (Par6 etal., 1991).
The principle of microwave use is that the sudden increase in temperature causes the
cell walls of the essential oil glands to rupture and release their oil to the solvent. Spiro
and Chen (1995) examined the kinetics of this process and found that under the severe
thermal stress the oil glands of peppermint not only ruptured but also totally disinte-
grated into aggregates of powdery fragments.

High-pressure extraction

Using modern high-pressure extraction techniques can successfully solve the main
problems of the conventional extraction. A great number of low boiling temperature
solvents can be used for this purpose, however, carbon dioxide (CO,), is the most suitable
material in various food applications. All dry botanicals with an oil or resin content can
be extracted with CO,. This pressured solvent behaves during extraction in a similar
way to any of the other solvents. As a solvent it has some significant advantages compared
to alternatives.
Commercially, C 0 2 can be used in two distinct modes of extraction, which are
dependent on its operation above or below the critical point in the phase diagram for
CO,. From this point of view, C 0 2 as a solvent can be used for the extraction in sub-
critical and supercritical states (PC> 73.8 bar, Tc > 31.3 OC). The main advantages of
CO, are the following (Moyler, 1991; Lawrence, 1995):

* odourless, colourless, tasteless and non-toxic;

* non-combustible;
inexpensive and readily available;
easily removed leaving no solvent residue;
because of a low viscosity it can readily penetrate comminuted dry plant material;
* by varying the temperature and pressure it can be used in a more selective manner.

However, wider commercial application of the supercritical C0,-extraction is limited

by economical reasons. The capital costs of the equipment are still rather high and the
process can be used only for the high-added value products, e.g. hops and coffee.
 Thyme -processing of raw plant material 23 1

Table 8.3 Solub~lityof botanical components in l ~ q u i dCO,

Very soluble Spa~inglysolable Al7nost insolable

Low M W aliphatic hydrocarbons, Higher M W aliphatic hydrocar- Sugars, protein, polyphenols,

carbonyls, esters, ethers bons, esters, etc.; substituted terpe- waxes, inorgan~csalts; high
(e.g. 1,8-cineole), alcohols nes and sesquiterpenes; carboxylic M W compounds, e.g.
monoterpenes, sesquiterpenes acids and polar N and SH com- chlorophyl, carotenoids,
pounds; saturated lipids up to C12 unsaturated and higher than
C12 lipids
M W up to 250 M W up to 400 M W above 400

Note
MW; Molecular weight.
Source: Moyler, 1987.

A comparison with traditional forms of extraction shows that CO, is a versatile

solvent. By using traditional isolation procedures we can obtain either essential oil
(distillation) or oleotesin (solvent extraction). By using C02-extraction we obtain oleo-
resin which can be fractionated into essential oil and resin.
Using extraction conditions of 50-80 bar pressure and 0 to + 10 OC, it is commer-
cially viable to extract essential oils as an alternative to steam distillation. The energy
savings of C 0 2 offset some of the capital expenditure of the extraction equipment. The
solubility of natural compounds in liquid C 0 2 are provided in Table 8.3 (Moyler,
1987). In order to increase the yield of the extracts, sometimes C 0 2 is used together with
some entraining solvent. For T. vulgaris, Calame and Steiner (1987) used supercritical
conditions with hexane as entrainer at 150 bar and 40C with subsequent subcritical
fractionation at 50 bar and 9 OC to obtain a similar yield of 2 per cent to that of steam
distillation. Thyme leaf was extracted by supercritical C 0 2 and ethanol and the reported
yield was 2.1 per cent (Moyler, 1993). Oszagyan etal. (1996) carried out supercritical
fluid extraction (SFE) of T. vulgaris under different extraction conditions. A stepwise
increase of the extraction pressure resulted in the fractionation of the extracts into liquid
and pasty products. SFE of thyme gave a product which contained 10-1 5 per cent thymol
and 30-35 per cent carvacrol, while steam distillation produced an oil containing 48-50
per cent thymol and only 8-10 per cent carvacrol.

Characterisation of different thyme extracts

The quality of the extracts depends on various factors as previously pointed out. This
can be illustrated by some experiments targeted on the investigation of different prop-
erties of thyme extracts. In early 1952 Chipault etal. assessed the antioxidant activity of
different herb and spice products and found that the antioxidant index of a thyme
fraction soluble in petroleum ether was two times lower than the antioxidant index of the
fraction soluble in alcohol (Table 8.4). Somewhat similar results were obtained with
other herbs, excluding savoury.
T. vzllgaris was extracted by C 0 2 in several studies with different purposes. Cardoso
etal. (1993) compared supercritical fluid carbon dioxide (SFC) extraction to steam and
hydrodistiliation in a Clevenget type apparatus. In thyme extracts, higher production
yields were always obtained by the conventional distillation method. Using SFC extraction
high yields were obtained but the extracts included other kinds of compounds. With
2 32 Petras R. Venskutonis

Table 8.4 Antioxidant properties of ground spices and of petroleum ether and alcohol-soluble fractions

Spice Antioxidant index determined by active oxygen method at 78.6 OC, employing as substrate
prime steam lard with a stability of 6.5 h

Ground spice Petroleum ether-solublefrdctivn Alcohol-soluble frdction

Thyme 3.0 1.5

Rosemary 17.6 2.2
Sage 16.5 2.2
Oregano 3.8 1.4
Savory 1.6 1.3

Source: Chipault etal., 1952.

Table 8.5 Yields of the isolates obtained from dried thyme and their antioxidant activity as evaluated by
the ,&Carotene Bleaching Test

Type of the extract Yield, g/kg Antioxidant activity,

dry matter 0 (low)/5 (high = BHT)

Essential oil
Deodorised acetone extract
Deodorised water extract
Acetone oleoresin 35.0
Methanol-water extract 73.9
CO, extract (300 bar, 40 OC, 3 min) 54.6

Source: Dapkevitius et al., 1998

respect to chemical composition, steam and hydrodistilled extracts showed similar

profiles. Supercritical extracts presented besides the same components as that of steam
distillation, non-volatile and non-aromatic compounds. Thymol was extracted at similar
levels by all the tested methods. Howevet,p-cymene, which was present in a high content
on the other extraction procedures (11 per cent for Clevenger, 5.6 per cent for steam
distillation and 7.2% for 20MPa) was almost absent in the SFC extract obtained at
l0MPa (0.07 per cent) and was very low in the product obtained at 15 MPa (1.5 per cent).
In general, the choice of the extraction method depends on the composition and charac-
teristics of the required products.
In another study T. vulgaris extracts were prepared by different methods to test their
antioxidant properties (Dapkevicius etal., 1998). The results obtained are summarised
in Table 8.5. The yield of the extract obtained by C 0 2 at 300 bar, 40C, 5 min was
54.6glkg and it possessed significant antioxidant activity. The experiments were further
expanded separately with thyme stems and leaves by using 120 and 450 bar pressure.
The antioxidant activity of the extracts obtained at different pressures was similar;
however, the concentration of active substances in the stems was considerably lower
than in the leaves (Figure 8.3).
A significant effect of the extraction conditions on the quality of extracts is clearly dem-
onstrated in Figure 8.4. By selecting the solvent and optimising the extraction procedures,
it is possible to isolate the largest amount of the substances of interest. For instance, by
 Thyme -processing of raw plant material 23 3

Im I20 atm
8 450 atm

Stems Leaves and

blossoms

Figure 8.3 Antioxidant activity of CO, extracts from different parts of thyme

Figure 8.4 Antioxidant activity and y~eldsof thyme extracts.

Lith.th., Tbymnus vulgaris grown in Lithuania; Dut.th., Thymz~svulgarzs grown in The Netherlands; AO, acetone
oleoresin; sh., extracted by shaking dried plant material with solvent; soxl., extracted in the Soxhlet apparatus;
MWE, methanol-water extract; DAO, deodorised acetone oleoresin; AO(C02),A 0 after C 0 2 extraction.

using methanol-water it was possible to obtain the highest extract yield although the anti-
oxidant activity of the extract was very low (about five times lower than the antioxidant
activity of butylated hydroxytoluene (BHT)). The yields of acetone extracts obtained in a
Soxhlet apparatus were lower, however their antioxidant activity was comparable to that of
BHT.
Nguyen etal. (1991) patented a method for extracting antioxidants from Labiate herbs,
which settles the following extraction and fractionation parameters for T. vulgaris: extractor
234 Petras R. Venskzttonis
500 bar195 OC; 1st separator 120 bar180 OC; 2nd separator 35 bar11 5 OC. These conditions
being applied, the yield of the essential oil fraction was 0.7 per cent while the yield of
the antioxidant fraction was 2.0 per cent.

INFLUENCE OF PROCESSING PROCEDURES O N THE QUALITY

OF THYME AND THYME PRODUCTS

Processing procedures are known to change some characteristics of herbs and spices.
This problem must be mainly focused on

(a) the essential oil content and its chemical composition and consequently the sensory
profile of the product aroma;
(b) the composition of other (non-volatile) constituents and consequently the properties
as regards taste and nutritional value;
(c) the structure of natural pigments and consequently the colour of the product;
(d) microbial contamination.

Usually the flavour impact of the freshly cut herb is appreciably higher and of a different
character from that of its dried counterpart. This is due to the loss or modification of
the low boiling fractions of the oil. The clean characteristic top notes associated with
the freshly cut green herb are, in the dried material, overlaid with a dull hay-like aroma
(Heath and Reineccius, 1986). Drying can be carried out by different methods, which
were in general described in Chapter 7. Literature data indicate that the changes of
aroma compounds during drying depend on the drying method as well as on the character
of the herbs and spices.
The treatments of foods with ionising radiation to reduce bacterial counts and thus
to prevent the spread of food-borne diseases and to improve the shelf life of the food
itself or the processed products has become a matter of much greater importance in the
past years. Results on the consequences of irradiation are available for about 50 different
spices. Some of these have been examined several times by different authors (Schiittler
etal., 1991). Most of the papers concerning irradiation of herbs and spices deal with
bacteriological decontamination, shelf life and detection of irradiation. So far as irradi-
ation is a concern of food legislation, the methods of the detection of irradiated herbs
and spices is also briefly outlined in the present section.

Effect of drying o n t h e composition of volatile c o m p o u n d s

Dehydration is still a very important process for the preservation of spices and aromatic
herbs. A great number of studies have been carried out on the effect of drying methods
and parameters on the quality of spices and aromatic plants, particularly focused on
Labiatae family herbs. The publications on the quality of thyme itself are not very
numerous, at least in the international journals and other available sources. Therefore,
some results on the effect of drying on the quality of other, particularly Labiatae family
herbs have also been included in this section. Considering biological similarities
between Labiatae plants this seems to be reasonable.
In general, two main approaches can be used in the assessment of volatile constituents
in herbs and spices as well as in other foods. These are: (1) the analysis of the total
 Thyme processing of raw plant nzaterial 23 5

Table 8.6 The content of essential oil, thyrnol and carvacrol in dried thyme

Characteristia Freeze-dried Oven-drzed

Essential oil (pllg) 15.2

Thyrnol (rngig) 9.6
Carvacrol (mglg) 0.5

Source: Bendl etal., 1988.

concentration of aroma compounds in the product matrix (essential oil in case of

thyme); (2) analysis of the headspace volatiles, which are above the matrix and which
are in close relation with the dynamics of release of volatiles (i.e. equilibrium between
aroma compounds in the matrix and above it) and consequently with the sensory profile
of the product. Therefore, the method of the analysis of volatile constituents is quite
important. Comparing several methods for the isolation of volatile compounds from
aqueous model systems Leathy and Reineccius (1984) concluded that headspace is very
dependent on the volatility of aroma compounds, whereas simultaneous distillation1
solvent extraction (SDE) exhibited reproducible recoveries.
Different methods of isolation for the investigation of the effect of drying on the
aroma changes in herbs and vegetables were applied: extraction (Huopalahti etal.,
1985, Nykanen and Nykanen, 1987); SDE (Kaminski etal., 1986; Kirsi etal., 1989);
headspace method (Koller, 1988). The latter enables one to determine the changes in
vapour phase, which can be much more related to the sensory aroma profile of the prod-
uct. The differences in the percentage composition of volatile compounds in the sam-
ples obtained by different methods could be significative as it is demonstrated by
Huopalahti etal. (1988) in the case of dill.
In general, headspace samples are dominated by the more volatile components,
steam distilled concentrates additionally contain some higher boiling compounds,
whereas extracts consist of both volatile and non-volatile fractions. Chialva etal. (1982),
Venskutonis and DapkeviFius (1995) compared the composition of the headspace over
fresh herbs with that of steam distilled essential oil and observed significant differ-
ences, e.g. some very volatile components were present in headspace and absent in the
essential oil. Jennings and Filsoof (1977) conclude that no single sampling system can
be regarded as uniformly satisfactory, but that, depending on the sample and what the
investigator wishes to study, one or another system may be superior.

Oil yield and oil composition

Oil yield and composition are the most important parameters defining flavour proper-
ties of a particular oil-bearing plant. Drying is the most critical process due to the vola-
tility and susceptibility to chemical change of the contained volatile oil. Several
important studies have been carried out to determine the effects of drying methods and
parameters on the volatile oil content of thyme.
Bendl etal. (1988) studied the effect of freeze-drying of sage and thyme on the con-
tent of essential oils and their characteristic flavour components in comparison to dry-
ing at increased temperature (40C). The examination showed (Table 8.6) that the
content of essential oil in the spice samples was higher in the freeze-dried products
236 Petras R. Venskutonzs

Table 8.7 Changes in volatile oil yield* with increasing drying temperature

Plant Temperature (OC)

Thyme
Savory
Basil
Marjoram
Rosemary
Sage
Tarragon

Note
" Percentage of volatile oil expressed as yield (vlw) dried plant matter.
Source: Deans et al., 1991.

Table 8.8 Comparison of the major peaks from G C analysis after warm air oven
and microwave-drying (%)

Constituent Oven-dried Microwave-dried

Thyme Savory Thyme Savory

Thymol 47.77 - 18.42 -

y-Terpinene 16.77 10.40 12.06 19.90
p-Cymene 11.91 22.05 3.04 0.00
Carvacrol - 37.61 - 66.66
New peaks - - 20.25; 6.66; 6.62 -

Source: Deans etal., 1991

compared to those dried at increased temperature. The concentration of the most

important components for aroma of thyme, thymol and carvacrol was also higher in the
freeze-dried herb.
Deans etal. (1991) studied T. vulgaris and six other culinary herbs dried by warm-air
and microwave ovens. The volatile oil content (Table 8.7) of seven plant species was
determined by Gas Chromatography (GC) following drying at temperatures from
40-100 "C, revealing that at temperatures >GO OC, most of the volatile constituents
were lost. The yield of volatile oils was substantially decreased in all microwaved herbs.
The qualitative and quantitative changes in the volatile oil profiles were profound. The
results on the main constituents of thyme and savoury, which are similar herbs in terms
of chemical composition, are presented in Table 8.8.
It is interesting to note that the percentage of thymol in thyme significantly
decreased after microwaving (most likely, due to the formation of new compounds),
whereas the content of carvacrol, a thymol isomer, in savoury considerably increased
(most likely due to the loss of more volatile constituents). Rather controversial results
were obtained with two other common compounds both for thyme and savoury,
p-cymene and y-terpinene. For instance, contrary to thyme, p-cymene was not detected
in microwaved savoury (in the oven-dried herb it constituted 22.05 per cent), whereas
the percentage of 7-terpinene was almost two times higher in the essential oil distilled
 Thyme -processing of raw plant nzaterial 23 7

Table 8.9 Effect of drying on the Indian thyme essential oil content (% on moisture free basis) and its
relative concentration (%)

Constituents Fresh Freeze Cross flow Throughflow Shade

Monoterpene hydrocarbons 29.3 29.6 29.6 23.9 0.1

Oxygenated compounds 2.9 2.4 2.4 2.7 0.0
Thymol 60.1 59.5 60.5 64.9 83.5
Sesquiterpenes 3.5 2.8 2.0 3.2 9.5
Essential oil 1.54 0.95 0.90 1.10 0.37

Source: Raghavan et dl., 1995.

Table 8.10 Content of some volatile compounds in fresh, air-dried and freeze-dried thyme (mgikg)

Constituent Fresh Air-dried Freeze-dried

Myrcene
a-Terpinene
y-Terpinene
Thymol
P-Caryophyllene

Note
a-b values with same letter w ~ t h i nsame row are not significantly different
Source: Venskutonis eta/., 1996.

from microwaved savoury than from the oven-dried herb. The results obtained by
Deans and co-authors clearly show that the behaviour of particular plants even belong-
ing to the same family (Labiatae in this case) can be significantly different.
Raghavan etal. (1995) compared the effect of cross flow drying, through flow drying,
freeze-drying and shade drying on the Indian thyme (T. vulgaris) essential oil content
and its composition. At temperatures of 50 and 6 0 OC losses from 50-75 per cent were
registered, therefore these temperatures proved not to be suitable. The results obtained
in this study, which are summarised in Table 8.9, also show that drying in the shade
was very ineffective and long (120 h). Other methods were comparable and, considering
the time of drying and the flavour quality of the dried herb, the authors concluded that
flow drying (40C, 8.5 h) should be the method of choice.
Venskutonis etal. (1996) studied the effect of air and freeze-drying on the content of
volatiles and their composition in thyme. Air-drying was carried out at 30 OC and air
velocity of approx. 3.3mIs for 25 h. The final moisture content of the air-dried herb
was 8.5 per cent. Freeze-drying was completed in 4 0 h with the final moisture content
of 5.5 per cent. Volatile constituents were isolated by simultaneous SDE procedure in
a Likens-Nickerson apparatus.
- - The reduction in the total content of volatile constituents
after drying was of approx. 1-3 per cent and no differences between the two drying
methods were found. This is less than for basil and marjoram and approximately the
same for wild marjoram (air-drying, room temperature, Nykiinen and Nykanen, 1987).
In Table 8.10 the content of some compounds are expressed that underwent more
considerable changes, from fresh to air-dried and freeze-dried thyme. In general, the
levels slightly decreased during drying, except for P-caryophyllene and thymol, which
238 Petras R. Venskzltonzs

0 JI I I
I,
Fresh 30 "C 60 "C Freeze-dried

Fresh 30 "C 60 "C Freeze-dried

Figure 8.5 Changes of the total content of aroma constituents in thyme during drying, in arbitrary
units (a.u.). (a) Simultaneous distillationiextraction (SDE), (b) headspace (HS); (Venskuto-
nis, 1997).

increased (although statistical evaluation did not give significant differences for the latter
compound in fresh and dried samples).
In another study, thyme was dried in the oven at temperatures of 30C and 60C
and in the freeze-dryer (Venskutonis, 1995, 1997; Venskutonis etal., 1996). The
changes of the total amount of SDE volatiles are demonstrated in Figure 8.5a. Very
close concentrations of volatiles were determined in fresh and oven-dried at 30 OC herb.
However, the reduction of the total amount of SDE compounds in oven dried at 6 0 OC
herb was 43 per cent. It is worth mentioning that the weight of thyme during 4 h
 Thyme - proce~singof r a w p l a n t material 23 9

Table 8.1I Composition of thyme essential oil extract (SDE) and headspace volatiles (HS), arbitrary units

Conzpound or SDE HS
retepztion tinze
Fresh Dried herb Freeze Fresh Driedherl. Freeze
herb herb
30C 60C 30C 60C

11:50
14:46
E-2-hexenal
a-Thujene
a-Pinene
Camphene
1-Octen-3-01
P-Pinene
Myrcene
a-Terpinene
p-Cymene
Limonene
1,8-Cineole
y-Terpinene
tr-Sabinene hydrate
Linalool
Isoborneol
4-Terpineol
Thymol
Carvacrol
P-Caryophyllene
Total

Source: Venskuton~s,1997

drying at 60C reduced 3.4 times. Koller and Raghavan (1995) obtained very close
results with rosemary: 30 per cent of the essential oil was lost during air convection drying
at 50C. The total amount of SDE volatiles in the freeze-dried thyme even increased
approximately by 20 per cent. Considerable increase in the content of the major
compound thymol (by 33 per cent) was the main contribution to the total increase.
Some interesting observations were made concerning the changes of individual flavour
constituents (Table 8.11). Most of the thyme SDE volatiles during oven-drying at
30 OC and freeze-drying did not undergo significant changes. Their reduction during
oven-drying at 60 OC depended on the volatility and chemical origin of the constituent.
For instance, the concentration of the quantitatively major compoundsp-cymene, y-terpinene
and thymol were reduced by 2.24, 2.57 and 1.56 times respectively. The amount of
P-caryophyllene in the oven-dried at 30 OC and freeze-dried thyme was found to have
increased by 29 and 37 per cent respectively. Very close results were obtained in a
previous study (Venskutonis etal., 1996). One more tendency in the changes of vola-
tiles is evident from the results obtained: the losses of non-oxygenated terpenes during
oven-drying at 60 OC were considerably higher than that of oxygenated compounds,
particularly terpene alcohols. Most likely, two reasons could be responsible for this
tendency: the differences in the volatility and the formation of oxygenated compounds
during drying.
 Fresh

Freeze-drying

Thyme Sage

Figure 8.6 Influence of drying temperature on the composition of the headspace gas over thyme and
sage (Koller, 1988).

Headspace ( H S ) constituents

Influences of drying methods and temperatures on the composition of the HS of thyme

and sage were analysed by Koller (1988). The so-called histograms (the result of
gas-chromatographic "odour" tests) presented in Figure 8.6 showed that the profile of
volatile compounds changed even by freeze-drying. However, freeze-drying and air-drying
at 50 "C were comparable, while air-drying at 8 0 "C was not suitable for both herbs due
to loss of volatile constituents.
Later some new results of HS analysis were obtained, which also revealed some inter-
esting peculiarities of the influence of drying on the aroma constituents in thyme, Table
8.11, Figure 8.5b (Venskutonis, 1997). The total amount of the collected volatiles in
thyme herb HS was the highest in the case of thyme oven-dried at 6OCC:it was 4.2 times
higher than in the fresh one, 19.4 times higher than that oven-dried at 30C, and 12.9
times higher than the freeze-dried one. It was obvious that the intensity of sensory odour
perception of the thyme herb oven-dried at 60cC was evidently stronger than that at
30 OC.
 Thyme -processing of raw plant material 24 1
It is interesting to note that similar experiments were carried out with another Labiare
herb, sage, and it was found that the total content of the absorbed compounds on Porapack
during dynamic HS purging of the samples was the largest in the case of fresh herb,
middle in freeze-dried, and lowest in oven-dried. The results were comparable for 30 OC
and 6 0 OC drying temperatures, when the total content of absorbed HS volatiles was
lower in comparison with fresh herb 4.6 and 3.7 times, respectively. It means that the
influence of the drying method on the rate of the release of flavour compounds can be
very particular for each given herb. For instance, the amount of volatiles in HS of fresh,
oven-dried and freeze-dried sage significantly exceeds that of thyme, however, when
the temperature of 6 0 C was applied, the intensity of aroma release from thyme was
"activated" 4.2 times (as compared with fresh herb) in terms of total increase of the
absorbed HS volatiles. It could be supposed that thyme leaves undergo significant
changes in their botanical structure during drying at higher temperatures. From this
point of view, sage leaves could be considered to be more resistant against the effect of
drying at higher temperatures.

Assessment of drying effects on thyme aroma

The comparison of data obtained by simultaneous SDE and HS allows one to assess every
volatile constituent of herb in terms of its total content (m) and the rate of its release
(v). The latter can be related to the concentration of a particular constituent in HS,
which depends on the morphological and anatomical characteristics of the structures
containing the essential oil in the plant. In this case a certain aroma potential (AP) of
every particular volatile constituent can be considered as a function of these two param-
eters and odour threshold value (c) (Venskutonis, 1997):

Certainly, such a function is rather conditional and depends on the parameters of SDE
and HS analysis. However, by using standardised conditions it is possible to have some
mathematical tool, representing a certain aroma potential of a particular volatile con-
stituent in aromatic herb.
In the case of thyme it is demonstrated that the major constituent thymol in SDE
concentrate constitutes 42-48 per cent, while in HS only 2.5-4.8 per cent (Figure 8.7).
Such volatile compounds as p-cymene, y-terpinene, and myrcene prevail in HS vapours
of thyme. The ratio of the percentages of SDEIHS characterises as a certain coefficient of
efficiency (C,) of a particular constituent in aromatic herb. To some extent, it represents the
activity of the participation of such a compound in the creation of the odour. In Table
8.12, percentage concentrations of some major thyme volatile constituents and their C,
coefficients are tabulated. It is interesting to notice that for some similar compounds
these coefficients are different in thyme and sage. For instance, C, of P-caryophyllene in
fresh thyme is 2.8 times higher than in fresh sage.
C, coefficients were also calculated for dried herb. The figures obtained can be informa-
tive for the evaluation of the degree of disbalance of the fresh aroma during drying. For
some constituents of thyme the changes of C, after drying are represented in Figure 8.8.
The diagrams show that the changes of C, depend on the chemical origin of the constit-
uent. For instance, C, of linalool significantly increased after oven-drying at 60 OC,
while that of P-caryophyllene was reduced several fold.
 Fresh Oven diced at 30 'C
mvrcene 3 4 mvicene. 3 7

gamma-teipinene. I5 I
Rest, 26 4

bets curyophylleile. 2 I
Thymol. 2 8
alpha-lhujene.2 2

Fzgzlre 8 . 7 Changes of the percentage content of the main SDE and HS constituents in thyme during
drying (Venskutonis, 1997).

Effect of drying on colour

The phenomenon of colour changes during drying is quite common for most of the
leafy plants, because the chlorophylls define the colour of green herbs and vegetables.
They are sensitive to many factors, which cause the shift of maximum absorbance and
therefore their natural green colour changes to less desirable colours. Therefore when
 Thynze - processing of vazu plant materzal 243

Tabb 8.12 Percentage content of volatile compounds and their

coefficients of efficiency (C,) in fresh thyme

Covzpou?zd SDE HS Cc

Camphene
,O-Pinene
Myrcene
1,8-Clneole
7-Terp~nene
p-Cymene
Linalool
Thymol
,8-Caryophyllene

Source: Venskutonis. 1997

T I Ki Fresh I
2.5

2 H Freeze-dried
.-
0
$ 1.5
0

i;' 1

0.5

Figure 8.8 Changes of the coefficients of aroma efficiency (C,) of some thyme volatile constituents
during drying (Venskutonis, 1997).

optimising the drying process the parameters should be kept to minimise both the
losses of volatile compounds and the change of a natural green colour.
Analyses concerning colour in Thyvzw are very scanty, however, there are some studies
dealing with colour changes in Labiate herbs. So far as the process of discolouration andlor
colour changes during drying can possess similarities between various aromatic plants, it
was considered reasonable to provide a few examples of the relevant investigations.
Takruri and Daqqaq (1984, 1986) studied the effects of storage and the methods of
drying on the carotene values in mint, Jew's mallow, thyme, and parsley. They found
that a range of 47-92 per cent of the carotenoid content was retained in these plants
when dried by the traditional methods of sun-drying and shadow-drying. In addition,
244 Petras R. Venskutonis

Table 8.13 Effect of drying on weight loss and microbiological quality of thyme

Treatnzent* Weight loss PCA Media V R B A BCSA B-PA

Initial microflora 1.62 x lo5 4 . 0 lo2

~ 2.0 x 10' < 10
After 48 h of drying oven at 38 OC 68.2 1 . 0 5 ~ 1 0 ~ 1 <10 110
After 5 min microwave-drying 46.5 7.35 x 10"l 1.0~10' <I0
After 7.5 min microwave-drying 52.9 6 . 1 0 ~ 1 0 ~ <1 1.0~10' <lo

Notes
:': Counts per gram fresh rnarer~al;

PCA, Plate Count Agar.

VRBA, Violet Red Bile agar for coliforms.
BCSA, B a n l l z ~ jrereits select~veagar for Bailll//i i-ereus.
R-PA, Baird-Parker agar for Staphylo~uic/liaxrezu.
Source: Deans et d l . , 1991.

44-69 per cent of the carotenoid values in the dried plants were detected after storage
for 1 year. The drying method and the drying temperature had varying effects on the
carotenoid value. Thus in mint, the percentage recoveries using shadow-drying,
sun-drying, and oven-drying at 40 OC, 6 0 OC and 100 OC were respectively 79 per cent,
76 per cent, 71 per cent, 74 per cent and 54 per cent. For the oven-dried samples,
increasing the drying time or the drying temperature over 10O0C resulted in greater
losses in the carotenoid content. It is apparent, therefore, that these plants dried by
traditional means, or in ovens at temperatures not exceeding 100 OC, remain good sources
of carotenes even after one year storage.
Miiller etal. (1989) studied drying of medicinal and spice plants with solar energy in
a plastic film covered greenhouse. In their experiments with mint, sage and hops, they
found that solar drying was much superior to conventional drying with regard to
colour, texture and contents of active ingredients.
The process of rapid heating and cooling of herbs and spices developed by Hosokawa
Micron Europe BV minimise loss of colour and essential oil. Special equipment for this
process has been developed (Spook, 1993; Anonymous, 1993).
Rocha etal. (1993) used steam blanching and surfactant pretreatment to increase
drying rate of basil and found that both pretreatments resulted in better retention of
the green coloration of the leaves. Steam blanching was shown to enhance chlorophyll
retention. Low air-drying temperatures were needed for samples that were not pretreated,
while high air temperatures were acceptable for pretreated samples.

Effect of drying on microbial contamination

Usually herbs and spices are heavily contaminated with microorganisms. Microbio-
logical quality also depends on drying method and conditions. Deans etal. (1991)
studied the microbiological quality of the raw and dried material of T. vulgaris and
six other culinary herbs dried by warm air and microwave ovens. The authors deter-
mined total bacterial counts, Staphylococcus aureus, Bacillus cereus and coliforms. Expo-
sure of herbs to microwaves was evaluated as a method of both drying and reducing
the microbial load present on the plants. The microflora was reduced by two or three
logarithmic cycles (Table 8.13).
 Thyme processing of raw plant nzaterial 245
Influence of irradiation processes
The use of ionising radiation has been extensively studied over the past 30 years as a
mean of sterilising spices. In all cases, microbial populations have been reduced (Eiss,
1984). The reduction of the microbial population both after irradiation and during storage
of irradiated herbs and spices is the most important task in post-processing treatment.
The studies have resulted in the establishment of optimal irradiation doses for various
herbs and spices. Ten kGy has been found to be effective in destroying bacterial spores,
while 5 kGy has been sufficient to eliminate mould contamination (Munasiri etal., 1987).
The treatment with 5-7.5 kGy was proposed as a sufficient dose for decontaminating
thyme (Farkas, 1988). Comparing irradiation with other sterilisation methods, e.g.
Vajdi and Pereira (1973), JosimoviC and JovanoviC (1982) found out that y-irradiation
was more effective than ethylene oxide in reducing the bacterial population of spices
(pepper, paprika, oregano, allspice, celery seeds, garlic, marjoram, cardamon, caraway and
parsley).
The microbiological data of some non-irradiated and irradiated herbs and spices are
provided in Table 8.14. The dose of 10 kGy reduced standard plate count in thyme by
3750 times, moulds more than 30 times and completely destroyed maximum probable
number (MPN) coliforms. Very good effects were achieved with other treated herbs and
spices as well.
In foods irradiation can result in the creation of new chemicals, called unique radi-
olytic products. These include known carcinogens like benzene, formaldehyde and
certain peroxides. However, the FDA has concluded that the amount of any toxic

Table 8.14 Microbiological data: irradiated splces

Spice Dose kGy Standard Yeast Mould M P N coliJoruzs

plate count

Thyme 0 150.000 0 300 30

10 40 0 <10 0
Allspice 0 2.278.000 < 10 0 0
10 < 10 < 10 0 0
Greek oregano 0 1214400 40000 9000 2636
10 <10 < 10 < 10 0
Black pepper 0 32.000.000 0 0 25
10 60 0 0 0
Garlic powder 0 414.000 < 10 7800 0
10 700 <10 < 10 0
Egyptian basil 0 3.000.000 >30.000 400 > 11.000
10 1000 <10 <10 0
Mexican oregano 0 1.500.000 >30.000 10.000 5000
10 30 < 10 <10 0
Domestic paprika 0 1.000.000 0 0 0
10 100 0 0 0
Spanish paprika 0 2.200.000 0 0 410
10 260 0 0 0
Celery seed 0 440.000 1500 200 25.000
10 < 10 < 10 < 10 0
Crushed red pepper 0 130.800 < 10 0 0
6.5 < 10 < 10 0 0

Source: Eiss, 1984.

246 Petras R. Venskztonzs
chemical created by allowable levels of irradiation is too small to be significant; foods
cannot be tested to determine that the proper amount of radiation was used. Strictly
from the scientific point of view, no ceiling should be set for food irradiated with doses
greater than the currently recommended upper level of 10 kGy by the Codex Alimenta-
rius Commission. The food irradiation technology itself is safe to such a degree that as
long as sensory qualities of food are retained and harmful microorganisms are destroyed,
the actual amount of ionising radiation applied is of secondary consideration.
In the case of irradiation of herbs and spices, this need for a greater average dose has
already been recognised in several countries. France permits an average dose of 11 kGy
for the irradiation of spices and dry aromatic substances, whereas Argentina and the US
permit a maximum dose of 30 kGy for this purpose.
The radiation effects on biological material are ascribed to the sum of two processes,
direct (chemical events occurring in the target molecule as a result of energy depos-
ition) and indirect (consequence of reactive, diffusible, free radicals formed from the
radiolysis of water: OH-, e,,, H', H2, H 2 0 2 )action. Experiments indicate that herbs
and spices with water contents of 4.5-12 per cent are very resistant to physical or
chemical change when irradiated. Sensory and food applications analyses indicate no
significant difference between irradiated samples and controls for all spices tested
(Eiss, 1984).
Another important point is that sterilisation by ionising radiation is a cold treatment.
Irradiation as high as 50 kGy will only increase the temperature of irradiated food by
12 OC. Therefore, there is little danger of the loss of volatile components of the spice.
Irradiation does not require the addition of any chemicals, liquid or gas. In addition,
no other methods of processing, e.g. heating, freezing or drying need to be employed
(Eiss, 1984).
The effect of irradiation on the sensory profile and chemical composition of herb and
spice essential oils has been thoroughly examined. Thyme was also selected as a testing
material in some studies. Most of them show that for the microbial decontamination
required, irradiation doses do not affect total essential oil content and the sensory profile
of the herb. For instance, marked reduction in bacterial count with no deterioration in
organoleptic quality of thyme, coriander and paprika was determined after irradiation
with 0-10 kGy by Van Dijk in 1970. Some workers compared the effect of irradiation
with that of ethylene oxide. The investigations on the effect of irradiation on some
widely used Labiate herbs, including thyme, are provided in Table 8.15 (Farkas, 1988).
The effect of irradiation on the chemical composition of herbs and spices has been
investigated by using different methods. Venskutonis (Venskutonis, 1994; Venskutonis
etal., 1996) studied the effect of 3, 10 and 30 kGy 7 and 0-irradiation on the chemical
constituents of the oven and freeze-dried thyme isolated by a simultaneous SDE proce-
dure in a Likens-Nickerson apparatus and analysed by capillary GC and GClmass-spec-
trometric (MS) methods. The quantitative content of the main constituents in air-dried
herb before and after irradiation at different doses is tabulated in Table 8.16. It was
shown that the concentration of y-terpinene has increased after P-irradiation. The
tendency for reduction ofp-cymene after 30 kGy for both types of irradiation was also
observed. This tendency was also found for the freeze-dried samples. Statistical analysis
of the results showed that irradiation did not have any significant effect on the concen-
trations of the thyme compounds, except for y-terpinene.
The lack or small effect of irradiation on the thyme aroma compounds is in agreement
with other works. For instance, irradiation of spices including Spanish thyme with a
Table 8.15 Dose requirements for radiation decontamination of thyme and some other Lab~araeherbs as
compared to the retention of t h e ~ rvolatile oil content, and threshold doses of organolept~c
changes

Held Dose reqr~irement Rebtive yield of volatzle Threshold dme of

(~GY) oils':' a t 8-10 kGy (%) organoleptic changes (kGy)

Thyme 5-7.5 101 210

Bas11 4-10 99 -12.5
marjoram 7.5-10 100-103 5-10 but also > 16
Oregano 54 99-100 >10
Sage 4 10
Savory <5

Note
'k As percentage of the y ~ e l dof untreated sample
Source: Farkas, 1988.

Table 8.16 Concentrations of the main volatile constituents In air dried thyme before and after irradia-
tion (mglkg), none of the results were significant, except for -;-terpinene

Constituent y 3kGy y lOkGy y 30kGy P3kGy PIOkGy PjOkGy

a-Thujene
a-Pinene
Myrcene
a-Terpinene
p-Cymene
y-Terpinene
tr-Sabinene hydrate
Linalool
Borneo1
Thymol
Carvacrol
8-Caryophyllene
Caryophyllene oxide

Source: Venskutonis et al., 1996.

dose up to 50 kGy provided no evidence of noticeable changes of volatile compounds

(Eiss, 1984). Bug (1989) applied 29 kGy doses to different herbs including thyme and
also did not find any effect on the quantitative and qualitative composition of its
essential oil.

Detection of irradiated herbs and spices

As far as the treatment of food with ionising energy is finally becoming reality there is
a need of a reliable detection method of irradiated foodstuffs. Usually, before the radiation
treatment of spices and herbs can be undertaken on a commercial basis, the respective
health authority or regulatory body must give written approval. As the process of food
irradiation produces practically no change in appearance, shape or temperature of products,
it is controlled mainly by administrative means through requirements for documentary
248 Petras R. Venskutonis
records and labelling. Therefore, there is an interest to supplement administrative
control by developing identification methods for irradiated foods (Farkas, 1992). The
regulations concerning irradiated foods are very different even between European
countries (IAEA, 1988). This has led to increasing interest in methods for detecting
prior irradiation of foods. A current problem with the use of irradiation is the lack of
a specific method for identifying foods that have been irradiated.
DelincCe (1998) recently reviewed significant progress, with the development of
analytical detection methods using changes in food with an origin as the radiation
treatment. Five detection methods (electron spin resonance, thermoluminescence, lipid
hydrocarbons, 0-tyrosine and microbiological analysis) have been developed to detect
a large variety of irradiated foods and their reliability has been confirmed through a
series of collaborative trials.
Some studies have been carried out in establishing special detection methods for
irradiated herbs and spices. Goksu (Goksu and Regulla, 1989; Goksu etal., 1990)
showed that most of the natural products contain minute amounts of wind blown or
intruding dust, which can be separated and used to identify irradiated spices by measuring
its thermoluminescence (TL).
Thermoluminescent dust obtained from herbs and spices, including thyme, were
investigated and it was concluded that for reliable identification of irradiated spices by
TL, it is essential that adhering inorganic dust is used for measurements. It has advan-
tages over the use of whole spices. Dust which is inorganic gives no TL due to incandes-
cence and can be heated up to at least 400C. At this temperature more stable TL
peaks are accessible. Thus, the identification and quantitative absorbed dose assessment of
irradiated spices is possible even after some years.
Sjoberg etal. (1990) tested three types of methods for the identification of irradiated
spices as potential control methods: a) a microbiological, combining a direct epifluorescent
filter technique (DEFT) with a total aerobic plate count (APC), b) a chemoluminescence
method and c) chemical GC and GCIMS methods for the analysis of volatile oils. The best
methods for control purposes were the microbiological (DEFT+APC) methods combined
with chemoluminescence measurements. No differences were detected between the
irradiated and non-irradiated samples with the chemical methods.

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9 The genus Thymus as a source
of commercial products
Brian M. Lawrence and Arthur 0. Tucker

INTRODUCTION

The commercial products that are obtained from the genus Thymw include essential oils,
oleoresins, fresh and dried herbs, and landscape plants. The genus Thymw has an esti-
mated 350 species, but only five have achieved any real economic importance (although
not all for the same reasons): Thymus capitatas (L.) Hoffmanns. et Link (classified most
recently as Thymbra qbitata (L.) Cav., Spanish oregano or conehead thyme), T. vzastichina L.
(Spanish marjoram or mastic thyme), T. serpylhnz L. (wild thyme, mother-of-thyme),
T. vulgaris L. (common thyme) and T. zygis L. (Spanish thyme). Although essential oils of
each of these species are items of commerce, thyme oil is mainly obtained from T. zygis,
whereas both T. zygis and T. valgaris are the main sources of the dried and fresh herb.

PRODUCTION STATISTICS OF THYMUS OILS

Thywzz~soils have been used since the 16th century (Gildemeister and Hoffmann, 1990);
however, the data on their production amounts prior to the 1930s could not be found,
although it was probably in the 5-10 ton level for many years. These oils were valued
because of their aroma character and their richness in a specific constituent. For ex-
ample, oils of T. serpylhm, T. vulgaris and T. zygis are typically thymol-rich, T. capitatas oil is
typically carvacrol-rich, and T. nzastichina oil is typically 1,s-cineolellinalool-rich.
In the early part of the twentieth century, thyme oil (ex T. vulgaris) was available
from cultivated plants in Germany and wild plants collected from the mountainous
regions of southern France. As it became less economically viable to cultivate and distil
thyme, harvesting of wild plants became the norm initially in France and then for
T. zygis in Spain.
Spain is the main country of production for thyme oil from T. zygzs. The main Spanish-
producing areas for thyme oil are Almeria, Murcia and Albacete. The crop is harvested
from wild plants from July to mid-September. Spanish oregano oil from T. capitatus is
produced in Huelva and northern Murcia from wild plants harvested between mid-May
and August. Spanish marjoram oil (T. mastichina), which is also harvested from wild
plants between mid-May and August is produced primarily in Murcia and Albacete. In
contrast, wild thyme (T. serpyllum) is produced almost exclusively in Cuenca (Gavifia
Mhgica and Torner Ochoa, 1966). A summary of Spanish thyme oil production since
1930 can be seen in Table 9.1 (Lawrence, 1985; Miralles, 1998). Between 1990
and 1998, the amount of oil produced annually has fluctuated between 35 and 45 tons.
 The genw Thymus aj a source oJ co~~z~nerczaIprodz~~-ts
253

Table 9. I Thyme oil production in Spain (tons)

Year 1930 1935 1940 1945 1947 1958 1955

Amount 12 15 20 25 5 25 14
Year 1960 1965 1970 1975 1980 1985 1990
Amount 14 19 17 22 30 23 25

Table 9.2 Production of oils from other Thyvzzir species in Spain (tons)

Year 1936 1946 1947 1950 1970 1980 1993 1994 1995 1996

Spanish marjoram 13 6 15 25 40 30 20 25
Spanish oregano 20 9 3 5 10 10 24 24 15 2

A limited quantity of thymol-rich thyme oil is produced in France (0.6 tons) annually,
while smaller quantities are sometimes available from Albania, Algeria, Hungary, Israel,
Morocco, Portugal and Yugoslavia.
Wild thyme oil is available only from Spain with its annual production in the 1-3
tons level (Lawrence, 1985). The other two oils that are exclusively produced in Spain
are Spanish marjoram and Spanish oregano oils. A summary of their production statistics
(Miralles, 1998) can be seen in Table 9.2. Because of an interest in uncommon oils in
the aromatherapy trade, a very limited quantity of lemon thyme oil (< 100 kg) which is
obtained from T. x cztriodorus (Pers.) Schreb., has become an item of commerce.
Both red and white thyme oils are available commercially. Authentic thyme oil
distilled in Spain is usually red in color. This color is caused by the reaction between
thymol and the iron in the field stills. White thyme oil is produced from red thyme oil
by re-distillation of the red oil in stainless steel equipment. In this re-distillation or
rectification process there is generally a small loss of the more volatile materials with
a corresponding increase in the thymol content of the oil.
It is estimated that the North American demand for thyme oil is between 18-24 tons.
Because of the increasing availability of synthetic thymol, the natural thymol oil
demand has remained fairly stable for the past decade. It is postulated that the rising
labor costs associated with harvesting and distilling the oil in Spain suggests that
production volumes greater than the current levels are unlikely to increase. Nevertheless,
assuming usage levels remain constant, current producers should be able to meet the
annual oil demands for oils of the commercially important Thymus species.

MISCELLANEOUS USES OF THYME OIL

Natural cosmetics or phyto-cosmetics are one of the fastest growing niche markets in
Europe and North America (Purohit, 1994). Although most of these products were
originally sold in health food stores, they have now found their way into wider distri-
bution channels such as department stores, boutiques, discount stores, salons, etc. and
direct sale through the Internet. Within this category of products, materials can be
found in which the natural essential oils are purported to be the efficacious components
found within them. As a result, thyme oil is used for its antiseptic and aromatherapeutic
properties; however, this use has little impact on the production volumes of the oils.
254 Brian M. Lawvence and Arthur 0. Tucker
OIL SPECIFICATIONS

To determine whether an oil is a pure product, internationally accepted specifications for

the commercially important Thynzw oils have been developed. The main organizations
that have well-recognized specifications are International Organization for Standardiza-
tion (ISO, 1996a,b) TC-54 (Essential Oils Section), Association Fran~aisede Norrnalisa-
tion, French Essential Oils Standards (AFNOR, 1996a,b), the now-defunct Essential Oils
Association of the USA (EOA)/the Fragrance Materials Association of the United States
Standards (FMA, 1998) and the USA Food Chemical Codex (FCC), National Academy
of Sciences (1996). A summary of these specifications can be seen in Tables 9.3-9.6.

Table 9.3 Specification for thyme oil ex T. V I L L ~ Y Z S

Appearance A colorless, pale yellow or red mobile liquid possessing a characteristic

pleasant odour.
Specific gravity (25 OC) 0.9150-0.9350 (FCC)
Refractive index (20 'C) 1.4950-1.5050 (FCC)
O p t ~ c a rotation
l (20 OC) levorotatory, but not more than -3 O (FCC)
Solubility in 80% vlv aqueous 1:2 volumes (FCC)
ethanol (20 OC)
Phenol content 240% (FCC)
Heavy metals (as Pb) 10.02% (FCC)
Water soluble phenols Shake 1 ml of oil w ~ t h20 ml of hot water and after cooling pass water
layer through a mo~stenedfilter. On add~tionof 1 drop of ferric
chloride solution (9 g FeClj.6H20), no transient blue or violet color
should be produced (FCC)

Tabb 9.4 Specification for thyme oil ex T. zygzr

Appearance Red to very intense brown-red, almost black mobile liquid with a characteristic
phenolic, spicy aroma
Density (20 OC) 0.9120-0.9350 (ISOIAFNOR)
Refract~veindex (20 OC) 1.4950-1.5050 (ISOIAFNOR)
Optical rotation Because of colour, it could not be measured, generally levorotatory
Solubility in 80% vlv 1:2 vols. (ISOIAFNOR)
aqueous ethanol (20 OC)
Flash polnt (clc) +62 "C (ISO)
Phenol content 38-56% vlv (ISOIAFNOR)
G C analysis (ISO) a-thujene (0.5-1.6%), a-pinene (0.6-2.1%), myrcene (1.0-2.8%),
a-terpinene (0.9-2.6%), ;-terpinene (5.0-10.3%),p-cymene (1 5.0-2S.O%),
tvsabinene hydrate (trace-0.5%), linalool(4.0-7.0%), methyl carvacrol
(0.1-l.5%), thymol(36-55%), carvacrol(1.24.0%), 8-caryophyllene
(0.6-1.8%)
~ - ~ - --- -

Tabb 9.5 Specification for Spanish oregano oil ex T. capitatus (today Thyvzbra cupitata)

Appearance A yellowish to dark brown almost black m o b ~ l el ~ q u i dwith a characteristic

phenolic, splcy odor
Specific gravity (20 OC) 0.9380-0.9630 (FMA)
Density (20 OC) 0.9300-0.9350 (ISOIAFNOR)
0.9350-0.9660 (FCC)
Refractive ~ n d e x(20 OC) 1.5000-1.5 130 (ISO)
1.5020-1.5080 (FMA)
 The genus Thymus as a source oJconzme~~czalproducts25 5

Optical rotation (20 OC) -2 O to +3 O (FMA)

-10 O to +2 (ISO)
Phenol content 60-75 96 (ISO)
Solubility in 70% vlv 1:4 vols. (ISO)
aqueous ethanol 1:2 vols. (FMA)
Flash point (closed cup) +65 OC (ISO)
G C analysis (ISO) a-thujene (0.5-2.0%), a-pinene (0.5-I.>%), mycrene (1.0-3.0%),
a-terpinene (0.5-2.5%), y-terpinene (3.5-8.5%),p-cymene (5.5-9.0%),
linalool (0.5-3.0%), rerpinen-4-ol(0.5-2.0%), thymol (0-5.0%),
carvacrol(60-75%), pcaryophyllene (2.0-5.0%)

Table 9.6 Specification for Spanish marjoram oil ex. T 17zmtzchina

Appearance A colourless to pale yellow liquid with a characteristic, agreeable, sp~cy,

eucalyptus-like odour
Density (20 OC) 0.9000-0.9200 (ISOIAFNOR)
Refractive index (20 OC) 1.4620-1.4680 (ISOIAFNOR)
Optical rotation (20 'C) -6 " to +10 " (ISOIAFNOR)
-5 " to + l o 0 (FCC)
Solubility in 70% vlv 1:3 vols. (ISOIAFNOR)
aqueous ethanol
Phenol content 1 4 . 0 % (ISOIAFNOR)
1.8-Cineole content 40-65 % (ISOIAFNOR)
49-65% (FCC)
Heavy metals (as Pb) 10.002% (FCC)
G C analysis (AFNOR) a-pinene, camphene, 8-pinene, sabinene, myrcene, limonene, 1,8-cineole,
2-terpinene, p-cymene, linalool and a-terpineol

As the oil of wild thyme (T. sevpyllzm) is neither produced nor used in large quanti-
ties, no international standard exists for this oil at the present time.

OIL ADULTERATION

In the 1920s, adulteration of red thyme oil with turpentine to produce white thyme oil
that had a phenol content of 1-2 per cent was a common practice (Parry, 1925).
According to Guenther (1945), in the mid-1940s thyme oil was frequently adulterated
by the addition of terpenes or 'thymene' and synthetic thymol and carvacrol. Thymene
is the by-product mixture obtained from ajowan oil (ex. Tvachysper~?zzlnz coptzcu~a(L.) Link)
after removal of thymol. Prior to the advent of modern instrumental analytical techniques
and the use of column chromatography or thin-layer chromatography (TLC), the oil
was evaporated to yield crystalline thymol, which was free from a creosote-like off-odour
associa,-edwith synthetic carvacrol. Also, if the oil did not crystalize on evaporation the
use of synthetic carvacrol as an adulterant was concluded. Thyme oil adulteration is
practiced even today. Such evidence is especially true when white thyme oil can be
found on the market at prices lower than red thyme oil. This is an impossible situation
because red thyme oil is the crude product used to make white thyme oil by treatment
with tartaric acid and re-distillation.
In the past, Spanish oregano oil was also subjected to adulteration generally by the
addition of synthetic p-cymene and/or synthetic carvacrol. Again, the detection of a
256 Brian M . Lawrence a n d Arthur 0. Tucker

creosote-like off-odor associated with synthetic carvacrol on evaporation of the oil was
used to determine adulteration (Gildemeister and Hoffmann, 1990).
Since the early 1960s, the use of gas chromatography (GC) combined with other
techniques has been used to determine the composition of an oil. More recently the use
of GC with flame ionization detection, electronic integration, automatic injection,
capillary columns of a polar and non-polar nature for retention index determination and
GCImass spectrometry (MS) has led to a more accurate detailed analysis of an oil
composition. As a result, the addition of synthetic thymol, carvacrol or 1,s-cineole to
thyme oil, Spanish oregano oil or Spanish marjoram oil, respectively, is readily detected
because of the corresponding decrease in the minor oil constituents.
To further assist the analyst in determining the genuineness of an oil, the introduc-
tion and subsequent use of chiral GC columns for enantiomer separation has become
a more common technique. Considering thyme oil, although thymol is not optically
active, some of the other constituents are, for example, examination of enantiomers of
a-pinene, P-pinene and limonene in thyme oil by Hener etal. (1990) revealed the
following distribution:

(1s)-(-)a -pinene (89 per cent) : (1R)-(+)-a-pinene (1 1 pet cent)

(1s)-(-)-P-pinene (96 per cent) : (1R)-(+)-P-pinene (4 pet cent)
(4s)-(-)-limonene (70 per cent) : (4R)-(+)-limonene (30 per cent)

Analysis of a thyme oil whose enantiomeric distribution of particularly a- and P-pinene

falls outside the levels shown above is indicative of oil adulteration. In addition to the
monoterpene hydrocarbons of thyme oil, the monoterpene alcohols are optically active.
Using chiral GC analysis, Casabianca etal. (1998) revealed that adulteration of the oil
with a coupage (a mixture of components) containing synthetic linalool was detectable
from examination of the distribution of linalool enantiomers. The results of this study
are shown in Table 9.7. As can be seen from these results, two of the French commercial
samples of thyme oil appear to be adulterated.
Linalyl acetate is also present in T. vulgaris oil at a very low level ( ~ 0 . 2
per cent);
nevertheless, Casabianca etal. (1998) determined that its enantiomeric distribution was
as follows:

(3R)-(-)-linalyl acetate (93.8-99.2 per cent) : (3s)-(-)-linalyl acetate (0.8-6.2 per cent)

Table 9.7 Enantiomeric distribution o f linalool in thyme oils

Botanical orrgin (3R)-(-) Linalool (3s)-(+)-Linalool

T. vulgaris (France) (7 lab distilled samples)

Commercial sample 1
Commercial sample 2
Commercial sample 3
T . zygis (Spain)
T. zygis (Portugal)
T. serpyllunz (France) 1
T. serpyllz~nz(France) 2
 The genus Thymus as a source of commercialprodztcts 25 7
Again, the enantiomeric distribution of linalyl acetate along with linalool can be used
as one of the indicators to determine oil adulteration.
The adulteration of Spanish marjoram oil with a 1,8-cineole-rich eucalyptus oil is
not uncommon. Detection of this adulteration is not easy especially if the level of adul-
teration is less than 10 per cent. Nevertheless, if the hydrocarbons are separated from
the oxygenated compounds of an oil suspected to be adulterated and aromadendrene is
found in the hydrocarbon fraction, and tr-pinocarveol and globulol are found in the
oxygenated fractions, then the oil is probably adulterated with a 1,8-cineole-rich
eucalyptus oil.
In 1995, Ravid etal. determined that, although a-terpineol was only present in
T. capitatzfi (today Thynzbra capitala) oil as a minor component (ca. 0.2 per cent), its
enantiomeric distribution was as follows:

(4R)-(+)-a-terpineol(61.5per cent) : (4s)-(-)-a -terpineol (38.5 per cent)

If an oil of Spanish oregano oil possessed distribution of a-terpineol enantiomers different

to that shown above, this is probably indicative of adulteration.

OTHER EXTRACTIVES

In addition to the oils a small amount of thyme (ex T. vulgaris) oleoresin is also pro-
duced. It is impossible to obtain volumes on the production of this minor commodity
as production is done by spice oleoresin and seasonings manufacturers in the US and
Europe. No oleoresins appear to be produced from T. zygis, T. capitatus, T. mastichina, or
T. serpyllanz.

WHOLE LEAF A N D GROUND THYME

Although there is a concern about pesticideifungicide residues, aflatoxins and micro-

biological contamination found in imported spices and herbs, the major concern by
various government agencies around the world is cleanliness. As a result, contaminant
levels for animal hairs and excreta, insect fragments and foreign material, standards
have been established in many countries (Tainter and Grenis, 1993).
In the US, the American Spice Trade Association (ASTA) is the advisory organization
that helps the spice and seasoning industry develop acceptability standards for whole
and ground spices and herbs. Examples of the recommended physical and chemical
specifications of whole thyme leaves and ground thyme can be seen in Tables 9.8 and
9.9 (Tainter and Grenis, 1993).

DRIED HERB PRODUCTION STATISTICS

Although thyme (T. vulgaris) is native to South Europe, it is both collected from the wild
in France, Albania, Spain, Morocco, Lebanon, Syria, Turkey, Tunisia, Greece, Yugoslavia,
etc. and widely cultivated in France, Germany, Morocco, India, Spain, Bulgaria, Hungary,
Russia, Canada, US, etc. while the so-called Spanish thyme (ex T. zygir) is collected
25 5 Brzan M. Lawrence and A~,thzlr0.Tucker

Table 9.8 Whole thyme: pliysical and chemical specifications

Cleanliness specifications
Whole dead insects Rlkg
Mammal~anexcreta 2lkg
Other excreta 101kg
Mold (wlw) 1.O%
Insect infestedlcontaminated (wlw) 0.5%
Insect fragments ca. 325125 g
Rodent hairs ca. 2125 g
Chenzical spec$ications
Volatile oil 20.8%
Moisture 110.0%
Ash 510.0%
Acid insoluble ash 53.0%
Bulk index ca. 400 mgIl00 g

Table 9.9 Ground thyme: physical and chemical specifications

Cleanliness speci~cation~
Insect fragments ca. 925110g
Rodent hairs ca. 2110g
Chenzicdl spec$cations
Volatile oil 20.5%
Moisture 10.0%
Ash 110.0%
Acid insoluble ash 43.0%
Sieve test 95% through a 200 mesh
Bulk density 2j0m11100g

from the wild in Spain and Portugal. Accurate up-to-date export and import statistics
for whole thyme leaves are not accessible because most countries group their minor
spices and herbs together into one statistic. For example, thyme is often grouped with
laurel (bay) leaves, marjoram, oregano, etc. in published government statistics.
In Europe, France is the largest producer of cultivated herbs destined for the culinary
and seasonings trade as dried herbs. Maffei (1992) reported that in France 25 tons of
dried whole thyme leaves were produced from wild collection in DrBme, Var and
Languedoc-Roussilon, whereas 250-280 tons of dried leaves were produced from plants
mainly cultivated in the Provence-Alpes-C8te d'Azur areas. Over the same time period
700-770 tons of dried whole leaf thyme were imported into France.
It was further reported (Maffei, 1992) that in 1990 Germany imported 500 tons of
dried thyme while an additional 50 tons were produced internally. Most of the thyme
was imported from Spain, although smaller quantities were imported from Poland and
Morocco. It should be noted here that the wild thyme of Moroccan origin is obtained
from T. satureioides Cas. and Bal. and not T. vulgaris or T. zygis.
In 1990, the Netherlands imported 90 tons of dried thyme mainly from Spain, while
the UK imported 220 tons during the same time period (Maffei, 1992). Like the Neth-
erlands, Spain was the major source of UK thyme, although ca. 32 tons were produced
domestically.
 Thegenw Thymus as a source oJcom~~ze~*~zalprodz~cts
25 9

U.S. importation of crude thyme, 1922-1996

3500

1922 1942 1962 1982

Year

Figz~re 9.1 US ~rnportationof crude thyme, 1922-1996; data are mlssing for 1925 and 1954-1962.
In 1989-1996, statistics for crude thyme were reported together with bay laurel, so an
estimate of 213 of this total was used for crude rhvme alone.

Importation of crude thyme into the US, 1922-1996, is illustrated in Figure 9.1.
Although large quantities of thyme are imported into the US, there were also three
commercial herb producers in California in 1974 (Tyner, 1974) that grow a variety of
herbs on approximately 125 ha of which ca. 10-15 pet cent is devoted to thyme. This
production is then used in bottle or jar trade of grocery store herbs in the US for a more
up-market product.

FRESH HERB PRODUCTION

Thyme has been grown since medieval times as a garden herb which was used at that
time to flavor a potage which was served with meat for its dual purpose of adding flavour
and its curative properties (Freeman, 1943). In modern times, the production of fresh
and deep frozen thyme has become a moderate-sized business in the fresh herb trade. In
France, fresh thyme is grown on ca. 220 ha in the Rhone-Alpes region in the depart-
ments of Drtime, Ardeche, Loire, Vaucluse, Alpes de haute Provence, Essone, Yvelines
and Seine et Marne (Garnon, 1992). It is an integral component of 'bouquet garni'
along with laurel leaves and rosemary. In fact, it is sold in fresh bunches all over France
where it is the largest item in the fresh herb marketplace (Verlet, 1989). Further
cultivation of thyme in France is being promoted by ITEIPMAI (Bouverat-Bernier
1992; Institut Technique Interprofessionel des Plantes Medicinales, Aromatiques et
Industrielles, 1983) and the Office National Interprofessionnel des Plantes Aromatiques
et Medicinales colloquially known as ONIPPAM (Garnon, 1992).
Thyme is also grown in other European countries for fresh herb production. For
example, thyme is grown on ca. 2.5 ha in the Canton d u Valais in Switzerland (Rey,
1992). It is also grown in Austria on 4 0 ha (Dachler, 1992), and in the former East
Germany on 85 ha (Pank, 1992). Unfortunately, there are no statistics on the amounts
of fresh thyme produced.
260 Brian M . Lawrence and Arthur 0. Tucker
Since the mid-1980s fresh herb production in Israel including thyme has become a
more important minor industry (Purievsky, 1988). For example in 1989, 25 tons of
fresh thyme was exported to France (30 per cent), UK (30 per cent), Germany (20 per cent),
Switzerland (10 per cent) and other European countries. Although no new figures on
this production are available, it can safely be assumed that this market has not only been
maintained but has grown.
There is also a cottage-scale trade in fresh herbs including thyme for the restaurant
trade. The herbs are generally raised by small landowner-herb growers who reside in
small towns and villages close to the metropolitan areas in the US and Europe so that
there is a rapid delivery of fresh thyme to the restaurants.
Finally, organically grown herbs have started to appear in speciality markets in the
US; however, the amount of fresh thyme sold is minuscule compared to the amount of
dry thyme sold.

THYMUS SPECIES AS GARDEN PLANTS

In addition to the industrial and commercial uses of Thynzw species and their extractives,
various species have been and are still grown as decorative, aromatic border, pathway or
rock garden plants. As a result, a flourishing herb trade exists in North America and
western Europe in which numerous Thymus species and hybrids are traded for their
decorative foliage and diverse aromatic properties. The most popular Thymus species
and hybrids cultivated in the US (Flannery, 1982) are: T. broussonetii Boiss., T. caespititiw
Brot., T. camnphoratus Hoffm. et Link, T. capitatus Hoffm. et Link, T. carnosw Boiss.,
T. cherlerioides Vis., T. cilicicus Boiss. et Bal., T. conzptw Friv., T. doerferi Ronn.,
T. herba-barona Loisel., T. leucotrichus Halacsy, T.nzustichina L., T. menzbranaceus Boiss.,
T. numnzularius Bieb., T. pruecox Opiz, T.pzllegioides L., T. qz~inquecostatusCelak., T. richdrdii
Pers., T. vulgaris L., T.zygis (Loefl.) L., T . x citriodorus (Pers.) Schreb., T. 'Argenteus',
T . 'Broad-leaf English', T . 'Doone Valley', T . 'Longwood', T. 'Pinewood', T. 'Porlock',
T. 'Variegated English', T. 'Wedgewood English', T. 'Woolly-Stemmed Sharp' and
T. 'Woolly-Stemmed Sweet'.

REFERENCES

American Spice Trade Association (1960) Official Analytical Methods, American Spice Trade
Association, Englewood Cliffs, New Jersey.
Association Fran~aisede Normalisation (1996a) Huiles Essentielles. Tome 2, SpeciJications 5e Edn.
Huile essentielle de thym sauvage d'Espagne (Thymus mastichina Linnaeus). NFT 75-343, Avril
1987, AFNOR, Paris, pp. 4 8 3 4 4 1 .
Association Franqaise de Normalisation (1996b),Huiles Essentielles. Tome 2, Specifications 5e Edn.
Huile essentielle de thynz 2 thymol (Thymus zygis ( L o g . ) L., type Espagne, NFT 75-3459, Mars
1993, AFNOR, Paris, pp. 4 7 3 4 8 1 .
Bouverat-Bernier, J.P. (1992)Les travaux d'amelioration genetique a I'ITEIPMAI. In N . Verlet
(ed.), 32mes Rencontres Techniques et Economiques. Plantes Aromatiques et Medicinales, Nyons, 2-3-4
Decenzbre 1991. Centre Formation Professionnelle Promotion Agricole (C.F.P.P.A.) de Nyons,
pp. 140-155.
Casabianca, H., Graff, J.B., Faugier, V., Fleig F. and Grenier, C. (1998)Enantiomeric distribu-
tion studies of linalool and linalyl acetate. J. High Resol. Chronzatogr., 2 1 , 107-1 12.
 The genw Thymus as a source of commercial products 26 1
Dachler, M. (1992) La production des plantes medicinales et aromatiques en Autriche, en
particulier celle d u pavot et d u carvi. In N . Verlet (ed.), 3Bmes Rencontres Techniques e t
Economiqnes. Plantes Aronzatiques et Medicinales, Nyons, 2-3-4 Decembre 1991. Centre Formation
Professionnelle Promotion Agricole (C.F.P.P.A.) de Nyons, pp. 122-128.
Flannery, H.B. (1982) A Stndy ofthe Taxa ofThymus L. (Labiatae) Cnltivated in the UnitedStates.
Ph.D. thesis, Cornell Univ.
Fragrance Materials Association (1998), FMA Monographs. Vol. 2, Origanum Oil Spanish Type.
(replaces EOA #142), FMA, Washington, DC (02-06-98).
Freeman, M.B. (1943) Herbs for the Medieval Household for Cooking, Healing and Dzvers Uses,
Metropolitan Museum of Art, New York.
Garnon, P. (1992) 10 ans de production en France: Bilan et perspectives. In N . Verlet (ed.),
3imes Rencontres Techniques et Economiques. Plantes Aromatiques et Medicinales, Nyons, 2-3-4
Decembre 1991. Centre Formation Professionnelle Promotion Agricole (C.F.P.P.A.) de Nyons,
pp. 216-231.
Gaviiia Mligica, M. and Torner Ochoa, J. (1966) Contribuci6n al estudio de los aceites esenciales
espa%o;olesI. Aceites de la Provincia de Cuenca. Ministerio de Agricultura, Instituto Forestal de
Investigaciones y Experiencias, Madrid, pp. 257-265.
Gildemeister, E. and Hoffmann, F. (1990) The Volatile Oils. Transl. E. Kremers. Pharmaceutical
Rev. Publ. Co., Milwaukee, Wisconsin, p. 32.
Guenther, E.A. (1945) Survey of Spanish essential oils. Amer. Perfurn., 60, 43-44.
Hener, U., Kreis, P. and Mosandl, A. (1990) Enantiomeric distribution of a-pinene, P-pinene
and limonene in essential oils and extracts. Part 2. Oils, perfumes and cosmetics. Flavour
Fragr. J., 5, 201-204.
Institut Technique Interprofessionel des Plantes Medicinales, Aromatiques et Industrielles
(1 983) Donzestication de la production conditionnement et de;iinition dz thynz (Thymus vulgaris L.),
I.T.E.P.M.A.I., Milley-la-FBret, France.
International Organization for Standardization (1992), ISOITC 54 47298 Oil of Wild Thyme
(Thymus mastichina L.) (15-02-1992).
International Organization for Standardization (1996a), ISOITC54lSCN1684, Essential Oils. Oil
ofThymbra capitata ( L . ) Cav., Spanish type. (2-26-1996).
International Organization for Standardization (1996b), ISOITC54ISCN1680, Essential Ozls.
Thynze oil containing thymol (Thymus zygis (Loefl.) L.), Spanish type. (2-26-1 996).
Lawrence, B.M. (1985) A review of the world production of essential oils 1984. Pegurn. Flavor.,
10, 1-16.
Maffei, M. (1992) Dry culinary herbs: an overview of selected western Europe markets. In
N. Verlet (ed.), 3277~sRencontres Techniques et Economiques. Plantes Aronzatiques et Medicinales, Nyons,
2-3-4 Decenzbre 1991. Centre Formation Professionnelle Promotion Agricole (C.F.P.P.A.) de
Nyons, pp. 249-292.
Miralles, J. (1998) Spanish distilled essential oils. Today's production. In Sevilla 1997. IFEAT
Proceedings. IFEAT, London, pp. 17 1-1 83.
National Academy of Sciences (1996) Food Chenzical Codex (FCC). 4th (ed.) Natl. Acad. Press,
Washington, DC.
Pank, F. (1992) Medicinal and Spice Plant Production and Research in the Eastern part of
Germany. In N . Verlet (ed.), 3dnzes Rencontres Techniques et Econonziques. Plantes Aronzatiques et
Medicinales, Nyons, 2 - 3 4 Decembre 1991. Centre Formation Professionnelle Promotion Agricole
(C.F.P.P.A.) de Nyons, pp. 129-139.
Parry, E.J. (1925) Parry's Cyclopedia of Pegnmery M-Z. P. Blakiston's & Son, Philadelphia,
pp. 746-754.
Purohit, P. (1994) Phyto-cosmetics and the expanding market. In 4Smes Rencontres
Techniques et Economiques. Plantes Aromatiques et Medicinales, Nyons, 5-6-7 Decenzbre
1993. Centre Formation Professionnelle Promotion Agricole (C.F.P.P.A.) de Nyons,
pp. 80-82.
262 Brian M. Lawrence and A~thul.0. Tuder
Putievsky, E. (1988) Production of aromatic plants in Israel. In J.E. Simon and L.Z. Clavio
(eds), Proceedings ofthe Third National Her6 G~*owing and Marketing Conj%ence, Baton Rouge, Lou-
isiana. International Herb Growers and Marketers Association, pp. 130-1 33.
Ravid, U., Putievsky, E. and Katzir, I. (1995) Determination of the enantiomeric composition
of a-terpineol in essential oils. Flavour F1,ag1*.J., 10, 281-284.
Rey, Ch. (1992) Recherche et production des plantes medicinales et aromatiques en Suisse:
Situation actuelle. In N. Verlet (ed.), 3dmes Rencontres Techniques et Econonziques. Plantes Aronzat-
iqz~eset Medicinales, Nyons, 2 - 3 4 Decembre 1991. Centre Formation Professionnelle Promotion
Agricole (C.F.P.P.A.) de Nyons, pp. 116-121.
Tainter, D.R. and Grenis, A.T. (1993) Spices and Seasonings. A Food Technology Handbook. VCH
Publ. Inc., New York.
Tyner, G.E. (1974) The Dispersal of Culinary Herbs in Relation to Contenzpo~*a~.y Californza Her6
Industry. Ph.D. Thesis, Univ. California, Los Angeles.
Verlet, N . (1989) New markets for herbs in France and Europe. In J. E Simon, A. Kestner,
M.A. Buehrle (eds), Herbs 1989. Proceedings ofthe Fourth National Herb Growing and klavketing
Conference, SanJose, California. International Herb Growers and Marketers Association, pp. 80-84.
10 The medicinal and non-medicinal
uses of thyme
Antonio Zu~zueloand Espemnzu Crespo

INTRODUCTION

The uses of thyme, Thynzw vulgaris and other Thynzus species are well known, and
extensive parts of the world get benefit from this plant group in medicinal and non-
medicinal respects. Following the development of the medicinal uses of thyme we can
see that thyme has changed from a traditional herb to a serious drug in rational
phytotherapy. This is due to many pharmacological in vitro experiments carried out
during the last decades, and even a few clinical tests. The studies have revealed well
defined pharmacological activities of both, the essential oils and the plant extracts, the
antibacterial and spasmolytical properties being the most important ones. The use of
thyme in modern phytotherapy is based on this knowledge, whereas the traditional
use of thyme describes only empirical results and often debatable observations. There-
fore it seems necessary to present here the data available on the pharmacodynamics of
thyme and thyme preparations in order to substantiate the use of thyme in modern
medicine.
The non-medicinal use of thyme is no less important, because thyme (mainly
T. vulgurzs) is used in the food and aroma industries. It serves as a preservative for foods
and is a culinary ingredient widely used as seasoning in many parts of the world.
This use is due to the typical aroma of the plant which the essential oil is respon-
sible for, and most people are very familiar with its typical smell. The special aroma
also causes the role of the essential oil as a raw material in perfumery and in
everyday cosmetics. The use of thyme as a preservative of food can be put down to
the antioxidative effects of the plant extracts, which were of increasing interest in
the last years. The results of these studies may be of further importance assuming
that free-radical generation causes oxidative stress when it exceeds the capacity of
antioxidant defenses in the human body. This way it may significantly contribute
to pathogenesis.
Irrespective of the pharmacological and non-pharmacological effects when we exam-
ine and discuss the use of thyme we must take into account the chemistry of the plant.
Therefore most of the studies described here refer to the chemical components of rhyme.
The structural details of the compounds mentioned in this chapter are given in Chapter 3
of this book.
264 Antonio Zarzuelo and Esperanza Crespo
THE THERAPEUTICAL USES OF THYME

T h e Traditional Use of T h y m e in Folk Medicine

The widespread use of thyme dates back to ancient Egypt where various species of
thyme were grown to perfume unguents and for embalming and, one can suppose, for
medicinal purposes as well. The Greeks and the Romans used it in the same way, how
and for what, we know thanks to Plinius (First century), Dioscorides (First century) and
Theophrastus Bombastus von Hohenheim (Paracelsus 1493194-1541). However the
use of this plant did not extend further than the Alps until the eleventh century.
The first northern chronicles, relating to this period, can be read in the "Physika" by
the abbess Hildegard von Bingen (1098-1 179) and in the works of Albertus Magnus
(1193-1280). All the posterior works come from the Herbal written by the herbalist
P. Mathiolus (1505-1577). The knowledge of traditional medicine is based on his
works, in which the strength and efficacy of thyme were mentioned first. From then on
numerous therapeutical properties have been attributed to thyme on a more or less
empirical and debatable basis.
So many beneficial effects have been attributed to thyme that Bardeau (1973) stated
thyme to be "an indispensable plant which should be consumed to conserve health.
Furthermore, if one could replace one's morning cup of coffee with an infusion of
thyme, you would quickly appreciate its positive effects: animation of spirit, sensation
of lightness in the stomach, absence of morning cough, and its euphoriant and tonic
effect". The readership will understand that it is impossible and not useful to enumerate
all therapeutical benefits attributed to thyme which can be found in numerous popular
works in folk medicine. Therefore only a selection of external and internal applications
and indications shall be described, especially those which seem to be the most plausible
ones.
Externally, infusions and the essential oil of thyme is traditionally used in treating
injuries, bruises, infected ulcers, abscesses, cutaneous ulcers, various types of dermatitis,
and in certain cases pruritis (Schauenberg and Paris, 1977). Emulsions are useful when
used in massage on rheumatic types of pain (sciatica, arthritis, lumbago), gout and
neuritic pains (Furlenmeier, 1984). As regards the capillaries, thyme improves blood
flow and the oxygenation of the scalp, reduces seborrhoea, regenerates the capillary
glands, improves the state of the hair, prevents baldness, and is therefore useful in cases
of alopecia (Poletti, 1979). Baths of thyme possess invigorative and sedative properties,
also behaving as coadjuvants in slimming cures (Pahlow, 1979; Furlenmeier, 1984;
Volkk and Stodola, 1989).
The internal use of thyme is highlighted in the treatment of a variety of illnesses of
the respiratory tract because of its expectorant, spasmolytic and antiseptic properties,
such as flu, colds, sinusitis, chronic and acute bronchitis, tuberculosis, calming convulsive
coughs (pertussis), and irritable and spasmodic coughs (asthma) (Bardeau, 1973;
Pahlow, 1979; Poletti, 1979; Gonzilez and Mufioz, 1980; Fernkndez and Nieto, 1982;
Furlenmeier, 1984; Vol5k and Stodola, 1989). It is attributed as having general stimu-
lant properties, acting as a nervous tonic and being used in asthenic states. It is also
useful in combatting insomnia, anxiety and depression (Valnet, 1964; Perrot and Paris,
197 1; Bardeau, 1973).
Moreover it is used in a wide variety of gastro-intestinal troubles like dyspepsia (slow
digestion), colic, fermentation, flatulence, diarrhea, gastritis and gastric ulcers. It is also
 The medicinal and non-medzcznal wei of thyme 265
useful in treating bacterial and parasitical infectious processes (Ascaris, Oxyurzs, Tuenk)
(Perrot and Paris, 1971; Schauenberg and Paris, 1977; Gonzilez and Mufioz, 1980;
William and Thomson, 1980; Fernindez and Nieto, 1982; Futlenmeier, 1984). In the
genitourinary system the use of thyme for its diuretic, antiseptic and emenagogic prop-
erties is appreciated (Benigni etal., 1964; Bardeau, 1973; Pahlow, 1979; Gonzilez and
Mufioz, 1980). In the cardiovascular system thyme helps the circulation, behaving like
a hypocholesterolemic agent (Valnet, 1964; Brasseur, 1983).

T h y m e in phytotherapy
Although for thousands of years plants have been used as remedies, a controversial
discussion of their usefulness has arisen in the last two decades. This is due to the
knowledge that chemical substances are responsible for the pharmacological effects in
the human body and additionally due to the derailed knowledge on their pharmaco-
logical mechanisms in human cells, tissues, and organs. Therefore in modern medicine
the tendency has developed that only chemically defined and pure substances should be
applied, exclusively such substances whose positive (curing) effects have been proved in
clinical tests. Moreover, an optimal "package" for the chemicals is required which
guarantees the liberation of the drug after application meaning highly developed drug
technologies such as tablets, capsules, suppositoria, erc. Within this concept medicinal
plants and herbal drugs cannot easily find consideration, because their chemical compo-
sitions represent very heterogeneous systems and, additionally, these complex mixtures
are inadequately packed in vegetable cells.
However the increasing consciousness for health and environment results in the
fact that people have not forgotten the herbal drugs. Quite the opposite has hap-
pened, globally an increasing demand for herbal medicines can be registered. This
trend has also been accepted by the orthodox medicine remembering the benefit of
traditional medicine lasting for centuries; but in modern medicine one cannot stick
only to traditional uses. In order to concede the herbal remedies a "real-drugs" status,
many scientific efforts have been and have to be made, and phytotherapy has developed
into "rational phytotherapy", a part of a scientifically-oriented medicine.
Legal regulations in Germany and Europe for drug registration ask for a proof of
quality, safety and effectiveness. The latter has been evaluated for many herbal drugs, also
for the herbs of T. vzllgarzs (common thyme) and T. serpylhm (wild thyme). A critical
investigation of all the bibliography dealing with the chemistry and pharmacology of
these two herbal drugs resulted in two "positive" (approved) monographs elaborated by
a German expert group. The so-called Commission E Monographs represent a valuable
therapeutic guide to herbal medicines in phytotherapy (Blumenthal, 1998). Within
the paragraphs "Uses" of these monographs the application of thyme and thyme prep-
arations is only recommended for the treatment of some clearly-defined diseases, reading
as follows:

(Common) Thynze - Thymi herba. External use: bath additive as a supporting cure of
acute and chronical diseases of the upper respiratory tract; in addition against prur-
itus of detmatosis. Internal use: can be applied against symptoms of bronchitis and
whooping cough. Catarrhs of the upper respiratory tract.
266 Antonzo Zarzuelo and Esperanza Crespo
Wild thyme - Serpylli herbu. External use: bath additive as supporting cure of acute
and chronical diseases of the upper respiratory tract. Internal use: catarrhal infec-
tions of the upper respiratory tract.

Compared with the traditional uses of thyme the Commission E monographs obviously
allow only restricted applications of thyme. These regulations must be followed when
drug manufacturers apply for a registration of their phytotherapeutics as drugs.
However, in serious publications on phytotherapy (Wagner and Wiesenauer, 1995;
Schulz and Hansel, 1996; Reuter, 1997; Loew etul., 1999) the area of application of
thyme preparations is described similarly limited with special reference to the essential
oil which can be used as bath additive (in mixtures) or for inhalation. It is recom-
mended for a treatment of cough and sinusitis and its effects are described as secreto-
motoric, bronchospasmolytic and antibacterial. Thymol is proved to be responsible for
these effects.
The essential oil of thyme can be administered in diverse galenic forms when used
in therapy. Administered externally it can be applied directly by means of pomades,
emulsions, poultices, and liniments. Alternatively it can be ingested in liquid form
(drops, syrups and elixirs) or administered in solid forms (capsules). Recently the
pharmaceutical industry has developed new ways of administering essential oils to
facilitate their dosage and handling. These new ways come in the form of buffered
microcapsules and consist of powder impregnated with essential oil (40 per cent). The
fact that they are buffered gives them greater gastric tolerance. The essential oil of
thyme can be administrated rectally in the form of suppositories and microenemas;
vaginally, in the form of ovules; in sublingual administration in the form of solutions
and nasally, in the form of drops and pomades. Finally we must not forget the inhalers
and aerosols which are commonly used for the treatment of respiratory conditions
(Giienechea, 1992).
Thyme oils (from e.g. T. vulpris, T. serpylluna), especially thymol and carvacrol,
provide an antiseptic action when eliminated via the lungs (Didry etal., 1993), but also
has a mild irritant effect which stimulates the secretory cells of the mucosa and increases
the movement of the ciliated epithelium in the bronchial tree (bronchi). This produces
an increase of secretions which causes the decongestion of the entire respiratory system.
The spasmolytic properties which these essential oils possess, capable of relaxing smooth
bronchial muscle, determine their usefulness in the treatment of respiratory tract
obstructive processes, and along with its expectorant properties make them effective
against different types of coughs: cough caused by thick and viscous secretions, irritable
cough, spasmodic cough (Errera, 1978; Forster eta]., 1980; Schafer and chafer, 1981;
van den Broucke and Lemli, 1981; Furlenmeier, 1984).

Thyme in aromatherapy
A pleasant odour has always been, and still is, an important factor for people to feel
good, and feeling well is synonymous with good health. The most ancient way to treat
a patient in the sense of aromatherapy was the fumigation which was practised in all
ancient civilisations. Although this was done by religious worship, it was nevertheless
useful in the treatment of patients because the air became disinfected and the good
aroma induced calmness. Up to the nineteenth century, the disinfectant effect of fumes
was often used and people tried to banish the bad air in sick rooms by igniting good
 The nzedicinal and non-medicinal uses of thynze 267
smelling candles and the doctors tried to protect themselves against infections by
sniffing essential oils.
The term aromatherapy was coined in the late 1920s by the French cosmetic chemist
R.M. Gattefosse, who noticed the excellent antiseptic properties and skin permeability
of essential oils. Anticipating the trends of the 198Os, "New Age" and esoterics,
Tisserand revived this term by including it in a general natural healing method with
elements of wholistic, cosmic, anthroposophic and other phenomena (Tisserand, 1980).
Confusingly, aromatherapy is used whenever good smelling plants or drugs are used to
cure diseases not asking if the "aroma" is really responsible for the effect and additionally
it is often combined with mystic elements.
A scientific clarification was necessary and it is due to Buchbauer (Buchbauer,
1990, 1996) that nowadays aromatherapy has become a scientific discipline. It is
strictly based on his definition given as follows: "Aromatherapy: therapeutic uses of
fragrances or at least mere volatiles to cure and to mitigate or prevent diseases, infec-
tions and indispositions only by means of inhalation". T o avoid misunderstandings
here "aromatherapy" is used according to Buchbauer's definition, although we are
aware of the fact that in popular works "aromatherapy" is more often used in the
traditional definition.
Concerning the application of thyme, a big overlap of aromatherapy with phyto-
therapy is inevitable, because the essential oil (the "aroma") is the most important and
effective principle of the herbal drug in both cures. Essential oils of thyme hold a priv-
ileged position because they have been demonstrated to have various pharmacological
activities, the antimicrobial and spasmolytic ones being the most utilised in therapy.
The essential oil of thyme, whichever administration route (orally, rectally or cutaneously)
is chosen, is eliminated via the lungs (Penso, 1980; Arteche etal., 1994) and there it
develops its capacity to act on the respiratory system.

Thyme in homoeopathy
Homoeopathy is based on an independent principle of therapy which was founded by
the German doctor Samuel Hahnemann (1755-1843) who was teaching in Leipzig.
This principle can be understood as a therapy which targets the internal regulation
(stimulation) of the human body itself by a (special) drug whose reactivity corresponds
to each patient individually. The methodical concepts base on the so-called "Ahnlich-
keitsregel", the Simile principle - Similia similibus curentur, meaning that "similar can
be cured by something similar", not as a law of nature but as an instruction for behav-
ing. The feature of the remedy must be similar to the f e a t ~ ~ of
r e the illness concerning
location, form and character. Typically the drugs are administered in diluted forms
which in Hahnemann's idea corresponds to an increase in "potency".
In homoeopathy numerous plants and parts of plants have a use for a multitude of
indications. In order to preserve the homoeopathic remedies it became necessary, like in
phytotherapy, to review the knowledge on their effects. This was performed by a group of
experts in Germany (Commission D). The fresh aerial parts of Thyme of two species,
T. vulgaris and T.seqyllum, have traditionally been used in homoeopathy and both plants
are described in the German homoeopathic pharmacopoeia (Homoeopathisches Arzneibuch,
HAB 2000). The critical evaluation of published data in 1989 resulted in the decision that
both plants lack any substantial effectiveness in homoeopathy (negative monographs). That
means that the application of thyme in homoeopathy can no longer be recommended.
268 Antonio Zarzztelo and Esperanza Crespo
PHARMACOLOGICAL EFFECTS OF THYME

Antimicrobial effects of thyme essential oils a n d thyme

preparations

Antibacterial effects
The first researcher who attributed antibacterial properties to thyme (without specifying
the species) was Chamberlain in 1887, after observing the antibacterial effect of its
"vapours" on Bacillus anthracis. Since then, numerous studies with essential oils of dif-
ferent species of Thymw have been carried out. They were shown to inhibit a broad
spectrum of bacteria, generally Gram-positive bacteria being more sensitive than Gram-
negative bacteria. This became obvious in some screening studies administering
Thymus oils to a variety of bacteria (Blakeway, 1986; Farag etal., 1986; Deans and
Ritchie, 1987; Knobloch etal., 1988).
Recently the antibacterial activity of thyme ( T . vulgaris) oil against some important
food-borne pathogens, namely Salmonella enteritidis, Escherichia coli, Sta.hy~o~0cc.U~ aureus,
Listeria monocytogenes, and Campylobacter jejuni, was tested. The latter was found to be the
most resistant of the bacteria investigated (Smithpalmer etal., 1998). In another study
it was shown that the essential oil of thyme and especially its phenols, thymol and
carvactol, have antibacterial acivity against periodontopathic bacteria including Actino-
bacillus, Capnocytophaga, Fusobacteriunz, Eikenella, and Bacteroides species (Osawa etal.,
1990), and may therefore be suitable for plaque control, although few essential oils
have been found to possess clinical efficacy (Marsh, 1992). Furthermore, the essential
oil of thyme showed a wide antibacterial activity against microorganisms that had
developed resistance to antibiotics such as methicillin-resisting Staphylococcus aureus and
vancomycin-resisting Enterococcusfaeciuvz (Nelson, 1997).
Several studies have focused on the antimicrobial activity of the essential oils of thyme
in order to identify the responsible compounds. Thymol and carvacrol seem to play an
outstanding role. These terpene phenols join to the amine and hydroxylamine groups of
the proteins of the bacterial membrane altering their permeability and resulting in the
death of the bacteria (Juven etal., 1994). In addition, thymol and carvacrol were shown
to induce a decrease of the intracellular adenosine triphosphate (ATP) pool of Escherichia
coli and an increase of the extracellular ATP (Helander etal., 1998). Antibacterial activity
was also observed for the aliphatic alcohols, especially geraniol, and ester components.
A variety of activities was presented by the esters, in some cases they were more active
than their corresponding free alcohols, but sometimes less active (Megalla etal., 1980).
Crespo etal. (1990) have evaluated the antimicrobial activity exhibited by the main
chemical groups found in the essential oil of Thymw serpylloides ssp. gadorensis including
hydrocarbons, alcohols, acetates, and phenols (Table 10.1). Again the phenols turned
out to be the most effective against all microorganisms tested, the activity of the
alcohols was on lower levels. Hydrocarbons proved to be effective only against Bacillus
nzegaterium and Mycobactevium phlei, against the latter also the acetates showed weak
activity. The higher sensitivity of Bacillus nzegateriurn and Mycobacterium phlei to the
essential oil of T . serpylloides ssp. gadorensis may be interpreted as the joint effectiveness
of three and four active fractions respectively.
Studies on the structure-activity relationships of 32 terpenoids resulted in the
following observations (Table 10.2, Hinou etal., 1989): (a) a-isomers were inactive as
 The medicinal and non-?/zedzcinaluses of thyme 269

Table 10.1 Antibacterial activity of the main chemical groups in the essential oil of T. serpylloides ssp.
gadorensis

Test ?izicroorganis?iz Essential Hydrocarbon Alcohol Acetate Phenol

oil 17zixtz~re n7ixtufl 7?zixri~re mixture

Pseudomonas flz~oresceni 31 - 12 - 11
Eicherichia colz 32 - 13.6 - 28.5
Bacillz~snzegateriunz 48 7 17 - 40.3
Staphylococcus aurew 32 - 8 - 17.3
M icococcus lz~ter~s 60 - - - 35
Mycobacteriuvz phlei 75 13 10.5 14 67

Note
Data expressed in m m of growth inhib~tionin an agar overlay technique assay
(-) means an i n h ~ b ~ t ~ o n minor than 7 mm.
area
Source: Crespo et nl., 1990.

opposed to the /I-isomers which showed a pronounced activity, e.g. a-pinene; (b) cis-iso-
mers proved to be inactive contrary to the active tr-isomers, e.g. geraniol versus its cis-
isomer nerol; (c) compounds with a methyl-isopropyl cyclohexane ring like some alco-
hols and ketones were the most active, e.g. pulegone; (d) unsaturation of the cyclohex-
ane ring further increased the antimicrobial activity, e.g. terpinolene and acterpineol
which proved to be the most active of the compounds examined against all the bacteria
of the test. Negative results were found in case of a- or cis-isomers or when the com-
pounds lack the common terpene C10-structure, e.g. citronellol or nerolidol.
With respect to the botanical species one can classify the essential oils of thyme, in
general terms, into two main groups (Crespo etal., 1991): (i) The first group contains
those species in which phenols (thymol and carvacrol) are the predominant compon-
ents. These oils show remarkable antimicrobial activities. (ii) In the oils of the second
group phenols are scarce or lacking, whereas other components, such as monoterpene
hydrocarbons, non-phenolic oxygenated monoterpenes or even sesquiterpene hydro-
carbons, predominate. Such oils usually demonstrate lower antimicrobial activities than
those in the first group.
The results obtained by the evaluation of the antimicrobial activity of a non-phenolic
essential oil of thyme from Thymus granatensis may serve as an example of the above
statement (Cabo etal., 1986b). Although this essential oil presented activity against all
the germs tested, with the exception of Escherichia coli, it proved to be only weakly active,
in some cases practically inactive as was the case of Candida albicans and Pseudomonas
fluorescens (Table 10.3). Similar results were obtained when other non-phenolic essential
oils of thyme were tested, e.g. T. hyenzalis (Cabo etal., 1982), T. longzflorus (Cruz etal.,
1989a) and T. baeticus (Cruz etal., 1993). A remarkably stronger antimicrobial activity
was observed when typically phenolic oils, such as e.g. from T. serpylloides ssp. gadorensis
were administered (Crespo etal., 1990) (Table 10.3), a fact which was confirmed by
studies with further phenolic essential oils such as that from T. zygis (Cabo etal., 1978)
and from T. orospeddnw (Cabo etal., 1987).
Two research groups evaluated the different antimicrobial (antibacterial and anti-
fungal) effects of the essential oils of the seven chemotypes of T. vulgaris containing
1,s-cineole, geranioligeranyl acetate, linalool, a-terpineolla-terpinyl acetate, thymol,
Table 10.2 Antibacterial activlty of individual components of thyme oil

Co7izponents Psez/dotizonas Escherichia Staphyloroccz~s Bacillzis R@re?zces

aert~ginosa coli awez/s ceret~s

I-ly~lrocarbons
Myrcene Megalla et al., 1980
Ocimene + Megalla et al., 1980
Limonene Megalla et al., 1980
Dipentene Megalla et dl., 1980
Phellandrene Megalla er al., 1980
A3-Carene + Megalla etal., 1980
P-Pinene Hinou etal., 1989
a-Pinene Hinou etal., 1989
Camphene Hinou etal., 1989
Sabinene Hinou etal., 1989
Terpinolene Hinou etal., 1989
Caryophyllene Megalla etal., 1980

Alcohols
Octyl alcohol Hinou etal., 1989
Linalool Hinou etal., 1989
Geraniol +++ Hinou etal., 1989
Megalla et al., 1980
Nerol Hinou etal., 1989
Citronellol Hinou etal., 1989
Terpineol Hinou etal., 1989
Borneo1 ++ Megalla et dl., 1980
Nerolidol Hlnou etal., 1989
Farnesol ++ Megalla etal., 1980

Aldehydes
Citral - +++ +tt Hinou etal., 1989
Citronella1 + +tt + ++ Megalla et al., 1980
Myrtenal - - +++ Hinou etal., 1989

Ketones
Carvone - - +tt Hinou etal., 1989
a-Thujone - - - Hinou etal., 1989
Pulegone - +++ +++ Hlnou etal., 1989
Camphor - - - Megalla etal., 1980

Phenols
Thymol + t+t ++ +++ Megalla et al., 1980
Carvacrol + +++ +++ tt+ Megalla et dl., 1980
Hlnou etal., 1989
Eugenol + +tt + +++ Megalla et dl., 1980

Esters
Neryl acetate +++ - +++ Hinou etal., 1989
Geranyl acetate - - +tt Hinou etal., 1989
Linalyl acetate +++ +tt +++ Hinou etal., 1989
Bornyl acetate - - - Hinou etal., 1989
Isobornyl acetate - - tt+ Hinou etal., 1989
 The nzedicinal and non-~nedicinaluses of thyme 271

Terpenyl acetate - +++ +++ Hinou etal., 1989

Methyl benzoate + ++ + ++ Megalla et al., 1980

Oxides
Cineole - -
- Megalla et dl., 1980

Notes
Results are expressed as growth ~ n l i i b ~ t ~inoan
n agar overlay technique assay.
(-)area of ~ n h ~ b i t i ominor
n than 7 m m .
(+) area of ~ n h l b i t i o nbetween 7-10 m m .
(++) area of lohibition between 11-16 mm.
(+++) area of Inhibition greater than 16 m m .

Table 10.3 Antimicrobial activity of essential oils from Thymjir

gra?zatensisand Thy?/zwserpylloides ssp. gadorensis

Test mzcro-organzsnzs Thj~ttzz~s Thyrizzlr serpyllozdes

granatensts ssp gadorensis

Escherichza rolz (-) (+++)

Psezido?tzovtasJlzlo~e~cens (-1 (-+HI
Citrobacterfreundii (+) (+*)
Micrococczls lutei~i (+) (+++I
Staphylococcz~saurexs (+I (-+HI
Bactllzis cerez~s (+) (+*I
Bacilliis /nacerans (+) (+++)
Bacillz~snzegaterizo/z (+I (++I
Mycobacteriunz phlei I*( (*+)
Ca?zdidaalbzcans (-1 (+++I
Notes
Results are expressed as growth inhibition in an agar overlay technique assay.
(-)area of inhib~tlonminor than 7 mm.
(+) area of l n h l b ~ t ~ obetween
n 7-10 mm.
(++) area of inhibit~onbetween 11-16 mm.
(+++) area of inhrb~tiongreater than I 6 m m .
Source: Cabo e t al., 1986a; Crespo et al., 1990.

carvacrol, and tr-sabinene hydratelcis-myrcenol-8 respectively as main compounds.

Logically the oils of these chemotypes vary in their minimal inhibitory concentration
(MIC) values obtained. The most active oil was proven to be that of the thymol
chemotype followed by the carvacrol and geraniol type; the linalool type showed
similar activity to that of the geraniol type. The oils of the other chemotypes were
much less active (Simeon de Bouchberg etal., 1976; Lens-Lisbone etal., 1987).
When accepting that the chemistry of the essential oils is responsible for their anti-
microbial activities, as was explained above, it becomes obvious that all factors influ-
encing the chemical composition of essential oils within the plant indirectly influence
their antimicrobial activity. Climatic conditions are known to modify the chemical
composition of the essential oils and therefore, climatic conditions indirectly influence
the antibacterial activities. For example, Kowal and Kuprinska (1979) found large
differences in the MIC values for the essential oils of T. pulegzoides from different regions
272 Antonio Zarzuelo and Esperanza Crespo
in Croatia. Also Cabo etal. (1986b) have found considerable differences in the antimicro-
bial activity of the essential oils obtained from different populations of T. grandtensis
collected in southeastern Spain. Another factor determining the variability of the oil
composition of thyme is the stage of the plant growth during harvest. Arras and Grella
(1992) studied the influence of the harvest time of T. capitatw (Thymdra capitata) on the
fungisraric effects of its essential oil.
Although the antimicrobial action is to a large extent attributed to the essential oils,
non-volatile constituents also have been described to possess antimicrobial activity,
such as saponins and resins. These two constituents from T. capitatus (Thymbra capitata)
inhibited the growth of several bacteria and fungi (Kandil etal., 1994). It has recently
been demonstrated that the watery extract of thyme (T. vulgaris) showed a strong inhibi-
tion of Helicobacter pylory reducing both, its growth and potent urease activity (Tabak
etal., 1996). Clinical trials confirming this property would be of significant interest,
since this microorganism plays a role in the etiology of gasrroduodenal ulcers.
Finally we will refer to the techniques commonly used to evaluate antibacterial
activity. They can be classified depending on whether they require a homogeneous
dispersion in water or not (Janssen etal., 1987). (a) The agar overlay technique does not
require a homogeneous dispersion in water. Discs, holes or cylinders are used as reser-
voirs containing the essential oils to be tested and are brought into contact with an
inoculated medium and, after incubation, the diameter of the transparent zone around
the reservoir (inhibition diameter), where the microorganisms have been destroyed by
the action of the essential oil, is measured. The method can be modified when the reservoir
is placed in the lid of the Petri dish, thus excluding transport by diffusion (Pellecuer
etal., 1980). (b) Dilution techniques require a homogeneous dispersion of the oil. Since
essential oils are insoluble in the watery liquid culture medium, non-ionic emulsifying
agents such as Tween or Spans are needed (Allegrini and Simeon de Bouchbetg, 1972).
Although the addition of an emulsifying agent introduces an extra component with
respect to activity and possible interactions, this method has proved easily reproducible
(Janssen etal., 1987). However, other dilution methods that avoid the use of tensio-
actives have been proposed, such as the solution of the essential oil in DMSO (Melegari
etal., 1985), or in the formation of a stable suspension for 24 h in a sterile watery
solution of agar (Lens-Lisbonne etal., 1987).
The techniques to determine the qualitative bacteriostatic activity are based on the
agar overlay technique. Quantification of the antimicrobial activity is generally estab-
lished by determining the MIC values. The MIC values can be determined either by
using the agar overlay technique or the dilution techniques.

Antifungal effects
Several in vitro and in vivo screenings have shown that volatile oils, especially those of
the genus Thymus, may be used against fungal diseases (Roussel etal., 1973; Blakeway,
1986; Farag etal., 1986; Deans and Ritchie, 1987). Different screenings focused on
the essential oil of T. vulgaris and its effect on food spoiling yeasts (Conner and
Beuchat, 1984; Ismaiel and Pierson, 1990), especially Aspergillas (Farag etal., 1989), on
various dermatophytes (Janssen etal., 1988), and on some phytopathogenic fungi, e.g.
Rhizoctonzd solani, Pythium ultimum, Fwarium solani, and Calletotrichum lindemthianunz
(Zambonelli etal., 1996). Not only the oil of T. vulgaris but also the oils of other
Thymus species showed antifungal activity, e.g. that of T. zygis against Botrytis cinerea
 The medicznal and non-nzedicinal uses of thyme 27 3
(Wilson etal., 1997). The oil of T . serpyllum was found to be highly active against various
species of Penicillium, Fusarium and Aspergillus (Agarwal and Mathela, 1979; Agarwal
etal., 1979). Various oils, namely the oils of T . zygis (Cabo etal., 1978), T . hyemalis
(Cabo et al., 1982), T. vulgaris (Menghini etal., 1987), T. serpylloides (Crespo etal., 1990),
and T . baeticus (Cruz etal., 1993), inhibited the growth of Candida albicans.
The essential oil of T . vulgaris inhibits both mycelial growth and aflatoxin synthesis
by Aspergilhs parasiticus (Tantaoui-Elaraki and Beraoud, 1994) at only 0.1 per cent in
the medium. Therefore it is used as a preservative in agriculture, completely inhibiting
aflatoxin production on lentil seeds up to eight weeks of incubation (El-Maraghy,
1995). In addition, the essential oil of T. vulgaris exerts a protective effect in corn
against Aspergillusflavus, without producing phytotoxic effects on germination or corn
growth (Montes and Carvajal, 1998).
According to Agarwal and Mathela (1979) and Agarwal etal. (1979) the antifungal
activity of the essential oil of T . serpyllunz is attributable to thymol and carvacrol. They
cause the degeneration of the fungal hyphae which seems to empty their cytoplasmic
content (Zambonelli etal., 1996). The terpenic alcohols as well as the aldehydes,
ketones and some esters, also presented considerable activities, whereas the hydro-
carbons showed only low activities (Table 10.4). Terpenic alcohols which display
monoterpenic structure and hydroxyl group at terminal carbon (i.e. geraniol, nerol and
citronellol) have shown the highest activity. No difference was observed in the antifungal
activity between cis or trans isomer forms of these molecules. The components which
display carbonyl groups were also active in inhibiting fungal growth, showing the alde-
hydes (i.e. citral and citronellal) a higher activity than ketones. Similarly in terpenic
alcohols, this effect could be attributed to the presence of the functional group at a
terminal carbon.

Antiviral effects
Only few studies demonstrate the antiviral effects of thyme extracts. In 1967, Herrmann
and Kucera reported on the antiviral effects of Thymus serpyllunz and Spanish and French
thymes (Thynzus sp.) against Newcastle disease virus (NDV). The antiviral activity was
concentrated in the tannin fraction although non-tannin extracts also showed effects
attributed to the polyphenol precursor compounds of tannins. However the activity
was smaller compared to that observed with Melissa officinalis extracts. More recently
other studies failed to demonstrate antiviral effects of Thyme extracts against Rubella
virus (Zeina etal., 1996). The antiviral activity recently observed in other members of
the Labiatae family has been attributed to new di- and tri-terpenoid compounds that
appear to be specific inhibitors of HIV-1 protease (Min etal., 1998, 1999). Those com-
pounds have not yet been detected in the genus Thymas.

Spasmolytic effects
The spasmolytic properties are commonly considered as the major action of thyme
preparations. In this regard T . vulgaris is the most representative species. Therefore
many publications have focused on the effects of thyme preparations on smooth muscles,
especially rat and guinea pig intestines, such as duodenum and ileum, guinea pig trache2
seminal vesicles and rabbit jejunum. Two different protocols are typically followed:
(i) The isolated smooth muscle is first contracted using several agonists (acetylcholine,
Table 10.4 Antlfungal act~vityof individual components of the essential oils of thyme

Fz~sa~*iunz Aspergillus Trichoderfna References

nzonil$or?ize aegyptiacz~s viride

Hydrocarbons
Myrcene Megalla et al., 1980
Ocimene Megalla et al., 1980
Limonene Megalla et al., 1980
Dipentene Megalla etal., 1980
Phellandrene Megalla et al., 1980
A3-Carene Agarwal and Mathela,
1979; lMegalla etal., 1980
Agarwal and Mathela,
1979
Agarwal and Mathela,
1979
Agarwal and Mathela,
1979; Megalla etal., 1980
Caryophyllene Megalla et al., 1980
Alcohols
Linalool - - Megalla etal., 1980
Geraniol + +++ +++ Agarwal and Mathela,
1979; Megalla et dl., 1980
Nerol +++ +++ Megalla et al., 1980
Citronellol +++ +++ Megalla et al., 1980
Terpineol + - - Agarwal and Mathela,
1979; Megalla etal.,
1980
Borneo1 + + Megalla et al., 1980
Farnesol ++ ++ Megalla et al., 1980
A ldehydes
Citral +++ ++ Megalla et al., 1980
Citronella1 +++ +++ Megalla etal., 1980
Ketones
Carvone ++ ++ Megalla et al., 1980
Camphor + + Megalla et al., 1980
Phelzols
Thymol +++ +++ +++ Agarwal and Mathela,
1979; Megalla et al., 1980
Carvacrol +++ +++ +++ Agarwal and Mathela,
1979; Megalla etal.,
1980
Eugenol +tt +tt Megalla etal. , 1980
Esters
Llnalyl acetate +++ + Megalla etal., 1980
Geranyl acetate - - LMegalla etal., 1980
Oxides
Cineole - ++ + Agarwal and Mathela,
1979; Megalla etal., 1980

.kites
f ( g ~I-..~ resre:! as grc,\:.ih ini:i:,iiico 11' no agar c:i.,ari-y technique assay.
. . & .L ,.. .- 1.- ->~r.3%:
l7 .:c-:!la~ 7 m1n.
( I-) hzc!o of iniiibition be:Lr,c.cn 7--!C niiii
("-+' liiai3 o i ini:rb?tio~;beri~ri-eo1!--I 6 r - 1 s .
(+++) halo of inhibition greater thhn i G mm.
 The medicinal and non-medicinal wes oftbynze 27 5
histamine, adrenaline, nicotine and BaC1,) and the thyme preparations are subsequently
added until maximum relaxation is achieved. The spasmolytical effect is evaluated by
measuring the maximum relaxant effect and the ED50 (contraction that produces 50
per cent of the maximum spasmolytic response). (ii) The isolated smooth muscle is first
incubated with the thyme preparations; the modification of the dose-response curves
produced by the contracting agents are calculated. In this protocol, the relaxant agent
remains in the bath throughout the experiment. The use of various spasmogens with
different mechanisms of action causing muscle contraction can provide information on
the pharmacological basis of the spasmolytic properties. As reference substances atropine,
papaverine, and isoprenaline are used.

Spasmolytic activity of the essential oil of thyme ( T .vulgaris)

Debelmas and Rochat (1964) have shown that thyme oils in which phenols are the
predominant components have antispasmodic activity on intestinal smooth muscle
contracted by several agents. Later the same authors (Debelmas and Rochat, 1967)
studied the spasmolytic activity of the oils of different plants using various isolated
smooth muscles and contractor agents (Table 10.5). They found that thyme oil was the
most active, presenting an antispasmodic action of an unspecific type inhibiting the
contractions induced by all agents tested. These initial studies were carried out with
water saturated with the essential oil, trying to overcome the problems to bring the
hydrophobic essential oil into contact with the isolated organ.
Reiter and Brandt (1985) studied the effects of the volatile oils of 22 plants from 11
different families on tracheal and ileal smooth muscles of the guinea pig (Table 10.6).
All the oils showed relaxant effects on the tracheal smooth muscle developing shortly
after addition. The most potent oils were (in the order of potency): angelica root, clove,
elecampane, basil and balm. Sixteen oils inhibited the phasic contraction of ileal prep-
arations with ED50 values between 4.5 and 76 mgll. With regard to the relaxant effects,
the majority of oils were 1.4-8.4 times more potent on the ileal than on the tracheal

Table 10.5 Relaxant effect of volatile-oil-saturated water from different plants, versus the contractions
induced by several contractor agents

Aninzal Rat Guinea pig Rabbit

Isolated 77zuscle Seminal ve~icles Duodenu77z Duodenzlnz Ileutiz Aorta Jejunu7iz

Contractor agent Adrenalnze Acetylcholine BaCl, Hz~ta??zine Adrenaline Nicotine

Plants
Chenopodium 0
Clove i
Caraway 0
Sage 0
Thyme ++
Balm 0

Notes
k,< 2 0 % of inhibition.
+, 2 0 4 0 % of inhibition.
++, 40-60% of inhibition.
+++, > 6 0 % of ~ n h ~ b i t i o n .
Source: Debelmas and Rochat, 1967
27 6 Antonio Zarzuelo and Esperanza Crespo

Table 10.6 Relaxant effect (ED50) of d~fferentvolatile oils on tracheal and ileal
longitudinal smooth muscle of the guinea pig

Species; pharnzaceutical Trachea EDSO Ileum EDSO EDSO tracheal

preparation (&I) (mgll) ED50 ileunz

Thynzus vulgaris L.
thyme oil
Melissa oficinalis L.
balm leaf oil
Mentha piperita L.
peppermint oil
Ocinzum basilicum L.
basil oil
Saluia oficinalis L.
sage oil
Reference drug trachea ED50 ileum ED50 ED50 tracheal
(nmolil) (nmol/l) ED50 ileum
Isoprenaline 3.9F0.7 21 F 1.0 0.19
Papaverine 240 f 0.9 3700f 1.6 0.06

Note
Voiatiie oils were solubilised in water by means of polyoxyethylene fatty ester, Arlatone 285
Source: Reiter and Brandt, 1985.

muscle. This ratio was almost eight-fold for the essential oil of T. vztlgurzs. The authors
divided the 22 volatile oils investigated into three groups according to their different
effects on the mechanical behaviour of the stimulated ileal mysenteric plexus-longitu-
dinal muscle preparations. Together with 15 others thyme oil belongs to the first group
which had predominantly relaxing effects.

Spasmolytic activity of the essential oils of different Thymus species

Between 1985 and 1990 our research group carried out several studies aimed at studying
spasmolytic activities of a variety of essential thyme oils from plants collected in eastern
Andalusia, relating them with their components. In Table 10.7, the ED50 values of
these essential oils and the chemical compositions (only functional groups) are given.
All the oils produced a relaxant effect against acetylcholine-induced contractions in
isolated rat duodenum. However marked differences existed which might partly be
explained by the differences of the chemical composition. One may highlight the
following:

Oils containing higher portions of phenolic components have shown higher spas-
molytic potency, namely the oils of T. zygis (Cabo etul., 1986a) and T. orospedunus
(Cabo etal., 1987).
The oil of T. grunutensis although lacking phenolic compounds showed a powerful
relaxant effect (Cabo etal., 1986a). This could be due to the high content of terpene
hydrocarbons, which also may explain the high effect of T. orospedunw containing
both, phenols and hydrocarbons.
 The medicinal and non-medicinal wes of thyme 277

Table 10.7 Relaxant effects (ED50) versus contractions induced by acetyl-

choline in isolated rat duodenum and quantitative composition of
different essential oils of thyme when collected in full bloom

Sample ED50 Components %

(Pg/ml)

T. zygis 6.5 Hydrocarbons

Alcohols /esters
Ketones
Aldehydes
Ethers
Phenols
Hydrocarbons
Alcoholslesters
Ketones
Aldehydes
Ethers
Phenols
Hydrocarbons
Alcohols/esters
Ketones
Aldehydes
Ethers
Phenols

Alcoholslesters
Ketones
Aldehydes
Ethers
Phenols
T. longiflorus Hydrocarbons
Alcoholslesters
Ketones
Aldehydes
Ethers
Phenols

Note
Volatile 011s were emulsified in water with Tween 20 at a proportion of 911 pip

Essential oils lacking phenolic components and with low hydrocarbon levels are
less potent, documented by the results obtained with oils of T. baeticzts (Cruz etal.,
1989b) and T. long$Iorus (Zarzuelo etal., 1989).
The lower spasmolytic effect of T. longzflorzts (Zarzuelo etal., 1989) in comparison
with that of T. baeticzts (Cruz etal., 1989b) may be due to a higher level of ethers
(1,s-cineole).

The above results prompted our group to investigate the spasmolytic potency in more
detail (Table 10.8, Cabo etal., 1 9 8 6 ~ )Taking
. all the data obtained into consideration
one can say that indeed the phenolic components (thymol and carvacrol) as well as the
terpene hydrocarbons (myrcene and caryophyllene) presented higher spasmolytic
278 Antonio Zarzzlelo and Esperanza Crespo

Table 10.8 Relaxant effects (ED50) of different components of essential oils versus contractions induced
by acetylcholine and BaC12 in isolated rat duodenum. The relative potency was calculated in
comparison to papaverine

Agontst Antagonist ED30 (pM) Relative potency

Acetylcholine Carvacrol 6.67 i 0.86

Thymol 4.88 10.74
Myrcene 5.48 i 1.08
Caryophyllene 9.00k 2.14
Camphor inactive
Papaverine 8.55 i 1.09
Carvacrol +
7.73 1.22
Thymol 7.25 i 0 . 8 1
Myrcene inactive
Caryophyllene 1.34k0.25
Camphor inactive
Papaverine 3.18f 0.68

Note
The components of volatile oils were emulsificied in water by means of tween 20 at a proportion of 911 pip.
Source: Cabo et al.,1 9 8 6 ~ .

potency. Camphor was shown to be inactive whereas 1,s-cineole acted as a spasmogenic

and showed to increase rat duodenum contractions up to a maximum of 75 per cent of
acetylcholine induced contractions. This effect was competitively antagonised by atropine.
Consequently these results favour the hypothesis that 1,s-cineole acts as a partial agonist
at the level of the acetylcholine receptors (Zarzuelo etal., 1987; Ggmez etal., 1990) and
that the presence of 1,8-cineole in the oils diminishes their spasmolytic capacity.
The mechanism of the spasmolytic effect was studied by the modification of the
dose-response curves induced by acetylcholine and CaC12.All the essential oils included
in Table 10.7 modified the dose-response curves induced by acetylcholine, showing
a dosage-dependent decrease in the maximum effect suggesting a non-competitive
mechanism (Cabo etal., 198621; Cruz etal., 1989b; Zarzuelo etal., 1989). Such oils are
capable of inhibiting the contractions induced by CaC12 in high-KC1 ca2+-free
solution, also diminishing their maximum effect in a dose-dependent way (Cruz etal.,
1989b; Zarzuelo etal., 1989). Godfraind etal. (1986) demonstrated that ca2+-induced
contractions of a KC1-depolarized smooth muscle are due to an increased ca2+ influx
through voltage-stimulated type-L ca2+ channels. Therefore, the inhibitory effects of
essential oils in these concentrations may also be explained by a) an inhibition of ca2'
entry through voltage-stimulated channels into the smooth muscle andlor b) blocking
release of intracellular bound ca2'.
Several authors have investigated the spasmolytic mechanism of some phenolic
compounds of essential oils. Van den Broucke and Lemli (1982) and Cabo etal. ( 1 9 8 6 ~ )
studied the antagonistic effect of thymol and carvacrol on guinea pig ileum and rat
duodenum against the contractions induced by carbachol, histamine and BaC12 and
concluded that contractions induced by these spasmogenic agents were inhibited by the
phenols in a non-competitive antagonistic way.
There are very few studies which demonstrate the relaxant effect of the essential oil
of thyme on vascular smooth muscle. In experiments on mice, guinea pigs and rabbits
 The medicinal and non-medicinal uses of thyme 279
Guseinov etal. (1987) found that the essential oil of Kochi Thyme (T. kotchyanw) was
non-toxic and produced hypotensive effects in rabbits at concentrations of 1 mglkg.

Spasmolytic a c t i v i t y of T . vulgaris extracts

The therapeutic value of the thyme herb, indeed, depends on the quantity of phenols in
the essential oil (see above). Therefore, for all the Thymzls species, one has to prefer those
that contain a high concentration of thymol andlor carvacrol. However the presence of
a non-volatile principle in the Thymus species has always been supposed. This was
established by Van den Broucke and Lemli (1981) who examined the correlation
between the phenol content of T. vulgaris liquid extracts and the spasmolytic activity on
the muscles of the guinea pig ileum and trachea. The extracts showed a high spasmolytic
action, but no correlation between phenol content and activity could be observed. The
thymol and carvacrol concentration of extracts was much too low (<0.001 per cent) and
could not be responsible for the antispasmodic activity.
The authors continued their experiments testing flavonoids isolated from T. vulgaris
(Van den Broucke and Lemli, 1983) in vitro for their spasmolytic activity on the smooth
muscles of the guinea pig ileum and of the rat vas deferens (Table 10.9). Both the
flavones and the thyme extracts inhibited responses to agonists which stimulate specific
receptors (acetylcholine, histamine, noradrenaline) as well as to agents whose actions
are not mediated via specific receptors (BaC12). Flavonoids appeared to act as musculo-
tropic agents. Musculotropic spasmolysis is complex and the results of this study could
not clarify the events in the muscle completely. However, inhibition of ca2+ induced
contractions in K+ depolarized muscles pointed to a possible decrease in the avaibility
of ca2+. Flavones induced relaxation of the carbachol-contracted tracheal strip without
stimulation of the P,-receptors, which were blocked by propranolol. This relaxation

Table 10.9 Relaxant effects (pD'2) and potency relacwe to papaverine of thymonin, 8-methoxycirsilineol,
cirsilineol, luteolin, apigenin, papaverine and phentolamine on the guinea pig ileum and the
rat vas deferens versus different smooth muscle agonist

Antagonist Guinea pig ileum Rat was defeens

Carbachol Histamine BaC12 Noradrenaline

Thymonin 4.55k0.12
30
8-MeOH-Cirsilineol 4.46 k 0.23
27
Cirsilineol 4 . 6 9 k 0.10
46
Luteolin 4.76k0.10
54
Apigenin 4.75 2 0.23
52
Papaverine

Phentolamine

Source: Van den Broucke et al. 1982, Van den Broucke and Lemli, 1983
280 Antonio Zarzzlelo and Esperanza Crespo
could probably be due to an inhibition of the phosphodiesterase, followed by an
increase of the intracellular c-AMP level (Beretz etal., 1980).
The flavonoid pattern of T . satureioides differs from that of the other Thymus species
in the high portion of polymethoxylated flavones. The in vitro tracheal relaxant activity
of thyme extracts prepared from T. satureioides compared with that of the pure flavones
(same quantity of flavones), supports the action of a thyme extract to be almost
completely explained by the content on flavones independently from their degree of
permethoxylation (Van den Brouke etal., 1982). The affinity of methoxylated flavones
in the guinea pig ileum does not differ significantly from that of luteolin and apigenin.
However, methylation of the hydroxy groups of the flavone skeleton increases the relaxant
activity.
Further evidence of the spasmolytic effects of flavonoids was provided by Capasso
etal. (1991a). They screened 1 3 flavonoids for their effects on contractions in guinea
pig ileum induced by prostaglandin E,(PGE,), Leukotriene D4 (LTD*), acetycholine
and BaC1,. The flavonoids showed spasmolytic effects that may be due to a non-specific
action since they were found to be active against contractions induced by several
agents. Flavonoids have also been shown to inhibit electrically-induced contractions
(transmural electrical stimulations) (Capasso etal., 199lb). Flavonoids inhibit the con-
tractile responses probably through a reduction of calcium influx by way of calcium
channels and through inhibition of calcium release from intracellular stores, decreasing
the calcium concentration available for contractile machinery (Gilvez etal., 1996).

Spasmolytic activity of extracts of different Thymus species

Blizquez etdl. (1989) examined the spasmolytic activity of T . webbianw and T. leptophyllzs
extracts. Air-dried leaves and stems of these species were extracted with 70 per cent
aqueous methanol. The methanol was removed and the remaining aqueous fraction was
successively treated with diethyl ether, ethyl acetate and butanol. The results of this study
showed that the extracts of both plants have significant spasmolytic effects on isolated
rat duodenum. In general, a difference in the responses produced between differently
polar extracts was observed. In fact, the diethyl ether extracts (low polarity) produced
a dose-dependent reduction in acetylcholine-induced contractions at 1, 10, 100pglml
concentrations, whereas the ethyl acetate extracts (middle polarity) and the butanolic
(high polarity) were inactive at the same concentrations, but active only at higher
concentrations. T . webbianus consistently proved to be more active than T . leptophyllus.
Zafra-Polo etal. (1990) isolated two steroidal compounds with relaxant properties from
the diethyl ether extracts of T. webbianus. The compounds could be considered as derivatives
of stigmastenone and its isomer 0-sitostenone.
Blizquez etal. (1995) investigated the effects of a diethyl ether extract of the leaves
of T, leptophylhs on rat uterine and aorta strip muscle. It inhibited the contraction of
uterine smooth muscle at lower concentrations than those observed for aorta strips. Rat
uterus experiments with and without extracellular calcium yielded similar ED50 values
suggesting a non-specific mechanism for the relaxant activity. In the presence of extra-
cellular calcium, the extract inhibited the contractile response of rat aorta induced by
K+ depolarising solution, and had a lower inhibitory effect on noradrenaline-induced
contraction.
An ethanolic extract of the leaves of T . orospedanus has been shown to significantly
decrease arterial blood pressure in Wistar rats, at a dose of 150 mglkg during the first hour
 The nzedicinal and non-medicinal zlseJ of thyme 28 1
following the administration, whilst the 300mglkg dose continued to show hypotensive
effects for up to 5 h (Jimknez etal., 1988). Flavonoids have also been shown to have
vasodilator effects in isolated rat and rabbit vascular smooth muscle (Duarte etal.,
1993a; Herrera etal., 1996). This vascular smooth muscle relaxation has been attributed
to several mechanisms including decreased transmembrane 4 5 ~ a 2uptake
+ (KO etal.,
1991) and inhibitory effects on CAMPand cGMP-phosphodiesterases (Beretz etal., 1980)
or on protein kinase C (Duarte etal., 1993b).

Antioxidant effects
It was only in the 1970s that scientists realised that the human body constantly creates
free radicals and eliminates them by a series of antioxidant defense mechanisms. When
free-radical generation exceeds the capacity of antioxidant defenses, the result is "oxidative
stress". It occurs in many human diseases and sometimes makes a significant contribution
to their pathogenesis. In literature several publications are dedicated to the antioxida-
tive effects of plant extracts with phenolic compounds (Alscher and Hess, 1993).
Recently Chung etal. (1997) studied the effects of methanol extracts from 51 plant
species on OH-radical scavenging. Mustard varieties, thyme, oregano, and clove all
exhibited strong scavenging activity.
There are several papers showing that both the essential oil and the flavonoids of
T. vulgaris are potent antioxidant agents. Dorman etal. (1995) studied the antioxidant
properties of the essential oil of T. vulgaris (0.75-100ppm) among others. The antioxidant
properties were evaluated in three avian thiobarbituric acid reactive substances (TBARS)
assays using egg yolk, one-day-old chicken liver or muscle from mature chicken. T. vul-
garis was one of the most effective antioxidants in the egg yolk assay besides Monarda
citriodora var. citriodora and Myristica fragrans.
Deans etal. (1993) investigated the protection of polyunsaturated fatty acids within
the liver of old mice by ingestion of culinary and medicinal plant volatile oils obtained
by hydrodistillation. This protection effectively reverses the normal trend in polyunsat-
urated fatty acid metabolism during aging where a decrease in level is concomitant with
a reduction in tissue function and integrity. The essential oil of thyme was overall the
most effective agent in this protective effect.
Ternes etal. (1995) showed that carvacrol, thymol and p-cymene-2,3-diol, all the
three components of thyme oil, exhibit antioxidant activities. The authors determined
the concentration of each substance in different foodstuffs containing thyme extracts
as well as their stabilities at different temperatures. Schwarz etal. (1996) assessed the
antioxidant activity of thyme phenols (Rancimat method 110 OC, Schal test 60 OC)
and showed that p-cymene-2,3-diol was the most active one being more active than
a-tocopherol and butylated hydroxyanisole. Five thyme species (T. vulgaris, T. pseudola-
nuginosw, T. citriodorus, T. serpylhm and T. doerfleri) were analysed by means of High-
Performance Liquid Chromatography (HPLC) for all 3 compounds. The highest amounts
were found in T. vulgaris.
Pearson etal. (1997) investigated the potential antioxidant activity of various plant
phenolics, namely carnosic acid, carnosol, and rosmarinic acid (in rosemary extracts), thy-
mol (in thyme extracts), carvacrol (in origanum extracts), and zingerone (in ginger
extracts), using aortic endothelial cells to mediate the oxidation of low-density lipoprotein
(LDL). The extent of oxidation was determined spectrometrically by measuring the
absorbance at 234 nm of the conjugated dienes. Their relative antioxidant activities
282 Antonio Zarzuelo and Esperanza Crespo
decreased in the order carnosol > carnosic acid approximate to rosmarinic acid > thymol >
carvacrol > zingerone.
Flavonoids are abundant in diverse species of thyme. They are potent antioxidants
which ate responsible for many of the beneficial effects of these plants. The electron-
donating properties of flavonoids have been repeatedly emphasised as the basis of their
antioxidant action. Common antioxidative flavonoids, like luteolin and quercetin, isolated
from various species of thyme, have shown potent antioxidant activity in viva as much
as in vitro (Gglvez etal., 1995a,b).
From leaves of T. vzllgaris Haraguchi etal. (1996) isolated two antioxidative com-
ponents by a bioassay-directed fractionation: a biphenyl compound, 3,4,3',4'-tetra-
hydroxy-5,5'-diisopropyl-2,2'-dimethylbiphenyland a flavonoid, eriodictyol. Their
antioxidant effects on biological systems were studied in three different biological
systems: inhibition of superoxide anion production in the xanthineixanthine oxydase
system, inhibition of microsomal peroxydation, inhibition of hemolysis of human erythro-
cytes. Both the new biphenyls as well as eriodictyol showed outstanding antioxidant
""
effects.

Further effects

Antiparasitic effects

An extract of T. valgaris shows antiparasitic properties against Leishmania nzexicana

(Schnitzler etal., 1995), because it inhibits the mitochondria1 D N A polymerase (IC50
value was 0.82 mgiml). Thymol was mainly responsible for this effect (Khan and Nolan,
1995). The essential oil of this species was also active against diverse phytonematodes,
the oxygenated compounds being partially responsible for the nematicidal effects
(Abd-Elgawad and Omer, 1995).
Perrucci etal. (1995) evaluated the in vitro ascaricidal properties of some natural
monoterpenoid constituents of several essential oils against rabbit mange mite (Psoroptes
caniczlli) by direct, external contact and by inhalation. The natural terpenoids assayed
were: hydrocarbons (limonene, myrcene, y-terpinene), alcohols (linalool, geraniol,
nerol, terpinen-4-01, a-terpineol) and phenols (thymol, eugenol), an ester (linalyl acetate)
and an ether (estragole). Because the test components represent different chemical classes,
it was also possible to discern in a preliminary fashion a correlation between chemical
structure and ascaricidal activity. All the monoterpene hydrocarbons, either acyclic (i.e.
myrcene) or cyclic (i.e. limonene and y-terpinene) did not show any miticidal activity
at the doses tested (1.0 per cent, 0.25 per cent and 0.125 pet cent). The double-bond
position andlor number seems to be unimportant for this kind of biological activity.
In contrast, the terpene alcohols, such as linalool, geraniol, nerol, menthol, terpinen-4-01,
and a-terpineol, were able to kill nearly 100 per cent of the mites at the dose tested.
Therefore, the oxygenated functional groups potentiate the ascaricidal properties among
these compounds. Neither the acyclic (i.e. linalool, geraniol, nerol) nor cyclic (i.e. menthol,
terpinen-4-01, a-terpineol) nature of the compound appeared to influence the miticidal
activity. Similarly, the site of linkage to the ring or to a side chain, as well as the nature
of the hydroxyl group (primary, secondary, or tertiary), does not influence the activity.
The cisltrans isomerism represented by nerol and geraniol seems to be important.
Thymol and eugenol killed nearly 100 per cent of the parasites at all dosages assayed
in the direct contact test, indicating that a phenolic function can enhance the miticidal
characteristics of terpenes. The low susceptibility of mites to linalyl acetate, particularly
 The medicinal and non-medicinal uses of thyme 283
at the lowest doses, could be related to the esterification of the oxygenated function.
Estragole, structurally close to eugenol, but with a methylated phenolic group, exhibited,
at a concentration of one per cent, an activity comparable to that of the same dose of
eugenol. However, this action decreased (63 per cent) at 0.25 per cent and disappeared
completely at 0.125 per cent. These results indicate that the best miticidal activity of
the monotetpenes examined in the direct contact test can be related to compounds with
free alcoholic or phenolic functional groups.

Insecticidal effects

Recently it was demonstrated that aromatic plants present a double insecticidal effect:
by direct toxicity on adult insects and by inhibiting reproduction. The most efficient
plant in this regard belongs to the Labiatae family (Regnaultroger and Hamtaoui,
1997). Therefore one can profit using the essential oils of T. vulguris and T. serpyllum in
addition to a fumigant against Anathoscelides obtectus Say (Coleoptera, Bruchidae), a fre-
quent pest that damages its host plant, the kidney bean (Phaseolzs vulgaris L.) in the
field and during storage (Regnaultroger etal., 1993). The oils have a toxic effect on adult
insects and also inhibit the reproduction through ovicidal and larvicidal effects (Reg-
naultroger and Hamraoui, 1994). This insecticidal action is also produced by other
components of the species such as non-volatile phenols, non-proteinic amino acids, and
flavonoids (Regnaultroger and Hamtaoui, 1995).
The essential oil of T. vulgaris and thymol shows activity against Tetranychus urticae.
Thymol was shown to be more potent than thyme oil as a deterrent factor for reducing
egg laying by the mite. Mortality percentage reached 100 per cent with both materials
used; however, at low concentrations the effect again was more pronounced applying
thymol than applying thyme oil (El-Gengaihi etal., 1996).
Karpouhtsis etal. (1998) have demonstrated the genotoxic effect of thymol on the
somatic mutation and recombination test on Drosophilu and that this effect could con-
tribute to the insecticidal action of essential oils such as T. vulgaris. Other components
of the oils such as carvacrol, y-terpinene and p-cymene have been found to be ineffective.
Another pest species sensitive to the essential oil of T. vzlgaris is Spodoptera littoralis.
Feeding larvae with leaves treated with the essential oil reduced the successful develop-
ment and egg production (Farag etul., 1994).
Lee etal. (1997) evaluated the acute toxicity of 34 naturally occurring monoterpenoids
against three important arthropod pest species: the larvae of the Western corn rootworm,
Diabrotica virgzJera LeConte, the adult two-spotted spider mite, Tetranychus urticue
Koch, and the adult house fly, Musca domesticu L. Thymol was the most topically toxic
against the house fly, and citronellol and thujone were the most effective on the Western
corn rootworm. Most of the monoterpenoids were lethal to the two-spotted spider mite
at high concentrations; terpinen-4-01 was especially effective.

PHARMACOKINETICS OF THYMOL A N D CARVACROL

Data about the pharmacokinetics of essential oils are scarcely available, but there can be
found a few on the phenolic terpenes, thymol and carvacrol. In an early publication
Schroder and Vollmer (1932) described thymol and carvacrol to redistribute rapidly to
the blood and kidneys following oral administration. These observations were made in
experiments with animals.
284 Antonio Zarzuelo and Esperanza Crespo
The metabolism of carvacrol and thymol has been studied in rats. It was found that
the urinary excretion of metabolites was rapid and only very small amounts were excreted
after 24 h. Although large quantities of carvacrol and, especially, thymol were excreted
unchanged (or as their glucuronide and sulphate conjugates), extensive oxidation of the
methyl and isopropyl groups also occurred. This resulted in the formation of deriva-
tives of benzyl alcohol and 2-phen~lpropanoland their corresponding carbox~licacids.
In contrast, ring hydroxylation of the two phenols was a minor reaction (Austgulen
etal., 1987).
Takada etal. (1979) investigated the metabolism of thymol in rabbits and humans.
Thymol glucuronide featuring an intact aglycone was isolated from the urine of thymol-
medicated rabbits and identified as an acetyl derivative of methyl glucuronate. The
hydroxylated product of thymol, thymohydroquinone, was detected in small amounts
in the urines of thymol-medicated humans. It was presumed that thymolhydroquinone
is excreted as an ethereal sulfuric acid conjugate.

TOXICOLOGY OF THYME OIL

Toxic effects of the vegetable parts of thyme, T. vulgaris, have not been published, but
it is important to mention that a certain level of toxicity can be found in the essential
oil of thyme (acute oral LD50 = 4.7 giKg rat). This toxicity has been attributed by
some authors to thymol and carvacrol (Dilaser, 1979), their acute oral LD50 being
0.88-1.8 giKg and 0.1-0.18 giKg, respectively. Furthermore, these phenols cause skin
irritations and especially irritations of the mucosa, which precludes patients with
gastroduodenal ulcers from the use of the essential oil. Thus undiluted thyme oil was
found to be severely irritating to both mouse and rabbit skin, however it produced no
irritation on human subjects when tested at 12 per cent (Tisserand and Balacs, 1995).
Hypersensitivity reactions have also been reported for this essential oil (Tisserand and
Balacs, 1995; Benito etal., 1996; Lemier etal., 1996); therefore it is strongly recom-
mended to perform a tolerance test prior to attempting internal administration.
Various essential oils from Thymus contain considerable quantities of camphor and
other terpenic ketones, which are known to produce convulsions and epilepticlneurotoxic
crises (Dupeyron etal., 1976; Steinmetz etal., 1980). Limonene, which is a common
component of thyme oil, is capable of diminishing the incidence of tumors in experi-
mental animals treated with tumor-inducing agents (Tisserand and Balacs, 1995). How-
ever upon oxidation of the molecule the risk of carcinogenesis is increased and it
behaves as a catalysing agent. Therefore it is important to use fresh, non-oxidized essential
oil of thyme in phytotherapy and aromatherapy.
Due to the toxic effects the use of the essential oil during pregnancy and lactation is
contraindicated (Peris etal., 1995).

THE NON-MEDICINAL USE OF THYME

T h y m e as a food preservative
Due to their antimicrobial and antioxidant qualities numerous aromatic plants, such
as thyme, have been used and are still being used as food preservatives (Shelef, 1983;
 The medicinal and non-medicinal uses of thynze 285
Nakatani, 1992; Amr, 1995). As was described before, the essential oils of thyme
present a marked antimicrobial activity. This activity has been demonstrated to
include bacteria responsible for alterations in food (Essen and Karapinar, 1986;
Akgul and K i v a n ~ 1988a).
, Aureli etal. (1992) carried out a study on the antimicro-
bial activity of diverse essential oils of plants widely used in the food industry
against Listeria monocytogenes (bacteria implicated in alterations in food). Only the
essential oils of cinnamon, clove, marjoram, pepper and thyme presented antimicro-
bial activity.
Researchers have also demonstrated that a number of aromatic plants, including
thyme, have a marked antifungal activity against food spoiling fungi (Akgul and Kivan~,
1988b; Salmer6n etal., 1990). The high antimycotic activity of clove and thyme was
tested for their possible use as preservatives for agricultural commodities by El-Maraghy
(1995). Both species completely inhibited aflatoxin production in lentil seeds for an
eight week incubation period.
Antioxidant activity can also be responsible for a preservative activity, especially in
preventing oxidation of lipids in food. This was studied by Budincevic etal. (1995) who
tested ethanol extracts of T. marschallianus using tallow and lard as the substrates, at
60 OC in the Rancimat apparatus. The extracts showed antioxidant effects with the sub-
strates processed at 60C but not at 100 OC. Adding citric and malic acid a synergistic
effect could be observed. Dorman et al. (1995) demonstrated the antioxidant activity of
the essential oil of T. vulgaris in TBARS using egg yolk, one-day old chicken liver or
muscles from mature chickens.
Botsoglou etal. (1997) evaluated the effect of dietary thyme on the oxidative stability
of egg shells over a 60-day refrigerated storage period. In addition, the influence of
dietary thyme and of the storage time on the oxidative stability of liquid yolks adjusted
to various p H values and agitated in the presence of light was investigated. Results
show that malonaldehyde was not produced during the storage of egg shells. It was also
evident that rhyme treatment reduced the oxidation of liquid yolk, which was signifi-
cantly increased by light and acidity. The authors proposed that thymol is the most
important antioxidant component of thyme, but that there must be other components
in thyme which act synergisricly with thymol.

The cosmetic uses of thyme

Thyme oil, in general, is used in many cosmetic preparations, such as deodorants, because
of its capacity to suppress smells (Gonz6lez and Muhoz, 1980; Brasseur, 1983) and for
its antimicrobial properties. The oil finds some use in soap perfumes where its power
and freshness can introduce a hint of medicinal notes, often desirable in certain types of
soap or detergent. The oil exerts an excellent masking effect over tarry odors (Arctander,
1960). Added to lotions perfumes or colognes in trace amounts, thyme oil may lend
body and sweet freshness. Therefore it is used in the composition of cosmetic creams
and milks, eau-de-cologne (frequently accompanied by lemon and bergamot), and soapy
solutions to disinfect surgeons' hands (Valnet, 1964). These cosmetic products are useful
to fight acne and skin complaints.
The essential oil of thyme and thymol are also used in the production of toothpaste
and mouthwashes (Marsh, 1992; Banoczy etal., 1995). This essential oil peroxidised
to 1 0 per cent in a soapy solution destroys the microbial flora in the oral cavity in
3 min.
286 Antonzo Zarzuelo and Esperanza Crespo
The culinary uses of thyme
Thyme is widely used as a seasoning especially in the Mediterranean kitchen. It has a
strong but agreeable aroma and is pleasant in greasy or fatty food, such as sausages,
bacon and other fatty meats and even strong cheeses. Along with rosemary it consti-
tutes a highly recommended seasoning for pizzas and similar products (Pahlow, 1979).
It can be added (with care) to sauces, soups, meat and fish dishes. The fresh leaves give
flavour to salads. In the liquor industry thyme is used to give flavour and aroma, T. moroderz
being the aromatic plant used to produce the 'Licor de Cantueso' and T. vzlgaris partic-
ipating in the formulation of several liquorices produced in the Spanish Eastern and
Balearic regions.

ACKNOWLEDGEMENTS

The authors would like to thank Dr Elisabeth Stahl-Biskup for her contribution to the
present chapter with the sections entitled "Thyme in Phytotherapy", "Thyme in Aroma-
therapy" and "Thyme in Homoeopathy", whose authorship is this way recognised.

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11 Thyme as a herbal
drug - pharmacopoeias
and other product characteristics

INTRODUCTION

Traditional medicine has been using thyme for many centuries. In the past the plants
were collected in the countryside and only the herb collectors were responsible for the
quality of the herbs, which differed considerably. Nowadays, in modern phytotherapy,
increasing requirements concerning the safety of drugs must be fulfilled. The current
status of drugs, including herbal drugs, has to take into consideration various legal
regulations so that the products can obtain the "drug status" and be sold in the pharma-
ceutical market according to the Medicines Acts of European and other countries. An
increased use of herbal medicines requires a thorough evaluation of the quality, overall
safety and effectiveness of phytomedicines.
The status of herbal drugs is not the same all over the world. In Europe, particu-
larly in Germany, herbs and phytomedicines are accepted and integrated into medi-
cine and pharmacy. In 1978, the German Ministry of Health established the
Commission E, a panel of experts charged with evaluating the safety and efficacy of
the herbs available in pharmacies for general use. The Commission reviewed over 300
herbal drugs and published its results in the form of monographs in the Bundesan-
zezger, the German Federal Gazette. These monographs provide guidelines for the
general public, health practitioners, and companies applying for the registration of
herbal drugs.
In the US herbs and phytomedicines are also experiencing explosive growth in phar-
macies and other mass-market retail outlets. It is said that the herb sector of the dietary
supplement market represents one of the biggest financial investment opportunities
since the advent of the high-technology industry. However, such a product cannot make
a statement that is deemed 'therapeutic' or imply that is useful to diagnose, treat, cure,
or prevent any disease. A petition by European and American phytomedicines manu-
facturers has requested that the Food and Drug Administration (FDA) grants well-
researched European phytomedicines the status of old drugs so that they would not
have to be evaluated by the prohibitively costly new drug application process, but the
FDA has not responded to this petition.
The edition of the German Commission E Monographs (English version: Blumenthal,
1998) is a result of intensive efforts to preserve and maintain the herbal remedies in the
status of drugs in accordance with the German Drug Law. Further intensive efforts on
herbal remedies were undertaken by the European Scientific Cooperative on Phytotherapy
(ESCOP) as well as by the World Health Organization (WHO). Both organizations
evaluated herbal remedies for their therapeutic benefit and safety in order to promote
harmonization in the use of herbal medicines with respect to levels of safety, efficacy,
and quality control. The herbal drug monographs of all three organizations consider
a clear definition of the herbal drug, the effectivity, the side-effects, interactions, toxi-
cological data and dosage. Neither the German Commission E nor the ESCOP mono-
graphs contain standards for assaying the quality and purity of herbal drugs. This is left
to the Pharmacopoeias, and quality standards can be found in the European Pharmaco-
poeia (3rd ed. 1997, Supplement 2001) or in the national Pharmacopoeias of Germany
(Deutsches Arzneibuch, DAB 2000, and Deutscher Arzneimittel Codex, DAC 2000),
the British Pharmacopoeia (BP 2000), the British Herbal Pharmacopoeia (BHP 1979),
the Pharmacopke Franfaise (PF X 2000), and the Swiss Pharmacopoeia (Pharmacopoea
Helvetica 8, Suppl. 2000). Pharmacopoeia1 summaries for quality assurance can also be
found in the W H O monographs.
Such a detailed introduction is necessary to understand the role of the monographs
quoted in the following paragraphs. The fact that thyme has found consideration in the
monographs of the Commission E, the ESCOP and the W H O as well as in the European
Pharmacopoeia reflects the importance of thyme among the herbal remedies. This is
also documented in the various sample product formulations which can be found on the
drug market.

MONOGRAPHS FOR THYME I N PHYTOPHARMACY

Commission E monographs
The work of the expert group of the Commission E (1984-1994) is closely connected
with the laws regulating the registration of drugs in Germany (German Drug Law).
The task of the experts was to evaluate scientific knowledge published on herbal rem-
edies resulting in a decision whether an herbal drug is approved ("positive monographs",
186 monographs, monopreparations only) or not approved ("negative monographs",
110 monographs). The latter category concerns herbals with no plausible evidence of
efficacy, or with potential benefits outweighed by safety concerns. Applying for the drug
registration of an herbal product the manufacturers can refer to the positive monographs
as a proof of effectiveness and safety of their products, but they have to complete their
documents by further publications or their own experiments, because the Commission
E monographs are no longer up to date.
Among the Commission E Monographs thyme is considered with two approved
(positive) monographs: Thymi herba (Thymw vulgaris) and Serpylli herba (T. serpyllum).
Both will be quoted in the following paragraphs. The category "uses" shows common
thyme to be therapeutically more beneficial than wild thyme.

Commission E: Thyme - Thymi herba (Thymiankrazlt)

Bundesanzeiger, published December 5 , 1984; revised March 13, 1990, and December 2,
1992.

Conzposition of dwg: Thyme is constituted of the stripped and dried leaves and flow-
ers of Thynzw vulgaris L., Thymus zygis L. (family Lamiaceae), or both species as well
 Thyme as a herbal dwg 29 5
as their preparations in effective dosage. The herb contains at least 0.5 per cent
phenols, calculated as thymol (ClOH140, M W = 150.2) based on dried herb.
Uses: Symptoms of bronchitis and whooping cough. Catarrhs of the upper respira-
tory tract.
Contraindications,side effects, interaction with other drzlgs: None known.
Dosage: Unless otherwise prescribed: 1 to 2 g of herb for 1 cup of tea, several times
a day as needed; 1 to 2 g fluid-extract, 1 to 3 times daily; 5 per cent infusion for
compresses.
Mode of adnzinistration: Cut herb, powder, liquid extract or dry extract for infusions
and other galenical preparations. Liquid and solid medicinal forms for internal and
external application. Note: Combinations with other herbs that have expectorant
action could be appropriate.
Actions: Bronchoantispasmotic, expectorant, antibacterial.

Commission E: Wild thyme herb - Serpylli herba (Quendelkraut)

Bundesanzeiger, published October 15, 1987; revised March 13, 1990.

Conzposition of dmg: Wild thyme consists of the dried, flowering, above-ground

parts of Thymus serpyllunz L. (family Lamiaceae), as well as its preparations in effective
dosage. The drug contains essential oil, principally carvacrol andlor thymol.
Uses: Catarrhs of the upper respiratory tract.
Contraindications, side effects, interaction with other dmgs: None known.
Dosage: Unless otherwise prescribed: Average daily dose: 6 g of herb; equivalent
preparations.
Mode of adnzinistration: Cut herb for infusions and other preparations for internal use.
Action: Antimicrobial

(Ed. Note: Commercially, Thymus pulegioides L. and T . praecox Opiz subsp. arcticus
(Dut.) Jalas are also offered as mixed with T . serpyllum L.)

ESCOP monographs
The ESCOP was founded in 1989 in Cologne in order to harmonise the evaluation critera
for phytomedicines in Europe. This scientific committee, which includes experts on
phytothetapy of all members of the European Union (EU), had the assignment to
develop monograph drafts on herbal drugs as guidelines for the European market of
herbal drugs. U p to date 60 monograph drafts in the form of Summaries of Product
Characteristics have been published in the time from 1994 to 1999, including one
monograph for thyme - Thymi herba. It is the result of 38 substantial publications on
thyme which are quoted at the end of the monograph.

ESCOP-proposal: Thyme - Thymi herba

Published by ESCOP, 1996.
Name of the medicinalproduct: T o be specified for the individual finished
product.

Qualitative and quantitative composition

Active ingredient: Thyme in the crude or processed state in appropriate dosage
units.
Definition: Thyme consists of the whole leaves and flowers separated from the pre-
viously dried stems of Thymw vulgaris L. or Thymus zygis Loefl. ex L. or a mixture of
both species. It contains not less than 1.2 per cent (Vim) of essential oil and not less
than 0.5 per cent of volatile phenols, expressed as thymol (ClOH140; M, 150.2),
both calculated with reference to the anhydrous drug.
The material complies with the European Pharmacopoeia. Fresh material may
also be used, provided that when dried it complies with the European Pharma-
copoeia.
Constituents: Essential oil containing phenols, predominantly thymol and/or car-
vacrol, and terpenoids (Weiss and Fliick, 1970; Adzet etul., 1977; Stahl-Biskup,
1991); glycosides of phenolic monoterpenoids, eugenol and aliphatic alcohols
(Skopp and Horster, 1976; Van den Dries and Baerheim Svendsen, 1989);
flavonoids, among which thymonin, cirsilineol and 8-methoxy-cirsilineol are
characteristic (Van den Broucke etul., 1982; Adzet etal., 1988) biphenyl com-
pounds of monoterpenoid origin (Nakatani etal., 1989); caffeic and rosmarinic acid
(Hegnauer, 1966; Litvinenko etal., 1975; Lamaison etal., 1990); saponins (Garcia
Marquina and Gallardo Villa, 1949; Hegnauer, 1966).

Pharmaceutical form
Crude or processed drug in appropriate dosage forms (to be specified for the indi-
vidual finished product).

Clinical particulars
Therapeutic indications: Catarrh of the upper respiratory tract, bronchial catarrh and
pertussis (whooping cough). Stomatitis and halitosis (Czygan, 1989).
Posology and method of administration: Dosage (internal use). Herb: Adults and chil-
dren from l year: 1 to 2 g of the dried herb or the equivalent amount of fresh herb
as an infusion several times a day (Van Hellemont, 1988; Czygan, 1989; Dorsch
etal., 1993); children up to 1 year: 0.5 to 1g (Dorsch etal., 1993). Fluid extract:
Adults and children: Dependant on the herb-extract ratio dosage to be calculated
according to the dosage to the herb (Hochsinger, 1931). Tincture (1: 10, 70 per
cent ethanol): 4 0 drops up to three times daily (Van Hellemont, 1988). Other
preparations accordingly.
Dosage (topical use). A 5 per cent infusion as a gargle or mouth-wash (Van
Hellemont, 1988; Czygan, 1989).
Method of administration: For oral or topical administration.
Duration of administration: N o restriction.
 Thynze as a herbal drug 297
Contraindications: None known.
Special warnings and special precautions for use: None required.
Interactions with other medicaments and other forms of interaction: None reported.
Pregnancy and lactation: N o data available. In accordance with general medical
practice, the product should not be used during pregnancy and lactation without
medical advice.
Effects on ability to drive and use machines: None known.
Undesirable effects: None reported.
Overdose: N o toxic effects reported.

Pharmacological properties
Pharmacodynamicproperties: In vitro experiments: Bronchospasmolysis is attributed
to the flavonoids, thymonin, cirsilineol and 8-methoxycirsilineol, shown to be
potent spasmolytics by in vitro experiments in guinea-pig trachea (Van den Broucke
etal., 1983; Van den Broucke and Lemli, 1983).
The essential oil is highly antibacterial and antifungal, when tested in Gram-positive
and Gram-negative bacteria, fungi, and yeasts, e.g. Candida albicans. The activity is
mainly attributed to thymol and carvacrol (Allegrini and Simeon de Bouchberg,
1972; Patgkova and Chlgdek, 1974; Simeon de Bouchberg etal., 1976; Farag etal.,
1986;Janssen etal., 1986; Lens-Lisbonne etal., 1987; Menghini etal., 1987; Deans
and Ritchie, 1987; Vampa etal., 1988; Janssen, 1989; Chalchat and Garry, 1991).
Thyme oil inhibits prostaglandin biosynthesis (Wagner etal., 1986). Rosmarinic
acid has anti-inflammatory activity due to inhibition of classical complement pathway
in rats and inhibition of some human PMN functions, when tested at several dosage
levels and by several application methods (Gracza etal., 1985; Englberger etal., 1988).
In vivo experiments: Rosmarinic acid exhibited inhibitory activity in three in vivo
models in which complement activation plays a role: reduction of oedema induced
by cobra venom factor in the rat; inhibition of passive cutaneous anaphylaxis;
impairment of in vivo activation by heat-killed Corynebacterium parvum of mouse
macrophages. Rosmarinic acid did not inhibit t-butylhydroperoxide-induced
paw oedema in rat, indicating selectivity for complement-dependent processes
(Englberger etal., 1988).
Pharmacokineticproperties: No data available.
Preclinicalsafey data: Acute toxicity: A concentrated extract produced decreased
locomotor activity and slight slowing down of respiration in mice in an acute
toxicity test. Oral doses were 0.5 to 3 . 0 g extractlkg body weight corresponding to
4.3 to 26.0g dried plant material and these effects were produced at all dose levels
(Qureshi etal., 1991). The LD50 of the essential oil is 2.84glkg body weight in
rats (Von Skramlik, 1959).
Subchronic toxicity: An increase in liver and testes weight was observed after oral
administration of a concentrated 95 per cent ethanol extract of plant material to
mice. A dose corresponding to 0 . 9 g dried plant was administered daily for three
months. 30 per cent of the male animals died while in the female and control
group only 10 per cent died (Qureshi etal., 1991).
 Mutagenicity: Thyme oil had no mutagenic or DNA-damaging activity in either
the Ames or Bacillus subtilis rec-assay (Zani et al., 1991).

W H O monographs
During the fourth International Conference of Drug Regulatory Authorities (ICDRA)
held in Tokyo in 1986, W H O was requested to compile a list of medicinal plants and to
establish international specifications for the most widely used medicinal plants and
simple preparations. ~ u i d e l i n e sfor the assessment of herbal medicines were subsequently
prepared by W H O . As a result of ICDRA recommendations and in response to requests
from W H O member States for assistance in providing safe and effective herbal medicines
for use in national health-care systems, W H O has published 28 monographs on selected
medicinal plants which are widely used and important in all regions, and for each
sufficient scientific information seemed available to substantiate safety and efficacy. One
of these monographs deals with thyme (Thymus vulgaris, T. zygis). In 1994 an advisory
group selected thyme to be an important plant in all W H O regions and recommended an
evaluation of 38 substantial publications (quoted within the monograph). The mono-
graph also includes the quality standards of the Pharmacopoeias (Part 1). In the following
the paragraphs concerning the clinical applications, pharmacology, contraindications,
warnings, precautions, potential adverse reactions, and posology are quoted (Part 2).

W H O Monograph: Herba Thymi ( P a r t 2 )

Published in 1999.
Definition: Herba Thymi is the dried leaves and flowering tops of Thymus vulgaris
L. or Thymus zygis Loefl. ex L. (Lamiaceae) (Pharmacopoeia Europaea 1995, Materia
medika Indonesia 1980).
Selected vernacular names: Common thyme, tomillo, farigola, garden thyme, herba
timi, herba thymi, mother of thyme, red thyme, rubbed thyme, ten, thick leaf
thyme, thym, Thymian, thyme, time, timi, za ater (Youngken, 1950; British
Herbal Pharmacopoeia 1979; Ghazanfar, 1994; Farnsworth, 1995; Pharmacopoeia
Europaea 1995; Deutsches Arzneibuch 1996).
Description: An aromatic perennial sub-shrub, 20 to 30 cm in height, with ascending,
quadrangular, greyish brown to purplish brown lignified and twisted stems bearing
oblong-lanceolate to ovate-lanceolate greyish green leaves that are pubescent on
the lower surface. The flowers have a pubescent calyx and a bilobate, pinkish or
whitish, corolla and are borne in verticillasters. The fruit consists of 4 brown ovoid
nutlets (Youngken, 1950; Mossa etal., 1987; Bruneton, 1995).
Major chemical constituents: Herba Thymi contains about 2.5 per cent but not less
than 1.0 per cent of volatile oil. The composition of the volatile oil fluctuated
depending on the chemotype under consideration. The principal components of
Hetba Thymi are thymol and carvacrol (up to 64 per cent of oil), along with linalool,
p-cymol, cymene, thymene, a-pinene, apigenin, luteolin, and 6-hydroxyluteolin
glycosides, as well as di-, tri- and tetramethoxylated flavones, all substituted in the
6-position (for example 5,4'-dihydroxy-6,7-dimeth~x~flavone, 5,4'-dihydroxy-6,7,3'-
trimethoxyflavone and its 8-methoxylated derivative 5,6,4'-trihydroxy-7,8,3'-
 Thyme as a herbal drug 299
trimethoxyflavone) (Youngken, 1950; British Herbal Pharmacopoeia 1979; Mossa
etal., 1987; Ghazanfar, 1994; Pharmacopoeia Europaea 1995; Deutsches Arz-
neibuch 1996).
Dosagefarms: Dried herb for infusion, extracts, and tincture (Pharmacopoeia Euro-
paea 1995).
Medicinal uses: Uses supported by clinical data: None.
Uses described in pharmacopoeias and in traditional systems of medicine:
Thyme extract has been used orally to treat dyspepsia and other gastrointestinal
disturbances; coughs due to colds, bronchitis and pertussis; and laryngitis and
tonsillitis (as a gargle). Topical applications of thyme extract have been used in the
treatment of minor wounds, the common cold, disorders of the oral cavity, and as
an antibacterial agent in oral hygiene (Youngken, 1950; British Herbal Pharmaco-
poeia 1979; Petersson etal., 1992; Bruneton, 1995; Twetman etal., 1995). Both the
essential oil and thymol are ingredients of a number of proprietary drugs including
antiseptic and healing ointments, syrups for the treatment of respiratory disorders,
and preparations for inhalation. Another species in the genus, T. serpyllzlm L., is
used for the same indications (Bruneton, 1995).
Uses described in folk medicine, not supported by experimental or clinical data:
as an emmenagous, sedative, antiseptic, antipyretic, to control menstruation and
cramps, and in the treatment of dermatitis (Farnsworth, 1995).

Pharmacology - experimental pharmacology

Spasmolyticandantitzlssive activities: The spasmolytic and antitussive activity of
thyme has been most often attributed to the phenolic constituents thymol and
carvacrol, which make up a large percentage of the volatile oil (Reiter and Brand,
1985). Although these compounds have been shown to prevent contractions
induced in the ileum and the trachea of the guinea pig, by histamine, acetylcholine
and other reagents, the concentration of phenolics in aqueous preparations of the
drug is insufficient to account for this activity (Van den Broucke, 1980; Van den
Broucke and Lemli, 1981). Experimental evidence suggests that the in vitro
spasmolytic activity of thyme preparations is due to the presence of polymethoxy-
flavones (Van den Broucke and Lemli, 1983). In vitro studies have shown that
flavones and thyme extracts inhibit responses to agonists of specific receptors such
as acetylcholine, histamine and L-norepinephrine, as well as agents whose actions
do not require specific receptors, such as BaC12 (Van den Broucke and Lemli,
1983). The flavones of thyme were found to act as non-competitive and non-specific
antagonists (Van den Broucke and Lemli, 1983); they were also shown to be ca2+
antagonists and musculotropic agents that act directly on smooth muscle (Van den
Broucke and Lemli, 1983).
Expectorant andsecretornotor activities: Experimental evidence suggests that thyme
oil has secretomotoric activity (Gordonoff and Merz, 1931). This activity has been
associated with a saponin extract from T. vzll,qaris
- (Vollmer, 1932). Stimulation of
ciliary movements in the pharynx mucosa of frogs treated with diluted solutions of
thyme oil, thymol or carvacrol has also been reported (Freytag, 1933). Furthermore, an
increase in mucus secretion of the bronchi after treatment with thyme extracts has
been observed (Schilf, 1932).
 Ant8ungal and antibacterial activities: In vitro studies have shown that both thyme
essential oil and thymol have antifungal activity against a number of fungi,
including Cryptococczls neoformans, Aspergillus, Saprolegnia, and Zygorhynchzls species
(Tantaoui-Elaraki and Errifi, 1994; Vollon and Chaumont, 1994; Perrucci etal.,
1995; Paster etal., 1995). Both the essential oil and thymol had antibacterial
activity against Salmonella typhimurium, Staphylococc.us aureus, Escherichia coli, and a
number of other bacterial species (Janssen etal., 1987; Juven etal., 1994). As an
antibiotic, thymol is 25 times more effective than phenol, but less toxic (Czygan,
1989).
Contraindications: Pregnancy and lactation (See Precautions, below).
Warnings: No information available.
Precautions: General: Patients with a known sensitivity to plants in the Lamiaceae
(Labiatae) should contact their physician before using thyme preparations. Patients
sensitive to birch pollen or celery may have a cross-sensitivity to thyme (Wiithrich
etal., 1992).
Carcinogenesis, mutagenesis, impairment of fertility: Thyme essential oil did
not have any mutagenic activity in the Bacilhs subtilis rec-assay or the Salmonella
microsome reversion assay (Zani etal., 1991; Azizan and Blevins, 1995). Recent
investigations suggest that thyme extracts are antimutagenic (Natake, 1989) and
that luteolin, a constituent of thyme, is a strong antimutagen against the dietary
carcinogen Trp-P-2 (Samejima etal., 1995).
Pregnancy: non-teratogenic effects.
The safety of Herba Thymi preparations during pregnancy or lactation has not
been established. As a precautionary measure, the drug should not be used during
pregnancy or lactation except on medical advice. However, widespread use of
Herba Thymi has not resulted in any safety concerns.
Nzlrsing mothers: (See pregnancy - non-teratogenic effects, above).
Otherprecautions: No information available concerning drug interactions, drug and
laboratory test interactions, paediatric use, or teratogenic effects on pregnancy.
Adverse reactions: Contact dermatitis has been reported. Patients sensitive to birch
pollen or celery may have a cross-sensitivity to thyme (Wiithrich etal., 1992).
Posology: Adults and children from 1 year: 1 to 2 g of the dried herb or the equivalent
amount of fresh herb as an oral infusion several times a day (Czygan, 1989; Dorsch
etal., 1993); children up to 1 year: 0.5 to 1 g (Dorsch etal., 1993). Fluid-extract:
dosage calculated according to the dosage of the herb (Hochsinger, 1931). Tincture:
(1:10, 70 per cent ethanol): 40 drops up to 3 times daily (Van Hellemont, 1988).
Topical use: a 5 per cent infusion as a gargle or mouth-wash (Van Hellemont,
1988; Czygan, 1989).

QUALITY CONTROL -THYME IN THE PHARMACOPOEIAS

According to all drug regulations, drugs (including herbal drugs) have to fulfil a high
qualitative standard and they are subject to a strict quality control. Quality standards
are given in the pharmacopoeias. The pharmacopoeias comprise pharmaceutical rules
 Thynze as a herbal dmg 30 1
acknowledged concerning quality, assay, storage, dispensation and labelling of drug
and drug raw materials. The fulfilling of these quality requirements is obligatory when
drugs are produced or treated respectively.
O n the pharmacopoeial level thyme is represented by 4 monographs. The Pharmaco-
poeia Europaea (3rd edition and Supplement 2001), which is in force in most of the
European countries, contains 2 monographs, namely Thymi herba (thyme herb) and
Thymi aetheroleum (thyme oil), the German Pharmacopoeia (DAB 2000) contains
another 2 monographs, namely Serpylli herba (Quendelkraut) and Thymi extractamflztidum
(Thymianfluidextrakt). In the Swiss Pharmacopoeia (Pharmacopoea Helvetica 8, Sup-
plement 2000) a monograph on Thymi extracturn liquidurn normaturn (Eingestellter
Thymianliquidextrakt) as well as one on Thymi sirupus (Thymiansirup) can be found.
The United States Pharmacopoeia (USP) has no record of thyme. As mentioned above
the WHO monograph on thyme also includes the pharmacopoeial quality standards
(Part 1).

European Pharmacopoeia (Ph. Eur. 2001)

Thyme (Thymi herba)

Definition: Thyme consists of the whole leaves and flowers separated from the
previously dried stems of Thymus vulgaris L. or Thymus zygis Loefl. ex L. or a mixture
of both species. It contains not less than 12 mllkg of essential oil and not less than
0.5 per cent mlm volatile phenols, expressed as thymol (CloHI4O;M 150.2), both
calculated with reference to the anhydrous drug.
Characters: Thyme has a strong aromatic odour reminiscent of thymol. It has the
macroscopic and microscopic characters described under identification tests A and B.
Identzfication: A. The leaf of Thymus vulgaris is usually 4 mm to 12 mm long and up to
3 mm wide: it is sessile or has a very short petiole. The lamina is tough, entire, lan-
ceolate to ovate, covered on both surfaces by a grey to greenish-grey indumentum;
the edges are markedly rolled up towards the abaxial surface. The midrib is depressed
on the adaxial surface and is very prominent on the abaxial surface. The calyx is
green, often with violet spots and is tubular; at the end are two lips of which the
upper one is bent back and at the end has three lobes, the lower is longer and has
two hairy teeth. After flowering, the calyx tube is closed by a crown of long, stiff
hairs. The corolla, about twice as long as the calyx, is usually brownish in the dry
state and is slightly bilabiate. The leaf of Thymw zygis is usually 1.7 m m to 6.5 mm
long and 0.4mm to 1.2 m m wide; it is acicular to linear-lanceolate and the edges
are markedly rolled towards the abaxial surface. Both surfaces of the lamina are
green to greenish-grey and the midrib is sometimes violet; the edges, in particular
at the base, have long, white hairs. The dried flowers are very similar to those of
Thymas vulgaris.
B. Reduce to a powder. The powder of the two species is greyish-green to greenish-
brown. Examine under a microscope using chloral hydrate solution. The epider-
mises of the leaves have cells with anticlinal walls which are sinuous and beaded
and the stomata are of the diacytic type; numerous secretory trichomes made up of
twelve secretory cells, the cuticle of which is generally raised by the secretion to
form a globular to ovoid bladder-like covering; the glandular trichomes have a
unicellular stalk and a globular to ovoid head; the covering trichomes of the adaxial
surface are common to both species; they have warty walls and are shaped as
pointed teeth; the warty covering trichomes of the abaxial surface are of many
types: unicellular, straight or slightly curved, and bicellular or tricellular, and
often elbow-shaped (Thymus vulgaris); bicellular or tricellular, more or less straight
(Thymus zygis). Fragments of calyx are covered by numerous, uniseriate trichomes
with five or six cells and with a weakly striated cuticle. Fragments of the corolla
have numerous uniseriate covering trichomes, often collapsed, and secretory trich-
omes with generally twelve cells. Pollen grains are relatively rare, spherical and
smooth with six germinal slit-like pores, measuring about 35 pm in diameter. The
powder of Thymus zygis also contains numerous thick bundles of fibres from the
main veins and from fragments of stems.
C. Examine by thin-layer chromatography, using silica gel with a fluorescent indi-
cator having an optimal intensity at 254 nm as the coating substance.
Test solution: To 1.Og of the powdered drug add 5 ml of methylene chloride and
shake for 3 min, filter through about 2 g of anhydrous sodium sulphate. Use the fil-
trate as the test solution. Reference solution: Dissolve 5 mg of thymol and 10 pl of
carvacrol in 10 ml of methylene chloride.
Apply separately to the plate as bands, 20 pl of each solution. Develop twice over
a path of 12 cm using methylene chloride. Allow the plate to dry in air and exam-
ine in ultraviolet light at 254 nm. Mark the quenching zones. The chromatograms
obtained with the reference solution and the test solution show in the central part
a quenching zone due to thymol. The chromatogram obtained with the test solu-
tion shows slightly above the zone due to thymol a prominent quenching zone and
other quenching zones in the lower third of the chromatogram. Spray with anisal-
dehyde solution using lOml for a plate 2 0 0 m m square and heat at 100C to
105 OC for 10min. The chromatogram obtained with the reference solution shows
in the central part a brownish-pink zone corresponding to thymol and, immediately
below it, a pale violet zone corresponding with the test solution to carvacrol. The
chromatogram obtained with the test solution shows these two zones in the central
part of the plate; they are more or less prominent, depending upon the species
examined. Between these two zones and the starting-line are four zones of similar
intensity; in order of decreasing Rf value these bands are: pink, violet (1,s-cineole
and linalool), greyish-brown (borneol) and violet-blue. Near the solvent front, an
intense violet-red to greyish-violet band is visible. Other bands are also present
adjacent to the starting-line.
Tests: Foreign matter: Not more than 1 0 per cent of stem. Stems must not be
more than 1 m m in diameter and 15 m m in length. Leaves with long trichomes
at their base and with weakly pubescent other parts are not allowed (Thymus
serpyllum L.).
Water: Not more than 10.0 per cent, determined by distillation of 20.0g of
powdered drug.
Total ash: Not more than 15.0 per cent.
Ash insoluble in hydrochloric acid: Not more than 3.0 per cent.
Assay: Essential oil: Carry out the determination of essential oils in vegetable
drugs. Use 30.0g of the drug, a lOOOml round-bottomed flask and 4 0 0 m l of
 Thyme as a herbal dwg 303
water as the distillation liquid. Distill at a rate of 2 mllrnin to 3 mllmin for 2 h
without xylene in the graduated tube.
Phenols: Taking care that as little water as possible is transferred, transfer the
essential oil obtained in the assay of essential oil to a 50.0ml volumetric flask with
the aid of small portions of alcohol (90 per cent VIV) rinsing the graduated tube of
the apparatus with the same solvent and dilute to 50.0ml with the same solvent. To
5.0 m! of the solution add 40 ml of alcohol (90 per cent (VIV) and dilute to 100.0 ml
with water. Place 5.0ml of the solution in a separating funnel and add 45 ml of
water, 0.5 ml of dilute ammonia and 1 ml of a 20gll solution of aminopyrazolone.
Mix and add 4 ml of a freshly prepared 20 gll solution of potassium ferricyanide
and mix again. Allow to stand for 5 min, add 25 ml of methylene chloride and
shake. Separate the methylene chloride layer and filter through a plug of absorbent
cotton moistened with methylene chloride into a 1OOml volumetric flask. Shake
the aqueous layer with two quantities, each of 25 ml, and with 1 0 m l of methylene
chloride, filter the methylene chloride layers through the plug of absorbent cotton.
Rinse the plug with methylene chloride and dilute to 100.0ml with the same sol-
vent. Measure the absorbance at 450 nm using methylene chloride as the compen-
sation liquid.
Calculate the percentage content of phenols, expressed as thymol, taking the
specific absorbance to be 805.
Storage: Store in a well-closed container, protected from light and moisture.

Thyme oil (Thymi aetheroleum)

DeJinition: Thyme oil is obtained by steam distillation from the fresh flowering
aerial parts of Thymus vzlgaris L., T. zygzs Loefl. ex L. or a mixture of the two
species.
Characters: A. clear, yellow or very dark reddish-brown, mobile liquid with a
characteristic aromatic, spicy odour, reminiscent of thymol, miscible with ethanol,
with ether and with petroleum ether.
Identzfication: A. Examine by thin-layer chromatography, using silica gel as the
coating substance.
Test solution: Dissolve 0 . 2 g of the substance to be examined in pentane and
dilute to lOml with the same solvent. Reference solution: Dissolve 0.15 g of thy-
mol, 25 pl of terpinen-4-01 and 4 0 p l of linalool in pentane R and dilute to 1 0 m l
with the same solvent.
Apply separately to the plate as bands of 20pl of each solution. Develop over
a path of 15 cm using a mixture of 5 volumes of ethyl acetate and 95 volumes of
toluene. Allow the plate to dry in air. Spray with anisaldehyde solution. Heat the
plate at 100 OC to 105 O C for 5 min to 10 min while observing. Examine in day-
light. The chromatogram obtained with the test solution shows three zones similar
in position and colour to those in the chromatogram obtained with the reference
solution: a violet zone corresponding to terpinen-4-01, a violet zone corresponding
to linalool and a brownish-pink zone corresponding to thymol and immediately
below it a pale-violet zone corresponding to carvacrol. It also shows a large violet
zone at the solvent front (hydrocarbons).
 B. Examine the chromatograms obtained in the test for "chromatographic profile"
(see below). The retention times of the principal peaks in the chromatogram
obtained with the test solution are similar to those of the peaks in the chromato-
gram obtained with the reference solution.
Tests: Relative density: 0.91 5 to 0.935.
Refractive index: 1.490 to 1.505
Chromatographic profile: examine by gas chromatography.
Test solution: The substance to be examined. Reference solution: Dissolve 0.15 g
of 0-myrcene, 0.1 g of y-terpinene, 0.1 g of p-cymene, 0.1 g of linalool, 0.2 g of
terpinen-4-01, 0.2 g of thymol and 0.05 g of carvacrol in 5 ml of hexane.
The chromatographic procedures may be carried out using:
a fused-silica column 25 m to 6 0 m long and about 0.3 mm in internal diameter
coated with macrogol - 20 000.
helium for chromatography as the carrier gas (other gases can also be used, the
author).
a flame-ionisation detector,
a split ratio of 1: 100,
maintaining the temperature of the column at 6 0 OC for 15 min, then raising the
temperature at a rate of 3 OC per min to 180 OC and maintaining at 180 OC; main-
taining the temperature of the injection port at about 200 OC and that of the detector
at 220C.
Inject about 0.2 pl of the reference solution. When the chromatograms are recorded
in the prescribed conditions, the components elute in the order indicated in the com-
position of the reference solution. Record the retention times of these substances.
The test is not valid unless: the number of theoretical plates calculated from the
p-cymene peak at 80 OC is at least 30 000; the resolution between the peaks corres-
ponding to thymol and carvacrol is at least 1.5.
Inject about 0.2 pl of the test solution. Using the retention times determined
from the chromatogram obtained with the reference solution, locate the compo-
nents of the reference solution on the chromatogram (Figure 1 1.1) obtained with
the test solution. Disregard the peak due to hexane.
Determine the percentage content of the components of the normalisation procedure.
The percentages range between the following values:
P-myrcene 1.O-3.0 per cent
y-terpinene 5.0-10.0 per cent
p-cymene 15.0-28.0 per cent
linalool 4.0-6.5 per cent
terpinen-4-01 0.2-2.5 per cent
thymol 36.0-55.0 per cent
carvacrol 1.0-4.0 per cent.
Storage: Store in a well-filled, air-tight container, protected from light and heat.

German Pharmacopoeia - DAB 2000

 Thyme as a herbal dmg 305

Figure 1 I . 1 Gas chromatogram of thyme oil (T. v?~lgaris)

according to the Pharmacopoeia Europaea,
on Macrogol 20 000, 30 m .
Notes
1, P-myrcene.
2, y-terpinene.
3, p-cymene.
4, linalool.
5 , terpinen-4-01.
6 , thymol.
7, carvacrol.

Definition: It consists of the whole or cut dried aerial parts of Thymus serpyllam L.
s.1. collected in blossom. It contains not less than 3 mllkg essential oil and not less
than 0.1 per cent phenols, expressed as thymol (C10H140; M 150.2), calculated
with reference to the anhydrous drug.
Characteristics: The herb has a characteristic, aromatic odout.
Identity: A. Morphological characteristics: Stem in the cross-section vaguely quad-
rangular to cylindrical, about 1.5 m m thick, weakly woody at base, side shoots
thinner. Leaves decussate, usually 3 mm to 12 mm long and up to 7 m m wide, entire,
lanceolate to ovate, wedge-shapedly ending in a short petiole, the edges are seldom
rolled up towards the abaxial surface; petiole and leaf basis often ciliate; on both
sides of the leaf glandular hairs (magnifying glass). Flowers crowded into a terminal
capitate inflorescence; tubular calyx has two lips, the lower with two teeth, the
upper one has three lobes; the calyx tube is closed by a crown of long, stiff hairs;
the corolla is bilabiate, purple to light-red, wrinkled; four protruding stamens or
only four rudiments.
B. Microscopical characteristics: see "Thyme herb" in the European Pharmacopoeia.
C. See "Thyme herb" in the European Pharmacopoeia; thin-layer chromatography of
the essential oil on silica gel; reference: thymol. Mobile phase: toluenefethyl acetate
(93+ 7). Detection UV 254 n m and daylight after spraying with anisaldehyde-
H,S04-reagent (a mixture of 0.5 ml anisaldehyde, l 0 m l acetic acid, 85 ml methanol,
and 5 ml H2S04).
Purity: Foreign matters: Not more than three per cent
Water: Not more than 10.0 per cent after 2 h at 100 to 105 OC.
Total ash: Not more than 10.0 per cent.
Acid-insoluble ash: Not more than 2.0 per cent
Assays: Essential oil and phenols: see Thyme herb in the European Pharmacopoeia.
Storage: In air-tight and light-protected containers.

Thymi extractum fhidum - Liquid thyme extract - Thymianfluidextrukt

Definition: Thymi extractum fluidum contains not less than 0.03 per cent phenols,
expressed as thymol (C10H140; M 150.2).
Production: Thymi extractum fluidum is produced from 1 part freshly powdered
thyme extracted with 2-3 parts of a mixture of 1 part ammonia 10 per cent (mlm),
20 parts glycerol 85 per cent, 70 parts ethanol 90 per cent (VIV) and 109 parts
purified water by maceration.
Characteristics: Dark brown liquid with an odour of thymol and a spicy, weakly
burning taste.
Identity: Thin-layer chromatography on silica gel of an extract from 5 ml liquid
thyme extract with 3 ml methylene chloride (application 20 pi); reference: thymol
(0.02 per cent in methylene chloride, application 20 pl). Mobile phase: methylene
chloride. Detection UV 254nm and daylight after spraying with anisaldehyde-
H,S04-reagent (see "Thyme herb").
Purity: Ethanol content (of the extract): 30-37 per cent (VIV)
Methanol, 2-propanol: content according to the permitted percentages in extracts
{see Pharmacopoeia Europaea, the author).
Assay: 20.0g liquid extract are mixed with 80ml water and distilled until 85 ml have
condensed in 10.0ml ethanol 96 per cent prepared in a 100ml volumetric flask. Top
up with water to 100.0ml. 5.0ml of this solution are mixed with 45 ml water, 0.5 ml
liquid ammonia, 1 ml aminopyrazolone (20 gll). Mix and add 4 ml of a freshly pre-
pared 20gll solution of potassium ferricyanide and mix again. Allow to stand for
5 min, add 25 ml of chloroform and shake. Separate the chloroform layer and filter
through a plug of absorbent cotton moistened with chloroform into a l 0 0 m l volu-
metric flask. Shake the aqueous layer with two quantities, each of 25 ml, and with
10 ml of chloroform, filter the chloroform layers through the plug of absorbent cotton.
Rinse the plug with chloroform and top up to 100.0ml with the same solvent. Mea-
sure the absorbance at 450 nm using chloroform as the compensation liquid.
The reference is prepared with 10.0 mg thymol in 25 ml Ethanol 96 per cent and
solved in water to 100.0 ml. 5 ml of this solution is treated as described above.
The calculation of the phenol content is performed according to the following formula:
 Thyme as a herbal drug 307

A, = Absorption of the test solution

A, = Absorption of the reference
m, = weight of liquid extract (g)
m, = weight of thymol (g)

(The blue colour develops according to the Emerson reaction, the author).
Storage: In tight vessels and light protected.

Swiss Pharmacopoeia - Pharmacopoea Helvetica (2000)

Thymi extracturn liqzidum normatum -preparative thynze extract - Eingestellter

Thymianextrakt
Definition: Thymi extractum fluidum notmatum contains not less than 0.025
and not more than 0.035 per cent phenols, expressed as thymol (C10H140; M
150.2).
Production: Thyme l0Og are macerated with 3 0 0 g of a mixture of 1.5 parts of
ammonia 10 per cent, 30 parts glycerol, 105 parts ethanol and 163.5 parts purified
water over 5 days at room temperature. The liquid is pressed and filtered. In the
filtrate the phenol content is quantified and adjusted with a mixture of 3.5 parts
ethanol 96 per cent and 6.5 parts purified water at the required content.
Characteristics: Brown to dark brown liquid with a characteristic odour of thymol
and a bitter taste. It can be mixed with water in a clear to opalescent liquid.
Identity: A. 1 ml of the extract is diluted with water to 50 ml. After the addition of
0.15 ml of a solution of potassium ferricyanide a green-brownish colour develops
and later a flaky, brown precipitate.
B. Thin-layer chromatography: (see "Thymi extractum fluidum", the author).
Test: Content of ethanol: 35 to 45 per cent (VIV).
Methanol, 2-Propanol: Not more than 0.05 per cent (VIV).
Assay: Spectralphotometric evaluation of thymol after reaction with aminopyra-
zolone and potassium ferricyanide (see "Thymi extractum fluidurn", the author).
Storage: In tight bottles, light protected.

Thymiansirup - Thyme syrup - Thymi sirupw

Definition: Thymi sirupus contains not less than 0.013 and not more than 0.017
per cent (mlm) phenols, expressed as thymol (C10H140; M 150.2).
Production: 5 0 0 g saccharose is dissolved in 3 0 0 g purified water by warming on a
water bath. After cooling the water loss must be replaced. Afterwards 1 5 0 g Thymi
extractum liquidurn normatum, and a solution of 0.10g thymol in 4 0 g ethanol 9 6
per cent is added.
Characteristics: Clear, light-brown liquid with an odour of thyme; miscible with
water, ethanol 70 per cent and ethanol 96 per cent.
Identity: A. Thin-layer chromatography: (see "Thymi extractum fluidum", the author).
 B. 1 ml sirup is mixed with 1 0 m l water. An aliquote of 0.05 ml of this solution is
warmed with 0.5 g resorcinol and 2.5 ml hydrochloric acid. After 10min a dark red
colour develops.
Test: Relative Density: 1.20 to 1.23
Refractive Index: 1.420 to 1.430
Preservative: not allowed.
Assay: Spectralphotometric evaluation of thymol after reaction with aminopyra-
zolone and potassium ferricyanide (see "Thymi extractum fluidum", the author).
Storage: In tight bottles, light protected.

WHO monographs
Herba (Part I )
Plant material ofinterest: dried leaves and flowering tops of thyme.
General appearance: Same test as in the Pharmacopoeia Europae.
Organolepticproperties: Odour and taste aromatic (Pharmacopoeia Europaea 1995,
Materia medika Indonesia 1980; British Herbal Pharmacopoeia 1979; Youngken,
1950).
Micrascopic characteristics: In leaf upper epidermis, cells tangentially elongated in trans-
verse section with a thick cuticle and few stomata, somewhat polygonal in surface
section with beaded vertical walls and striated cuticle, the stoma being at a right
angle to the two parallel neighbouring cells. Numerous unicellular, non-glandular
hairs up to 30 pm in length with papillose wall and apical cell, straight, or pointed,
curved, or hooked. Numerous glandular hairs of two kinds, one with a short stalk
embedded in the epidermal layer and a unicellular head, the other with an 8- to
12-celled head and no stalk. Palisade parenchyma of two layers of columnar cells
containing many chloroplastids; occasionally an interrupted third layer is present.
Spongy parenchyma of about 6 layers of irregular-shaped chlorenchyma cells and
intercellular air-spaces (Youngken, 195 0).
Powderedplant materiaI: Grey-green to greenish-brown powder; leaf fragments,
epidermal cells prolonged into unicellular pointed, papillose trichomes, 6Opm
long; trichomes of the lower surface uniseriate, two- to three-celled, sharp pointed,
up to 300 pm in diameter, numerous labiate trichomes with 8 to 12 secretory cells
up to 80 pm in diameter; broadly elliptical caryophyllaceous stomata. Six- to eight-
celled uniseriate trichomes from the calyx up to 400 pm long; pollen grains spheri-
cal; pericyclic fibres of the stem (British Herbal Pharmacopoeia 1979; Materia
medika Indonesia 1980; Pharmacopoeia Europaea 1995).
GeographicaldistribzItion: Indigenous to southern Europe. It is a pan-European
species that is cultivated in Europe, the United States of America and other parts of
the world (Youngken, 1950; British Herbal Pharmacopoeia 1979; Materia medika
Indonesia 1980; Van den Broucke and Lemli, 1983).
General identity tests: Macroscopic and microscopic examinations (Youngken, 1950;
Pharmacopoeia Europaea, 1995; ), and chemical and thin-layer chromatography
tests for the characteristic volatile oil constituents, thymol.
 Thyme as a herbal drug 309
Purity tests: Microbiology: The test for Salmonella ssp. in Herba Thymi products
should be negative. The maximum acceptable limits of other microorganisms are
as follows (Deutsches Arzneibuch 1996; Pharmacopoeia Europaea 1997; W H O ,
1998). For preparation of infusion: aerobic bacteria - not more than 107lg; fungi -
nor more than 105lg; Eschwichia coli - not more rhan 102lg. Preparations for oral use:
aerobic bacteria - not more than 105lml; fungi - not more than 104lml; enterobacteria
and certain Gram-negative bacteria - not more than 103lml; Escherichia coli - Olml.
Foreign organic matter: Not more than ten per cent of stem having a diameter up to
1 mm. Leaves with long trichomes at their base and with weakly pubescent other parts
not allowed. The leaves and flowering tops of Orzgdnum cretzcztm or 0. dictanznzls are con-
sidered adulterants (British Herbal Pharmacopoeia 1979; Youngken, 1950). Other
foreign organic matter, not more than two per cent (Materia medika Indonesia 1980).
Total ash: Not more than 15 per cent (Pharmacopoeia Europaea 1995).
Acid-insoluble ash: Not more than 2.0 per cent (Pharmacopoeia Europaea 1995).
Moisture: Not more rhan ten per cent (Pharmacopoeia Europaea 1995).
Pesticide residues: To be established in accordance with national requirements.
Normally, the maximum residue limit of aldrin and dieldrin in Herba Thymi is not
more than 0.05 mglkg (Pharmacopoeia Europaea 1997). For other pesticides, see
W H O guidelines on quality control methods for medicinal plants (WHO, 1998)
and guidelines for predicting dietary intake of pesticide residues (WHO, 1997).
Heavy metals: Recommended lead and cadmium levels are not more than 10.0
and 0.3 mglkg, respectively, in the final dosage form of the plant material.
Radioactive residues: For analysis of strontium-90, iodine-1 3 1, caesium-137, and
plutonium-239, see W H O guidelines on quality control methods for medicinal
plants (WHO, 1998).
Otherpurity tests: Chemical, alcohol-soluble extractive, and water-soluble extractive
tests to be established in accordance with national requirements.
Chemical assays: Herba Thymi contains not less than 1.0 per cent volatile oil, and
not less than 0.5 per cent phenols. Volatile oil is quantitatively determined by
wateristeam distillation, and the percentage content of phenols expressed as
thymol is determined by spectrophorometric analysis. Thin-layer chromatographic
analysis is used for thymol, carvacrol, and linalool (Pharmacopoeia Europaea 1995;
Twetman etal., 1995).

German Homoeopathic Pharmacopoeia - Homoopathisches

Arzneibuch 2000
The monographs of the Homoeopathic Pharmacopoeia of Germany characterise the
quality of ingredients of homoeoparhic drugs prepared from plants, animals, minerals
and also synthetic drugs. The test methods and quality requirements are identical with
those of the DAB and the Pharmacopoeia Europaea, respectively. In homoeopathy,
plants are mostly used in the form of tincture standards (German: Urtinktur) and
liquid decimal dilutions (German: Decimalporenz). Within the Homoeopathic Pharma-
copoeia thyme is represented with 2 monographs, T. vulgaris and T. serpylhm. The tincture
standards from both plants are prepared by a ten days maceration at room temperature
of homogenised fresh plant material with ethanol 86 per cent (depending on the water
content of the fresh plant from 1.0 to 1.3:1, ethanol content in the end product about
6 0 per cent). The D l dilutions are prepared by mixing 3 parts of the tincture standards
with 7 parts ethanol 62 per cent ( D l ) . D2 dilutions are prepared by mixing 1 part D l
with 9 parts ethanol 62 per cent, D 3 dilutions by mixing 1 part D2 with 9 parts etha-
nol 62 per cent, and so on. For the quality control of the plant material a description of
the plants is given more or less identical to the descriptions in the DAB and the Phar-
macopoeia Europaea, respectively.
The liquid standards of both plants are described as brown liquids with an aromatic
smell and taste. Identity control of the liquid standards is performed by mixing them
with water and iron(II1) chloride yielding a characteristic green colour. A mixture of the
liquid standards (0.5 ml) with water (10ml) develops a characteristic blue colour when
a ten per cent solution of sodium carbonate (0.1 ml) and a two per cent solution of dichlo-
rochinone chlorimide in ethanol (0.1 ml) is added. Further evidence of the identity is
given when a chromatographic separation of the apolar fraction on TLC according to the
methods described in the DAB and the Pharmacopoeia Europaea, respectively, is
performed. The assays concern the relative density of the liquid standards (for both herbs
0.900 to 0.920) and the residue on drying (1.4 per cent for T. vulgurzs, 1.2 per cent for
T. serpylLum). The liquid standards have to be stored light-protected.

CURRENT SAMPLE PRODUCT FORMULATIONS I N INDUSTRIES

The "Rote Liste 1999", a list of drugs sold on the German market, supplies about 100
"hits" of different drugs which contain thyme as an active principle. Such phytoprep-
arations not only contain thyme as an herb but also in the form of dry extracts, liquid
extracts (tincture, fluid-extract), semi-solid extracts, pressed juice from the fresh plant,
and homoeopathic tinctures. Also the essential oil of thyme alone can be a constituent
of those drugs. The uses of phytopreparations of thyme are mostly given with "for
treatment of catarrhs of the upper respiratory tract, of bronchial catarrh, pertussis and
hoarseness; they act as expectorants". Only four phytopreparations containing thyme
show other applications: in mixtures with other herbal drugs as a roburant and for
homoeopathic indications.

Internal application
For internal application liquids and syrups are the most usual formulations which are
administered drop by drop or by the spoonful depending on the concentration of the
active principle. For such formulations mostly fluid-extracts of thyme are processed
with a herb to liquid ratio of 1:2.5 or 1:3 according to the pharmacopoeia1 instructions
"Thymianfluidextrakt" in the German and Swiss pharmacopoeia (ethanol content
35-45 per cent). According to modern knowledge which prefers monopreparations to
combinations, many of the liquid phytopreparations (18 of 100) contain thyme extracts
as the only active compound. But there are also several combinations with other herbal
extracts from plants which are known to be effective against catarrhs of the upper
respiratory tract listed in Table 11.1. In this respect the most important herbs are
Primulu root, ivy leaf or Droseru herb. W e also can find thyme tinctures (herb to extract
ratio 1:5) as the active principle in liquids and syrups. One of these preparations
 Thyme as a herbal drug 3 11

Table 11.1 Thyme liquid extracts in product formulations including combinations with other herbal
products

Herbal products Drops, syrups, and liquids Bath additives

Thyme fluid extract x x x x x x x x x x x x x x x x x

Anise oil x x x
Camphor x x
Drosera x x
Eucalyptus leaf extract x
Eucalyptus oil x x
Fennel oil
Horsetail herb
Ivy leaf
Khella fruit
Liquorice root
Marshmallow root
Peppermint oil
Pine oil
Plantain herb
Pnmula root
Snail extract
Turpentine oil
Verbascum flower
Ephedrin
Guaifenesln
Gypsophila saponin
Castanea leaves
Number ofproduct formulations 17 1 1 1 1 1 2 5 2 1 2 1 1 1 1 1 1

Table 11.2 Thyme dry extract In product formulations including combinations with other herbal
products

Herbal products Tablets Teas Capsules Dragees Suppositories

Thyme dry extract X X X X X X X X

Anise oil X

Drosera X

Eucalyptus oil X
Ivy leaf X
Primula root dry extract X X X X X

Number of product formulations 3 4 1 1 1 1 1 1

combines thyme tincture with tinctures prepared from Grindelia herb, Quedracho bark,
Saponaria root and Primula root. One product on the market contains the pressed juice
from the fresh thyme plant, won by a very special procedure.
Dry extracts contain the effective principle of thyme in higher concentrations than
the liquid extracts, because the usual herb to extract ratio is 6 to 10:1 meaning a 6- to
10-fold accumulation. Water, ethanol 70 per cent, and ethanol 96 per cent are used as
the solvents. Dry extracts represent the effective principle of capsules, tablets, dragees, and
suppositoria or instant teas. The dry extracts are processed alone or together with other
plant extracts (Table 11.2). Semi-solid extracts of thyme can be found in 7 preparations
in the form of liquids, syrups, and pastilles (herb to extract ratio 4.5 to 8:1, mostly
5.5:l). Again combinations with other plant extracts, e.g. from Primula root, Drosera,
or combinations with the essential oils from anise, fennel, and Eucalyptus are common.
Perhaps due to the bitter taste of thyme, infusions (a hot tea prepared with thyme)
are not very common. In order to improve the taste teas are offered in mixtures with
other herbal drugs used for the same purpose, e.g. lime tree flowers and elder flowers,
fennel fruits, Primula flowers and plantain herb (Table 11.3). The most complex tea in
the market represents a mixture of thyme with fennel fruits, Iceland moss, mullein

Table 11.3 Herbal teas of thyme in product formulations

including combinations with other herbal teas

Herbal products Herbal teai

Thyme
Lime tree flowers
Elder flowers
Primula flowers
Plantain herb
Fennel fruit
Iceland lichen
Mullein flowers
White deadnettle flowers
Herb of black knotweed
Marigold flowers
Raspberry leaves
Number of product formulas

Table 11.4 Thyme oil in product formulations including combinations with other oils or chem~cals

Herbal products Bath additives Balsams Syrups Ointments instant Nasal

teas Ointments

Thyme oil X X X X X X X X X X X X X
Anise oil X X
Camphor
Coal tar
Clove oil
Conifer oil
Dwarf pine oil
Liquorice
Marshmallow
Peppermint 011
Pine oil
Pine oil, Sibirian
Rosemary oil
Prinzzlla root extract
Thymol
Turpentine
Levomenthol
Dihydrocodein
Numberofproduct formulas 3 1 1 1 1 1 1 1 1 1 1 1 1
 Thynze as a herbal d?*zlg 3 13
flowers, lime tree flowers, Primula flowers, white deadnettie herb, herb of black knot-
weed, marigold flowers, and raspberry leaves.

External application
For external applications thyme oil is exclusively used against colds in bath additives,
balsams, ointments and ointments for the nose. Especially the bath additives contain
other essential oils, mostly Eucalypt~lsoil or camphor (Tables 11.1; 11.4). During
application the oil is thought t o reach the nose when warmed on the skin and to pene-
trate into the upper bronchial tract, and there i t develops its disinfectant effect.

REFERENCES

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Index

Acacet~n 146 carvacrol 28 1

Acylated flavonoid aglycones 148 p-cymene-2,3-diol 281
T. hirtzls 148 eriodictyol 282
Adulteration, oil 255 thymol 281
Alelotrichous 11 T . vulgaris 281, 282
Alternantes, Subsection 26, 31, 36 Antiparasitic effect 282
Anatomy thymol 282
gland 7 6 T. vulgaris 282
trichome 76 Antiviral effect 273
Aneuploidy 20 T . serpyllam 2 7 1
Anomalae, Subsection 26, 29, 32 Apigenin 146, 149
Anther morphology 17 Application
Anthocyanidins 148 external 313
T . pdegioides 148 internal 310
T. vz~lgarzs 148 Aromatherapy 266
Antibacterial effect 268-272 Aromatic rerpenes, biosynthes~s 8 1
Thyme oil 270
Thymus oil 268 Biogeography 17
T. baeticas 269 Biology, T h y t z u 10
T. capitatzls 272 PBisabolene 79
T . granatensis 269, 27 1 Bloom 1 3
T. hyemalis 269 Borneo1 7 9
T . orospeda?zas 269 Bornyl acetate 7 9
T. serpj~lloides268, 269, 27 1 Bract 11
T. vz~lgaris 268, 269 morphology 12, 17
Antifungal effect 272
essential oil components 274 T-Cadinol 7 9
T . baetzcas 273 Caffeic acid 150
T . serpylloides 2 7 3 Calyx 11
T . serpyllanz 27 3 morphology 14
T. vulgaris 272, 273 Camphene 7 9
T. zygis 272, 273 Camphor 7 9
Antimicrobial effect 268 Carvacrol 7 9
T . graaatensis 269 biosynthesis 81, 8 4
T. hyenzalis 269 content, effect of storage 220
T. longz;ilor~is269 pharmacokinetics 283
T. orospedanzls 269 /3-Caryophyllene 7 9
T . serpylloides 269 Chemical polymorphism 125-143
T. vz~lgaris 268 essential oil 125
T . zygic 269 section Martichina 127, 130, 135
Antioxidant effect 281 section Micantes 135
biphenyl compound 282 sectlon Piperella 127, 131
3 18 Index
Chemical polymorphism (Continued) Ecological form 22
section Pseudothymbra 127, 130, Ecology 15
135 Endemic species 17
section Serpyllum 137 Eriodictyol 147
section Thymus 127, 129, 135 Essential oil
T . albzcans 136 biosynthesis 81-85
T . antoninae 131 chemistry 75-124
T . baeticus 131 components 79
T . caespititius 136 composition 86-105, 199-201, 235
T . camphoratus 135 content 199
T . capitellatus 13 5 enant~omericcomposition 8 1
T. carnosus 13 5 infraspecific variation 106
T . funkii 132 isolation 225
T. hyemalis 132 monoterpenes 77-80
T. longzflorus 132 non-terpenoid aliphates 8 0
T . lotocephalus 136 non-terpenoid aromatics 8 0
T . mastichina 132, 136 ontogenetical variation 108
T . menzbranaceus 133 phenylpropane derivatives 80
T . moroderi 133 seasonal variation 108
T. orospedanus 13 3 sesquiterpenes 77-80
T . piperella 13 3 sexual variation 107
T. praecox 138 summary table 85-105
T. pulegioides 140 Thynzw species 83-10>
T . serpylloides 134 yield 235
T. serpylhm 139 Ethylene oxide, sterilisation 210
T. villosus 135 Etymology 1
T . vulgaris 134 Evolution 15
T.zygis 134,135 Evolutionary characteristics 15
Chlorogenic acid 150 Evolutionary relationships 16
Chromosome 18 Extract, characterisation 227, 231
Chrysoeriol 146 Extraction 227-23 1
1,s-Cineole 79 atmospheric pressure 229
Cirsilineol 146 high pressure 230
Cirsileol 146 methods 227-231
Cirsimaritin 146 solvent 228,229
Citral 79 Exudate flavonoids 150
Coridothymuscapitatus 52 T . camphoratus 169
Corolla morphology 13, 14, 17 T. capitellatus 169
Cosmetic use, thyme oil 285 T. carnosw 169
p-Coumaric acid 150 T . funkii 168
Culinary use, thyme 286 T. herba-barona 168, 169
Culture medium components 192 T . longiflorus 168
Cyanidin 147 T. membranaceus 168
p-Cymene 79 T . moroderi 168, 169
T. piperella 169
Date, harvest 198 T. satureioides 169
5-Desmethylnobiletin 146
5-Desmethylsinensetin 146 tr-tr-Farnesol 79
3,5-Dicaffeoylquinic acid 150 Field culture 178
Dihydrocarvone 79 Field cultured Thymus
Dihydroflavonols 148 frost-resistance 186
Dihydrokaempferol 147 homogeneity 185
Dihydroxanthomicrol 147 productivity 182
Diosmetin 146, 149 Flavanones 148
Dispersion, fruit 11 Flavones 146
Distribution 17, 18 T. nzoroderi 146
Drying methods 212 T. piperella 146
 Index 119
Flavonoid aglycones 145 Hybrids
substitution pattern 146-147 list of 38
Flavonoid glycosides 148 number of 1 9
substitution pattern 1 4 9 p-Hydroxybenzoic acid 150
sugars 149 Hyphodromi, Section 26, 29, 3 3
taxonomic value 149
T. membranacez~s148 Ice Age, origin 1 8
T. moroderz 148 Illustrations 9
T. serpyllunz 148 In vitro culture 188
Flavonoids growth regulator 192
c h e m o t a x ~ n o m170
~ Indumentum 11
environment 169 Inflorescence 11
Flavonols 148 Insecticidal effect 283
T. moroderi 148 T. serpyllum 28 3
T. vulgari~ 148 T. vulgaris 283
Flower 1 1 Insulares, Subsection 26, 31, 34
bloom 15 Irradiation
Food preservative, use 284 detection 247
Fruit dispersion 1 1 sterilization 2 10
Isolepides, Subsection 26,3 5
Gardenin 146
Gardening 260 Kaempferol 146
Gene flow 5 5 Kotschiany, Subsection 26, 31, 34
Genetic control, sex 59
Genetic incompatibility 21 Ladanein 146
Genkwanin 146 Lamiaceae family 15
Geraniol 7 9 Leaf morphology 11, 12
Geranyl acetate 79 Limonene 79
Germacrandien-6-01 7 9 Linalool 7 9
Germacrene D 79 Linalyl acetate 79
Germacren-4-01 7 9 Luteolin 146, 149
Gland 11
anatomy 7 6 Machinery, harvest 198
morphology 1 3 Mastichina, Section 26,28, 31
Glandular trichome 11 Mediterranean, distribution 17
Globose gland 11 Mentheae tribe 16
Glycosidically bound volatiles 110 Methyl bromide, sterilisation 210
T. x citriodor~~s1 10 Mzcantes, Section 26, 27, 31
T. praecox 110 M~crobialcontamination 244
T. pulegioides 1 10 processing 245
T. vulgararic 110 Miocene, origln 1 8
Goniotrichous 11 Monoterpenes 77
Gynodioecy 15, 17 adaptive value 47
sexual polymorphism 45, 57 allelopathic effects 5 1
chemical defence 53
Habitus 10, 21 genetic control 4 6
Hair 1 1 skeletons 78
morphology 1 3 Morphology 10-14
Harvest, effects 199 anther 17
Heat sterilisation 2 11 bract 12, 17
Hedycaryol 7 9 calyx 14
Heliophyllous 15 corolla 13, 14, 17
Herbal tea 312 gland 1 3
High pressure extraction 230 hair 1 3
Holotrichous 11 leaf 11, 12
Homoeopathy 267 nutlet 1 7
Hybridization 19 stem 1 1
320 Index
Myrcene 7 9 T. alternus 15 1
Myrcenol-8 7 9 T. amictus 15 1
T. antoninae 15 1
Names T. aranjuezii 15 2
popular 20 T. ararati-minoris 152
vernacular 20 T. attenuatus 15 2
Naringenin 147 T. baeticus 152
Nepetoideae, subfamily 1 6 T . balcanus 152
Nerolidol 7 9 T. bashkinensis 15 3
Number T. borysthenicus 153
of chromosomes 20 T. bracteatus 15 3
of hybrids 21 T. cuespititius 15 3
Nutlet T. calcareus 15 3
fruit 15 T. callieri 15 3
morphology 17 T. camphoratus 15 3
T. capitellatus 15 3
Oil composition, processing 235 T. carnosus 154
Oil yield, processing 235 T. caucaszcus 154
Old world, distribution 17 T. cephalotos 154
Organic culture 177 T. cherlerioides 154
Ozone, sterilisation 2 11 T. ciliatissinzus 154
T. cinerascens 154
Packaging, post-harvest 216 T. circumcinctz~s 154
Paleocene, origin 1 8 T. collinus 154
Pasteurisation, sterilisation 21 1 T. cretacezls 154
Pebrellin 146 T. czernajevii 1 5 5
Pedicellate gland 11 T. dagestanicus 15 5
Pharmacokinetics 283 T. decussatus 15 5
Pharmacology 297, 299 T. desjatovae 15 5
Pharmacopoeia T. dimorphus 15 5
European 301 T. dolopicus 15 5
German 304 T. dzevanovskyi 15 5
German homoeopathic 309 T. elisabethae 15 5
Swiss 307 T. eupatoriensis 15 5
Phellandrene 7 9 T.fontqueri 15 5
Phenolic acids 149, 150 T. fonzinii 15 5
T. carnosus 149 T. funkii 155
T. webbianus 149 T. glandulosus 156
Phytopharmacy, monographs 294 T. granatensis 156
Phytotherapy 265 T. graniticus 156
Pilloin 146 T. herba-barona 156
a-Pinene 79 T. hirsutus 156
Piperella, Section 26, 28, 32 T. hirtelhs 15 6
Pollen 18 T. hirtus 157
production 14 T. hyemulii 157
Pollen grain 1 8 T. jajlae 157
morphology 19 T. jankae 1 5 7
Polychemism, essential oil 125 T. kalmiussicus 157
Polymorphism T.karamaria?zicus 158
essential oil 125 T. kostchyanus 158
flower size 67 T. lacaitae 152
secondary compound 4 4 T. latifolius 158
Polyphenols 15 1-168 T. leucotrichus 158
T. aestivus 15 1 T. littoralis 158
T. albanus 15 1 T. loevianus 158
T. algeriensis 15 1 T. longidens 15 8
T. alsariensis 15 1 T. longtjlorus 15 8
 T . loscosii 15 9 Quality 179
T. macedonicus 159 Quaternary, origin 18
T. marschallidnw 15 9 Quercetin 146
T . mastichina 159
T . mastlgophorus 159 Rosrnarinic acid 150
T.fnembranaceus 159
T . migricus 160 Sablnene 7 9
T. moesiacus 160 tr-Sabinene hydrate 79
T . nzoldavicus 160 Sakuranetin 147
T. nzoroderi 160 Salvigenin 146
T. nervosus 161 Scutellarein 147, 149
T . nz~mvnularit~s161 Section
T . orospedanus 1 61 Anonzali 9
T . pallasianus 162 Coridothymus 9
T . pannonicus 162 evolutionary relationships 16
T. pastoralis 162 Hyphodromi 18, 26, 29, 33
T . piperella 162 list of 27
T. platiphyllus 162 Mastichina 5, 8, 9, 26, 28, 31
7: podolicus 162 chemical polymorphism 127, 129, 135
T . polesicus 162 exudate flavonoids 168
T . praecox 162 principal component analysis 130
T . pseudogranitzcus 163 Mzcantes 9, 18, 26, 27, 31
T . pseudohumillimus 6 3 chemical polymorphism 135
T . pseudonumnzulariu 163 Orientales 9
T . pulegioides 163 Piperella 8 , 9, 26, 28, 32
T . rariflorus 163 chemical polymorphism 127, 129
T. richardiz 163 exudate flavonoids 168
T. ~d77~rei03de~ 163 flavonoids, chemoraxonomic value 171
T . serpylloides 164 principal component analysis 131
T . serpyllz~m 164 Pseudotbymbra 8 , 9, 26, 28, 32
T . sosnowskyi 165 chemical polymorphism 127, 129, 135
T . striatzts 165 exudate flavonoids 168
T . ~zdbn~pestri~ 165 flavonoids, chemotaxonom~cvalue 17 1, 172
T . tauricus 165 Serpyllttnz 5, 8, 9, 18, 26, 30, 34
T . tifIisiensis 165 flavonoids, chemotaxonomic value 17 1, 172
T. tosevii 165 Teu~riozdes 26, 28, 32
T. transcaucasicz~s 165 Thymastra 9
T . trautvetteri 165 Thynzzls 18, 26, 29, 32
T . ucrainicus 165 chemical polymorphism 127, 129, 135
T . villosus 165 exudate flavonoids 168, 169
T. vulgarzs 166 flavonoids, chernotaxonomic value 17 1, 172
T. webbianw 167 Vulgares 9
T. willkomii 167 zygis 8
T. zeleitzkyi 167 Seed germination 15
T . ziaratinz~s 167 Selection
T . zygis 168 method 182
Population structure 58 variety 180
Popular names 22 Serpyllastrum, Subsection 26, 30, 33
Processing, post-harvest 203, 234 Serpylli herba 304
Product formulation 3 11 Serpyllunz, Section 26, 30, 34
Protocatechuic acid 150 Serpyllum, Subsection 27, 31, 36
Pseudonzargznati, Subsection 27, 3 1; Sesquiterpenes 77, 7 8
36 Sideritoflavone 147
Pseudopiperellae, Subsection 26, 3 1, Solvent extraction 228
35 Sorbifolin 147
Pseudothyvzbra, Section 26, 28, 32 Spanish marjoram oil 253, 255, 257
Pseudothyvzbra, Subsection 29, 32 Spanish oregano oil 253, 254, 255,257
 322 Index
Spasmolytic effects 273-281 a-Terpinyl acetate 7 9
apigenin 279 Teucnoides, Section 26, 28, 32
camphor 278 Thecae, anther 17
carvacrol 278 Thymastpa, Subsection 2 6 , 29, 32
caryophyllene 278 Thymbra capitata 2 , 5 2 , 8 8 , 94, 95, 96, 97, 9 9 ,
cirsilineol 279 100, 1 0 4 , 1 7 0 , 2 5 4
luteolin 279 Thymbropsis, Subsection 26, 30, 34
myrcene 278 Thyme
thyme extract aromatherapy 266
thymol 278 cleaning methods 204
thymonin 279 cleaning, post-harvest 204
T . baetici~s277 colour 242
T . granatensis 276, 277 colour, processing 242
T . leptophylhs 280 comminution 208
T. longiforus 277 defects 205
T . orospedanzls 276, 277, 280 dried herb
T . satureioides 280 importation 259
T . vulgaris 273 production 257
T. webbiaws 280 specification 2 58
T . zygic 276, 277 drying 212,234,237-243
Species, list of 32 external use 265, 265
Specification, oil 254 extract 231, 239, 242, 306, 307
Statistics, production 252 antioxidant properties 233
Steam distillation 225 fresh herb production 259
Stem morphology 11 herbal drug 293-316
Sterilisation procedures 209 herbal tea 312
Storage, post-harvest 2 12 headspace volatiles 239, 240, 242
Sz~bbracteati,Subsection 26, 30, 33 homoeopathy 267
Subsection insect fragments 205
Alternantes 26, 31, 36, 171, 172 internal use 265, 266
Anomalae 26, 29, 32, 171, 172 irradiation 2 10, 244-247
Bracteatae 9 liquid extracts 3 11
Inszllares 26, 31, 34, 171, 172 medicinal use 263-283
Isolepides 26, 35 microbiological quality 244, 245
Kotschyani 26, 3 1, 34 non-medicinal use 284-286
i%fatszchznae 9 oil 227, 303
Piperella 9 adulteration 25 5, 257
Psezldomarginatz 28, 3 1, 36, 172 extraction methods 228
Pseudopiperellae 26, 31, 35, 171, 172 gas chromatogram 305
Psezldothymbra 26, 29, 32, 172 product formulation 3 12
Serpylla 9 toxicology 284
Serpyllastrum 26, 30, 33 pharmacological effect 268-282, 297
Serpyllz~m28, 3 1 , 36 phytorherapy 265
Subbracteati 26, 30, 33 product formulations 31 1
Thynzastra 26, 29, 32, 171, 172 production statistics 252, 253
Thynzbropsir 26, 30, 34 quality control 300-3 10
Thynzus 26, 29, 171, 172 red 253
Vulgares 9 specifications 254
Syringic acid 150 syrup 307
therapeutical use 264-267
Taxifolin 147 traditional use 264
Techniques, harvest 198 white 253
Tector hair 11 postprocessing treatments 209
Terpene biosynthesis 81-85 ethylene oxide 209
Terpinen-4-01 7 9 methyl bromide 209
a-Terpineol 7 9 ozone 21 1
-/-Terpinene 7 9 processing 224
 products 227 T. aruratz-nzinorzs 34
quality 234 polyphenols 152
quality control 300 T. x arcanus 38
therapeurical uses 264 T . x arczldtus 39
traditional use 264 T. argueus 29, 33, 100
Thymi aetheroleum 303 T. urmeniacus 34
Thymi extractum fluidum 306 T . x arnzuniae 39
T h y m ~extractum liquidum normatum 306 T . arsenijeuii 36
Thymi herba T . x arundunus 38
Commiss~onE monograph 265, 293,294 essential oil 8 8
European pharmacopoeia 301 gynodloecy 57
ESCOP monographs 295-297 T . aschurbajeuii 36
W H O monographs 308 T . asiaticz~s 37
Thymi sirupus 307 T . uttenuutus, polyphenols 152
Thymol 235 T. atticus 29, 33, 100
biosynthesis 8 1, 84 T. uznuuourii 30, 33, 100
content, effect of storage 220 T. baeticus 8, 10, 32
pharmacok~netics 283 chemical polymorphism 131
Thymonin 147 chemotypes 128
Thymus 1-3 16 essential oil 8 8
T . acinos 3 polyphenols 152
T . aestiuw principal component analysis 129
essential oil 92 T. bulcunus
polyphenols 15 1 essential oil 98
T . aztanue nothosubsp. dominguezii 40 polyphenols 152
T. x aitanae 40 T . bushhzriensis, polyphenols 153
T. alatauensis 36 T. x beltranii 40
T. albanus, polyphenols 15 1 T. x benitoi 39
T. ulbanu subsp. ulbunus 99 T . bihoriensis 3 1, 36
T . ulbicuns 5, 9, 12, 28, 32 T . bineruubtus 35
essential oil 88, 93 T. bitunzinosw 37
chemical polymorphism 135, 136 T . bleicherianus 28, 32
gynodioecy 57 T . bozssieri 29, 33
T. algarbienszs 9 T . x bonzchensis 39
T. algeriensis 19, 28, 29, 33 T. borgiae 88
bract morphology 12 T . bornmuelleri 3 1, 34, 100
essential oil 87, 104 T.borysthenicus 29, 33
leaf morphology 12 essential oil 97
polyphenols 15 1 polyphenols 153
T. x al7neriensis 40 T . x borzygzs 38
T. x alnzijarensis 38 T . bouei 9, 18, 30, 34, 104
T. alpestris 3 1, 36, 97 T.x brachychaetus 40
T. alpigenus 96 T. bracbychilus 29, 33
T. alpinus 3 T . bracbyphyllus 85
T. ultaicus 36 T. bracteatus 30, 33
T. ulsarensir, polyphenols 15 1 essential oil 8 8
T . alternuns 36 polyphenols 153
T. ulternus, polyphenols 151 T. bracteatus f. uiczosoi 40
T. amictus, polyphenols 15 1 T. bracteosus 29, 33
T. arnurensis 36 T . x bructichinu 38
T. antoninae 9, 10, 29, 33 T . broussonetii 8, 9, 30, 34
chemical polymorphism 131 essential oil 87
chemotypes 129 figure 8
essential oil 8 8 T. broussonetii subsp. broussonetii 34
polyphenols 15 1 T. broussonetii subsp. hunnonis 34
principal component analysis 130 T . bucharicus 37
T. urunjuezii, polyphenols 152 T.bulgaricus 3 1, 35
324 Index
Thymus (Continued) T. cimicznus 85
T. buschianus 36 T. cineraseens, polyphenols 154
T. caespititius 5, 9, 10, 11, 18, 19, 27, 31 T. czrumcinctus, polyphenols 154
calyx morphology 14 T. x citriodorus 96
chemical polymorphism 135, 136 T. clivorum 85
corolla morphology 14 T. collinu 3 5
figure 10 essential oil 102
essential oil 88, 93 polyphenols 154
polyphenols 153 T. comosus 3 1, 36, 98
T. calcarew, polyphenols 15 1 T. comptus 19, 29, 33
T. callieri, polyphenols 153 T. convolutus 29, 33
T. camphoratus 5, 12, 32 T. corilfoliu 35, 103
bract morphology 12 T. crenulatu 37
chemical polymorphism 131, 135 T. cretaceus, polyphenols 154
chromosomes 20 T. cuneatw 30, 33
essential oil 93 T. curtus 37
exudate flavonoids 169 T. czernjaevii 3 5
leaf morphology 12 polyphenols 155
polyphenols 153 T. dacicus 98
T.canoviridis 30, 33, 100 T.daenensis subsp. daenensis 3 5
T. capitatus 2, 6 , 4 1 , 227 T. daenensis subsp. Iancifolius 35
essential oil 88, 94, 95,96, 98,99, 100, 104 T. daenensis 35
enantiomeric composition 257 T. dahuricus 105
specification 254 T. dagestanicus 30, 3 3, 103
flavonoid composition 170 polyphenols 155
T. capitellatw 5, 6, 9, 29, 32 T. decussatz~s 18, 30, 34, 104
chemical polymorphism 135 polyphenols 155
chromosomes 20 T. desjatovae 35
essential oil 94 polyphenols 155
exudate flavonoids 169 T. x diazii 38
polyphenols 153 T. diminutw 37
T. cappadociczls 29, 3 3 T. dimorphus 35, 97
T. carzenszs 30, 34, 100 polyphenols 155
T. carmanicus 35, 104 T. disjunctus 36
T. carnosus 8 , 9, 10, 32 T. diversifoliw 37
chemical polymorphism 135 T.dolopzcus 29, 33
chromosomes 20 polyphenols 155
essential oil 88, 94 T. x donzinguezii 40
exudate flavonoids 169 T. dredtensis 3 1, 34
figure 17 T. dmcei 138
polyphenols 154 T. dzevanovskyz,polyphenols 155
T. x carolzpaui 40 T. eigii 30, 34, 100
T. x carrionii 40 T. x eliasii 40
T. caucasicus 36 T. elisabethae 35
polyphenols 15 1 T. eltonicus 85
T. x celtibericus nothosubsp. bonichensis 39 polyphenols 155
T. x celtibericus 39 T. x enicensis 38, 89
T. cephalotos 3, 6, 9, 85 T. eravinensis 37
polyphenols 154 T. erenzita 30, 33
T. cerebrifolius 37 T. ericoides 8 9
T. chanzaedrys 140 T. eriocalyx 35
T. chancoanus 37 T. eriophorus 35, 103
T. cherleriozdes 29, 3 3 T. eubajcalenszs 37
polyphenols 154 T. eupatoriensis,polyphenols 15 1
T. ciliatissinzus,polyphenols 154 T. extremus 37
T. ciliatus 87 T. fallax 3 1, 35, 100
T. cilicicus 30, 34, 100 T.fedtschenkoi 3 5, 103
 Index 125
T. fidtschenkoi vat. hundelii 10 1 T. x hieronynzi 40
T. flexilir 37 T. x hieronylni nothosubsp. hurtudoi 40
T. fominii 35 T. hivsutus, polyphenols 156
essential oil 103 T. hirtellus, polyphenols 156
polyphenols 155 T. hirtus 8, 9
T. fontunesii 9, 34 essential oil 87
T. fontqueri 30, 33 polyphenols 157
essential oil 89 T. holoserzceus 30, 34
polyphenols 155 T. x hurtudoi 40
T. x fontquenanu 38 T. hyemulir 2, 9, 10, 12, 19, 32, 201
T.froelichzunus 3 1, 36 calyx morphology 14
T. funkzi 9, 32 chemical polymorphism 132
chemical polymorphism 132 chemotypes 128
chemotypes 129 corolla morphology 14
essential oil 89 essential oil 8 9
exudate flavonoids 168 seasonal variation 199
polyphenols 155 figure 3
principal component analysis 130 leaf morphology 12
T. fi~nkiivat. nzurtineziz 40 pollen grain 19
T. x garcia-murtznoi 37 polyphenols 157
T. x genesidnus 40 principal component analysis 129
T. glubrescens 19, 3 1, 3 5, 98 T. hyemalis subsp. fumunifolius 32
T. glubrescens subsp. decipiens 35 T. hyenzulir subsp. hyenza1z.r 32
T. glubrescens subsp. glabrescens 35 T. hyemulzs subsp. nzilleflorir 32
T. glabrescens subsp. urumovii 35 T. hyenzalis x T. nzustichiuu subsp. ~nustichina
T. gbciulis 16, 36, 37 39
T. glundulosus T. x ibericus 39
essential oil 89 T. iljinii 37
polyphenols 156 T. inuequulir 37
T. gobiczlc 105 T. incertus 30, 33
T. godayanus 89 T. x indulicus 37, 8 9
T. granatensis 8 , 9, 20, 30, 33 T. integer 29, 33, 100
calyx morphology 14 T. zntercedens 35
corolla morphology 14 T. zrtyschensis 30, 33
essential oil 89 T. jujlue, polyphenols 157
habitus 21 T. jankae 98
polyphenols 156 T. jankae var. jankae
T. granatensis subsp. grunutensis 33 essential oil 99
T. grunutensis subsp. nzicranthus 33 polyphenols 157
T. graniticus, polyphenols 156 T. j u n k vat. pantotrichus
T. guberlinenszs 35 essential oil 9 9
T. x guerrue 40 polyphenols 157
T. guyonii 31, 34 T. jankae vat. putentipilus
T. hadzhievii 30, 33 essential oil 9 9
T. hartvigi 28, 32 polyphenols 157
T. hartvigi subsp. hurtvigi 32 T. jenisseensis 37
T. hartvigi subsp. macrocalyx 32 T. x jimenezzi 40
T. haussknechtii 30, 33, 101 T. x josephi-ungeli 40
T. helendzhicus 30, 33 T. x josephi-ungeli nothosubsp. edetunus 40
T. x henriquesii 38 T. jovinieni 40
T. herba-baronu 19, 3 1, 35 T. kalmiussicus,polyphenols 157
essential oil 95 T. ka~umurzanicus 35
exudate flavonoids 168, 169 essential 011 103
flavones 169 polyphenols 158
polyphenols 156 T. kurjaginii 30, 33
T. herba-burona subsp. bivalens 35 T. kirgisorunz 30, 33
T. herba-burona subsp. herbu-buronu 3 5 T. kjupuzi 35, 103
326 Index
Thymus (Continued) chemotypes 129
T. klokouii 35 corolla morphology 14
T. koeieanus 35 essential oil 90
T. komarovii 36 exudate flavonoids 168
T. kosteckyanus 97, 98 leaf morphology 12
T. kotschyanus 35 polyphenols 158
essential oil 103, 104 principal component analysis 130
polyphenols 158 T. loscosii 33
T. kotschyanus vat. glabrescens 101 essential oil 90
T. krylovii 87 polyphenols 159
T. lacaitae 10, 12, 30, 34 T. lotocephulus 2, 3, 6, 28, 32
bract morphology 12 bract morphology 12
essential oil 8 9 chemical polymorphism 135, 136
figure 26 essential oil 94
leaf morphology 12 leaf morphology 12
polyphenols 152 T. lusitanicz~s2, 8
T. laconicus 30, 34 T. nzacedonicz~s,polyphenols 159
T. ladjanuricz~s30 T. mucedonicu subsp. macedonicus 99
T. laeuigatus 18, 30, 35 T. nzagnus 105
T. x lainzii 38 T. majkopiensis 30, 34
T. lanceolatus 30, 34 T. maly 98
T. landjanzlricus 34 T. mandschuricus 37
T. latifolzus 35 T. x nzariae 38
polyphenols 158 T. nzarkhotensis 36
T. laurenkoanus 35 T. nzaroccanus 30, 34
T. leptophyllus 19, 30, 34 essential oil 87
polyphenols 158 figure 27
T. leptophyllus subsp. leptophyllus 90 T. maroccanus subsp. nzaroccanw 34
T. leucospermus 28, 32 T. maroccanus subsp. rhonzbicz~s34
T. leucostomus 30, 34 T. murschullianzl~
T. leucostomw var. argillaceus 101 essential oil 87, 97, 98, 103
T. leucostomw var. gypsaceus 101 polyphenols 159
T. leucostomw vat. leucostomus 101 T. x nzartinezii 40
T. leucotrichus 29, 3 3 T. nzastichina 2, 3, 9, 10, 19, 20, 28, 3 1
polyphenols 158 calyx morphology 14
T. linearis subsp. hedgez 36 chemical polymorphism 132, 135
T. linearis subsp. linearis 36 chromosome number 19
T. linearis 37 corolla morphology 14
T. lipskyi 30, 34 essential oil 90, 252
T. littoralis, polyphenols 158 figure 6, 22
T. longedentatus 19, 3 1, 35 gynodioecy 57
T. loevianus, polyphenols 158 polyphenols 159
T. longicaulic 19, 31, 36, 96, 98 T. nzastirhzna subsp. donyunae 28, 32
T. longicaulis subsp. chaubardii 36, 100, 101 essential oil 94
T. longicaulij subsp. longicaulis 36, 101 chemical polymorphism 136
T. longzcaulis var. longicaulis 96 T. mastichina subsp. mastichina 28, 31
T. longicaulis var. subzsophylhs 96, 101 essential 011 90, 94
T. longidens var. dassaretzcus chemical polymorphism 136
essential oil 9 9 T. mastichina var. b~uchychaetus40
polyphenols 158 T. x mastichinalzs 38
T. longidens var. lanicaulir T. nzastigophorus 29, 33
essential oil 99 chromosomes 20
polyphenols 158 essential oil 90
T. longifilorz~s8, 9, 12, 14, 32 polyphenols 158
bract morphology 12 T. membrunaceus 8 , 9, 10, 32
calyx morphology 14 chem~calpolymorphism 133
chemical polymorphism 132 chemotypes 129
 Index 327
essential oil 90, 91 chemical polymorphism 133
exudate flavonoids 168 essential oil 91
figure 24 polyphenols 161
polyphenols 159 principal component analysis 129
principal component analysis 130 T. oxyodontus 37
T. migriczl~ 35 T. palla~ianw 29, 3 3
essential oil 101, 103 polyphenols 162
polyphenols 160 T. pallasianus subsp. brachyodon 33
T. minussinensis 37 T. pallasianus subsp. pallasianus 3 3
T. x mixtus 39 T. palLescens 30, 34
T. x nzixtzls var. toletanus 40 T. pallidz~s 87
T. moesiacus T. pannonicus 3 1, 36
essential oil 9 9 essential oil 97, 98
polyphenols 160 polyphenols 162
T. moldavicus T. xparadoxus 38
polyphenols 160 T. parnasszcus 29, 33, 100
T. mongolicu 37, 105 T. pastoralis
T. x monrealensis 40, 9 1 essential oil 103
T. x monrealensis nothosubsp. garcza-vallejoi polyphenols 162
40,91 T. pectinatus 30, 34
T. x nzoralesii 39 T. pectinatus vat. pectinatus
T. x nzoralesii nothosubsp. cistetorum 39 essential oil 101
T. x moralesii nothosubsp. navarroi 39 seasonal variation 203
T. moroderi 2, 32 T. xpectinatzls 38
chemical polymorphism 133 T. perszcus 29, 33
chemotypes 129 T. petrueus 30, 34
essential oil 91 T. phyllopodus 37
exudate flavonoids 168, 169 T. piperella 2, 4, 5 , 9, 10, 20, 28, 29, 32
figure 4 calyx morphology 14
flavones 146, 169 chemical polymorphism 133
polyphenols 160 corolla morphology 14
principal component analysis 130 essential oil 91
T. x nzourae 39, 94 exudate flavonoids 169
T. nzunbyanus 29, 32 figure 23
T. nzunbyanus subsp. coLoratus 32 flavones 146, 169
T. nzunbyanus subsp. nzunbyanzls 32 habitus 21
T. murcicus 91 in vitro culture 190-195
T. naryrnensis 37 polyphenols 162
T. x navarroi 39 T. plasonii 29, 33
T. x pastoris 40 T. platypbyllus, polyphenols 162
T. nerczensis 30, 34 T. podolicus 36
T. nervoszls 3 1, 36 polyphenols 162
exudate flavonoids 168 T. polessirzls, polyphenols 162
T. nervulosw 37 T. portae 9
polyphenols 161 T. praecox 10, 12, 16, 18, 19, 31, 36
T. nitens 31, 35, 95 essential oil 97
T. noeanzls 38 polyphenols 162
T. nunzidiczls 30, 34 T. praecox subsp. arcticu
T. nzlmnzularizls 36 chemical polymorphism 137
essential oil 103 chemotypes 138, 139
polyphenols 161 essential oil 8 6
T. ochew 31, 36 exudate flavonoids 168
T. ochotensir 37 T. praecox subsp. praecox 36
T. odoratissimz~s85 T. praecox subsp. britannicus 36
T. oehnzianus 3 1, 36 T. praecox subsp. grossheinzii 36, 101
T. origanoides 9, 18, 30, 34 T. praecox subsp. grossheinzzz var. grossheivzii 10 1
T. orospedanus 32 T. praecox subsp. polytrichw 36, 96, 97
328 Index
Thymus (Continued) T. satureioides subsp. conznzutatus 3 1
T. praecox subsp. skorpilii 36 T. satureioides subsp. satureioides 3 1
T. praecox subsp. skorpilii vat. lanzger 101 T. schilnperi 18, 30, 35, 104
T. praecox subsp. skorpilii vat. skorpilii 101 T. schinzperi subsp. hedbergianzls 35
T. praecox subsp. zygzfir~nis 36 T. schimperi subsp. schz7zperi 35
T. proxinzw 37 T. schischkinii 37
T. przewalskii 36 T. semiglaber 36
T. pseudograniticus, polyphenols 163 T. x sennenzi 38, 40
T. pseudohumillimw, polyphenols 163 T. x sennenzi var. lez~codonthu 38
T. pseudonumnzzlaius 36 T. seravshanicz~s37
polyphenols 163 T. serpylhddes 33
T. pseudopulegioides 36, 102 polyphenols 164
T. pubescens 3 5, 102, 104 T. serpylloides subsp. gadorensis 3 3
T. pulchellus 36 chemical polymorphism 134
T. pulcherrinzus 36, 97 chemotypes 128
T. pulcherrimus subsp. carpathicus 36 essential oil 91
T. pulcherrimus subsp. pulcherrimzls 36 polyphenols 164
T. pulegzoides 3, 4, 10, 18, 3 1, 36, 199, 200 principal component analys~s 129
chemical polymorphism 137 T. serpylloides subsp. serpylloides 3 3, 9 1
chemotypes 140 essential oil 91
essential oil 86, 87, 94, 96, 97, 98, seasonal variation 199
exudate flavonoids 168 polyphenols 164
polyphenols 163 T. serpyllunz 3, 18, 31, 37
T. pulegioides subsp. chamaedrys 97 calyx morphology 14
T. puluinatus 29, 33 corolla morphology 14
T. pzmctatzls 35 enantiomeric composition 81
T. quinquecostatus 3 1, 37, 105 essential oil 87, 97, 99, 103, 104, 105, 252
T. quinquecostatus subsp. asiaticus 105 gynodioecy 57
T. quinquecostatus subsp. prazewlskii 105 harvesting date 198
T. x ramonianus 38 polyphenols 164
T. rariflorus T. serpyllunz praecox 95
essential oil 103 T. serpyllunz subsp. angustifolius 139
polyphenols 163 T. serpyllum subsp. serpylhvz 37
T. rewerdattoanus 37 chemotypes 139
?: revolutr*~29, 33, 102 chemical polymorphism 137
T. riatarunz 28, 31, 87 essential oil 86
T. richardii 12, 31, 35 T. serpyllum subsp. carnzolicus 85
leaf morphology 12 T. serpyllum subsp. tandensic 37
T. richardii subsp. ebusitanus 3 1, 34 chemotypes 139
polyphenols 163 chemical polymorphism 137
T. richardii subsp. nztidus 3 1, 33 essential oil 86
polyphenols 163 T. serpylhm var. mongolzcu 105
T. richardii subsp. richardii 3 1, 34 T. serrulatus 18, 30, 35, 104
T. x riojanzls 39 T. x seuerianoi 40
T. x riuas-molinae 38 T. sibiricus 37
T. roegneri 101 T. sibthorpii 3 1, 36
T. rohlenae 99 essential oil 100, 102
T. roseus 35 gynodioecy 57
T. x rubioi 39 T. sipylew 30, 34
T. x ruiz-latorrei 39 T. sipylew subsp. rosulans 34
T. sabulicola 9 T. sipyleus subsp. sipyleus 34
T. sanzins 29, 34 T. sipyleus subsp. sipyleus var. dauisianus 102
T. satureioides 28, 3 1 T. sipyleus subsp. sipylezls var. sipyleus 102
essential oil 87 T. sokolovii 37
exudate flavonoids 169 T. x sorianoi 39
flavones 169 T. sosnowskyi 30, 34, 165
polyphenols 163 T. spathuliJoliw 30, 34, 102
 Index 329
T . spinzllosus 27, 33 chemical polymorphism 135, 136
T. squarrosw 3 5, 8 5 essential oil 91, 94
T. stojanovii 3 1, 36 T. vzlloszls subsp. oretaniczis 32
T. striatzls 19, 29, 33 T. villoszls subsp. ~llosus32, 94
essential oil 96, 98 chemical polymorphism 135
polyphenols 165 T.vulgarzs 2 , 3 , 5 , 7 , 9 , 18, 1 9 , 2 9 , 3 2
T. smatzls var. mterrzlptzis 102 chemical polymorphism 134
T. szlhalpestvis, polyphenols 165 chemotypes 47, 128, 227
T. szlbcollinw 102 adaptive value 47
T. sylvestris 5 distribution 50
T. syriacus 30, 34, 102 spatial structure 4 7 , 4 9
T. talijevii 3 1, 37 cultivation 178
T. taz~riczls,polyphenols 165 essential oil 92, 95, 96, 104, 105, 252
T. tezlcrioides 28, 32 composition 202, 236
T. tezlcrioides subsp. aljjinus 32 enantiomeric composition 8 1
T. teucriozdes subsp. candiliczls 32 figure 5, 25
T. teucriozdes subsp. tezlcrioides 3 2 polymorphism 45
T. thracicus 3 1, 36 seasonal variation 199
T. thracicz~svar. longzdens 102 specification 254
T. t$lisiensis 36 storage 218, 219
essential oil 103 yield 235, 236
polyphenols 165 female frequency 61
T. thraciczls 36 frost damages 187
T. x toletanus 40 frost resistance 186
T. tomentotw 5, 9 gene flow 5 5
T. tonsilzs 37 germination 52, 53, 187
T. toseviz 100 glandular peltate trichome 75, 76
T. twevii subsp. szlbstndtus harvesting date 198, 199
essential oil 97 hermaphrodites 64, 66
polyphenols 165 in vitro culture 189, 190
T. tosevii subsp. tosevii 99 male fertility restoration 60
T. tosevii subsp. tosevii var. degenii monoterpenes
essential oil 99 genetic control 46
polyphenols 165 chemical defence 53, 54
T. tosevii subsp. tosevzi var. longlfronj phenotype, homogeneity 185
essential oil 79 polyphenols 166
polyphenols 165 principal component analysis 129
T. tosevii subsp. tosevii var. tosevii productivity 182, 183
polyphenols 165 seed production 186
T. transcaspiczls 35 selection 180, 182
T. transcauraszczls 3 1, 35 sex determination 67
essential oil 103 T. vulgaTi( subsp. aestivus 32, 92
polyphenols 165 T. vfdgaris 5ulLrp. wicoide~,pol yphenols 167
T. trazltvetteri 35 T. vulgaris subsp. vulgaris 32, 92
essential oil 104 T. webbianus
polyphenols 165 essential oil 92
T. tschernjajeviz 36 polyphenols 167
T. turczaninovii 36 T . x welwitschii 7, 38
T . ucrainicus, polyphenols 165 T . willdenowii 8, 28, 32
T. u5~11rien5is37 T. wilLhornnzii 3 1, 34, 92
T. x viciosoi 40, 94 polyphenols 167
T. vzllosw 3, 6, 9, 12, 32 T. x xilocae 37
bract morphology 12 T. zeleneitzkyi, polyphenols 168
leaf morphology 12 T. ziaratinzls 35
polyphenols 165 polyphenols 167
T. villoszls subsp. lzlsitanicus T. zygiozdes 19, 30, 34
32 T. zygiozdes vat. lycaunicus 102
330 Index
Thymus (Continued) chemical polymorphism 135
T . zygioides var. zygioides 102 essential oil 95
7.zygis 2 , 3 , 7 , 9 , 12, 1 9 , 2 9 , 3 2 polyphenols 168
chemical polymorphism 134, 135 T. x zygophov~ 40, 9 3
chemotypes 128 T h y m u , Section 18, 26, 29, 32
essential oil 88, 92, 252 Thynzus, Subsection 26, 29, 32, 171,
enantiomeric composition 8 1 172
habitus 21 Thymusin 147
leaf morphology 12 Thymus species, essential oil 85-10>
specification 254 Toxicology, thyme oil 284
seasonal variation 202 Traditional use 264
gynodioecy 57 Trichome 11
polyphenols 168 anatomy 7 6
principal component analysis 129
T. zygir subsp. gracilir 32, 92 Vanillic acid 150
T . zygk subsp. syluestris 32, 9 3 Vernacular names 20
essential oil 95
chemical polymorphism 135 Water distillation 225
polyphenols 168
T . zygis subsp. zygis 32, 9 Xanthomicrol 147
(Continued)

Volume 17
Tea, edited by Yong-su Zhen
Volume 18
Artemisia, edited by Colin W . Wright
Volume 19
Stevia, edited by A. Douglas Kinghorn
Volume 20
Vetiueria, edited by Massimo Maffei
Volume 2 1
Narcissas and Daffodil, edited by Gordon R. Hanks
Volume 22
Eucalyptus, edited by John J.W. Coppen
Volume 23
Pueraria, edited by Wing Ming Keung
Volume 24
Thyme, edited by E . Stahl-Biskup and F. SBez
Volume 25
Oregano, edited by Spiridon E. Kintzios
Volume 26
Citrus, edited by Giovanni Dugo and Angelo Di Giacomo
Volume 27
Geranium and Pelargonium, edited by Maria Lis-Balchin
Volume 28
Magnolia, edited by Satyajit D. Sarker and Yuji Maruyama
Volume 29
Lavender, edited by Maria Lis-Balchin
Volume 30
Cardamom, edited by P.N. Ravindran and K.J. Madhusoodanan