Biochemical Systematics and Ecology 31 (2003) 587–600
www.elsevier.com/locate/biochemsyseco
Leaf surface flavonoids in Iranian species of
Nepeta (Lamiaceae) and some related genera
Ziba Jamzad abc, Renée J. Grayer b, Geoffrey C. Kite b, Monique
S.J. Simmonds b,∗, Martin Ingrouille c, Adel Jalili a
a
c
Research Institute of Forests and Rangelands, P.O. Box 13185-116, Tehran, Iran
b
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK
School of Biological and Chemical Sciences, Birkbeck College, University of London, Mallet Street,
WC1E 7HX, UK
Received 17 August 2002; accepted 30 September 2002
Abstract
A HPLC survey of the leaf surface flavonoids of 38 species of Nepeta (Lamiaceae) and
four species of the related genera Agastache, Dracocephalum and Lallemantia revealed 14
different flavones, one of which is new (8-hydroxycirsiliol or 5,8,3⬘,4⬘-tetrahydroxy-6,7dimethoxyflavone). In addition, two flavonols (methyl ethers of kaempferol) were found in
Dracocephalum kotschyii. The most frequently encountered flavones in Nepeta were cirsimaritin (5,4⬘-dihydroxy-6,7-dimethoxyflavone); 8-hydroxycirsimaritin (5,8,4⬘-trihydroxy-6,7-dimethoxyflavone, also called isothymusin) and genkwanin (5,4⬘-dihydroxy-7-methoxyflavone).
Apigenin and the 4⬘-methyl ethers of cirsimaritin and 8-hydroxycirsimaritin (salvigenin and
8-hydroxysalvigenin, respectively) were also relatively common. The distribution of surface
flavones in the four genera provided some valuable data for the phylogenetic relationships at
generic level. The presence of surface flavones with a 5-hydroxy-6,7-dimethoxy A-ring (as
found in cirsimaritin and salvigenin) and the unusual 5,8-dihydroxy-6,7-dimethoxy A-ring substitution pattern (as found in 8-hydroxycirsimaritin, 8-hydroxysalvigenin and 8hydroxycirsiliol), can be considered as a characteristic chemotaxonomic feature typical of the
genus Nepeta. Cirsimaritin and 8-hydroxycirsimaritin were also detected in the one species
examined for Agastache, but the absence of genkwanin and the presence of acacetin in A.
barberi and in ten Agastache species studied previously, distinguished Agastache from Nepeta.
The presence of methoxylated flavonols and absence of 8-hydroxycirsimaritin and related com-
∗
Corresponding author. Tel.: +44-208-332-5328; fax: +44-208-332-5340.
E-mail address: m.simmonds@rbkew.org.uk (M.S.J. Simmonds).
0305-1978/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0305-1978(02)00221-1
588
Z. Jamzad et al. / Biochemical Systematics and Ecology 31 (2003) 587–600
pounds distinguished species of Dracocephalum and Nepeta, whereas lack of flavonoids with
6- and 8-oxygenation of the A-ring characterised species of the genus Lallemantia.
2002 Elsevier Science Ltd. All rights reserved.
Keywords: Nepeta; Catmint; Agastache; Dracocephalum; Lallemantia; Lamiaceae; External flavonoids;
8-Hydroxyflavones; Chemotaxonomy
1. Introduction
The genus Nepeta (catmint) comprises about 300 species, occurring in Asia and
Europe. The greatest diversity and richness of species is found in two areas, Southwestern Asia, especially Iran, and the Western Himalayas including Hindukosh
(Pojarkova, 1954). It is one of the largest genera of Lamiaceae in Iran where 75
species are found, of which 54% are endemic. Some species are used as medicinal
herbs in Iran (for example, N. ispahanica, N. binaloudensis, N. bracteata, N. pogonosperma and N. pungens), and N. crispa is used as a culinary herb. The medicinal
properties of Nepeta species are usually attributed to their essential oils and flavonoids.
Nepeta belongs to the subfamily Nepetoideae, tribe Mentheae (Cantino et al.,
1992). There are three global infrageneric classifications of Nepeta, those of Bentham
(1832-1848, 1848); Briquet (1896) and Budantsev (1993). These works are mainly
based on morphological characters, which are very variable and do not reflect phylogenetic relationships among the species and some ambiguities among these classifications are mainly the consequence of the paucity of reliable morphological characters. Because of the difficulty in using classical morphological and anatomical
characters to establish relationships among the species of Nepeta, chemical and molecular characters have been investigated to assess their potential value in phylogeny
reconstruction (Jamzad, 2001).
Among chemical features, surface flavonoids (also called external flavonoids) have
been shown to provide useful taxonomic characters at various levels of classification
(Tomás-Barbarán and Gil, 1992). These compounds, which are aglycones and therefore lipophilic constituents, are not as universal in occurrence in higher plants as the
more polar flavonoid glycosides, which occur in vacuoles in the plant cells. However,
surface flavonoids are common in the Lamiaceae, especially in the subfamily Nepetoideae. Their presence is often correlated with the production of other lipophilic secondary products such as essential oil terpenoids (Wollenweber, 1982). These terpenoid-flavonoid mixtures appear to function as UV screens, for heat reduction, as
anti-microbial agents or as insect-feeding deterrents (Porksch and Rodriguez, 1985).
In the Lamiaceae, at least 147 structurally different surface flavonoids have been
identified, and a correlation between certain flavonoid types and subfamilial or tribal
classification has been established (Tomás-Barberán and Gil, 1992). The significance
of surface flavonoids as taxonomic characters at the infrageneric level has also been
determined in a number of genera, e.g. Thymus (Hernandez et al., 1987), Teucrium
(Harborne et al., 1986) and Ocimum (Grayer et al., 2001).
Z. Jamzad et al. / Biochemical Systematics and Ecology 31 (2003) 587–600
589
This paper reports on a study of external flavonoids in Nepeta and some related
genera and evaluates their usefulness in determining the phylogenetic relationships
at species and generic levels.
2. Materials and methods
2.1. Plant material
Leaves of 38 species of Nepeta, one species of Agastache, two of Dracocephalum
and one of Lallemantia were investigated. Most plant species were collected from
their natural habitats in Iran. Seeds when available were collected from the different
localities, and these were germinated in a glasshouse and then grown outside in the
Royal Botanic Gardens, Kew. The remaining species were studied from recently
dried material, apart from a few species that were studied from old herbarium
material. Table 1 shows a list of the plant species that were investigated, the locality
from which they were originally collected and details of the vouchers deposited in
various herbaria. Details about the state of the leaves investigated (fresh, recently
dried or old herbarium material) are presented in the Results along with data about
the distribution of surface flavonoids (Table 3).
2.2. Extraction
Fresh leaves to a weight of 5 g were washed in 20 ml diethyl ether for 5 min and
then the extract was filtered and measured. One third of each extract was used for
the flavonoid identification, and this was evaporated to dryness (the remainder of
the extracts were used for other studies). Dried leaves and herbarium material (0.5–
1 g) were soaked overnight in diethyl ether and then processed in the same way as
the extracts of the fresh materials. The dried extracts were redissolved twice in 0.5
ml of 100% MeOH, and these two solutions were filtered into a HPLC autosampler
vial through a Gelman Nylon Acrodisc 13 filter (pore size 0.45 µm) before HPLC
analysis.
2.3. HPLC with Diode Array Detection (HPLC-DAD)
The flavonoid profile of each extract was examined using a HPLC system consisting of a Waters LC 600 pump and 996 photodiode array detector. A Merck LiChrospher 100RP-18 (5 µm) column was used (4.0 mm i.d. × 250 mm) and a gradient
profile based on two solvents denoted A and B was employed. A was 2% aqueous
HOAc and B was MeOH-HOAc-H2O, 18:1:1. Initial conditions were 75% A and
25% B, with a linear gradient reaching B ⫽ 100% at t ⫽ 20 min. This was followed
by isocratic elution (B ⫽ 100%) to t ⫽ 24 min, after which the programme returned
to the initial solvent composition. The temperature of the column was maintained at
30 °C and a flow rate of 1.0 ml min⫺1 was used. For each extract a 40 µl injection
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Z. Jamzad et al. / Biochemical Systematics and Ecology 31 (2003) 587–600
Table 1
Species studied and information about their source and voucher numbera
Species
Collecting data, voucher no. and herbaria
Agastache barberi (B.L.Rob.) Epling Cultivated in RBG Kew, Chase 10034 (K).
Dracocephalum grandiflorum L.
Cultivated in RBG Kew, Chase 10098 (K).
D. kotschyii Boiss.
Iran, Tehran, Alborz Mt., cultivated in RBG Kew, Chase
11158 (K).
Lallemantia peltata (L.) Fisch. &
Bakhtiari, Wastegan, Chahartagh excloser, 1900 m, Jamzad et
C.A. Mey.
al. 79930 (TARI).
Nepeta amoena Stapf
Iran, Azarbayejan,between Meshkinshahr and Ahar, Nowdouz,
1100 m, Wendelbo & Assadi 27882 (TARI).
N. assurgens Hausskn. & Bornm.
Iran, Kerman, Mt. Lalehzar, 3000 m, Jamzad et al. 80094
(TARI).
N. asterotricha Rech.f.
Iran,Yazd, Tezarjan, Barfkhaneh, 2850m, Froughi 5562
(TARI).
N. bornmulleri Hausskn. Ex Bornm. Iran, Kerman, 25 Km from Sirjan to Shahre Babak, 1680 m,
Mozaffarian 74229 (TARI).
N. bucharica Lipsky
USSR, cultivated at RBG Edinburgh, Chase 10032 (K).
N. cataria L.
Iran, Tehran, cultivated at RGB Kew, Chase 10035 (K).
N. cephalotes Boiss.
Iran, Tehran, NW of Tehran, Yadegar- Emam Free way, 1500
m, Jamzad 80475 (TARI).
N. congesta Fisch. & C.A.Mey.
Iran, Bakhtiari, Lordegan Chenar- e Mahmoodi,
var. cryptantha (Boiss.)
exclosure, 2100-2200 m, Jamzad et al.79954 (TARI).
Hedge & Lamond
N. crassifolia Boiss. & Buhse
Iran, Tehran, cultivated at RBG Kew, Chase 10033 (K).
N. crispa Willd.
Iran, Hamadan, cultivated at RBG Kew, Chase 10026 (K).
N. daenensis Boiss.
Hamadan, ca. 8 km E of Ganjnameh, 2750 m, Assadi and
Mozaffarian 36866 (TARI).
N. denudata Benth.
Iran, Tehran, W. of Tehran, Souleghan, Sangan, 2300 m,
Jamzad and Nikchehreh 80407 (TARI).
N. dschuparensis Bornm.
Iran, Kerman, Kuh-e Lalehzar, 3000 m, Jamzad, 80009,
(TARI).
N. fissa C.A. Mey.
Iran, Tehran,W of Tehran, cultivated at RBG Kew, Chase
10028 (K).
N. gloeocephala Rech.f.
Iran, Kashan,between Ghamsar & Reza-Abad, 2450 m, Assadi,
Jamzad and Azizian 80054 (TARI).
N. glomerulosa Boiss.
Iran, Kerman, cultivated in RBG Kew, Chase, 10037 (K).
N. grandiflora M.B.
Cultivated in RBG Kew, Chase 10097 (K).
N. isaurica Boiss. & Helder.
Turkey, cultivated at RBG Edinburgh, Chase 10096 (K).
N. ispahanica Boiss.
Iran, Tehran, c. 80 km to Qom, cultivated at RBG Kew, Chase
10023 (K).
N. kurdica Hausskn. & Bornm.
Iran, Kordestan, cultivated in RBG Kew, Chase 9951 (K).
N. menthoides Boiss. & Buhse
Iran, Azarbayejan, cultivated at RBG Kew, Chase 10025 (K).
N. microsiphom Boiss.
Iran, Fars, Kuh-e Dena, 2900 m, Riazi 7415 (TARI).
N. mirzayanii Rech.f.
Iran, Balouchestan, 18 km from Khash to Iranshahar, road to
Irandegan, 1500 m, Mozaffarian 42836 (TARI).
N. mussinii Spreng.
Cultivated in RBG Kew, Chase 10099 (K).
N. nuda L.
Cultivated in RBG Kew, Chase 9953 (K).
N. oxyodonta Boiss.
Iran, Fars, Bamu National Park, Jamzad et al. 69391 (TARI).
N. persica Boiss.
Bakhtiari, between Lordegan and Cenar-e Mahmoudi, 21002200 m, Jamzad et al. 79953 (TARI).
(continued on next page)
Z. Jamzad et al. / Biochemical Systematics and Ecology 31 (2003) 587–600
591
Table 1 (continued)
Species
Collecting data, voucher no. and herbaria
N. pogonosperma Jamzad & Assadi
N. pungens (Bunge) Benth.
Iran, Mazandaran, cultivated in RBG Kew, Chase 10029 (K).
Iran, Bakhtiari, Gandoman, Comunication Station, 2100 m,
Jamzad et al. 79960 (TARI).
Iran, Tehran, Touchal, Jamzad 79977 (TARI).
Afghanistan, Doab, 15 km W Doab, 1450 m, Hedge and
Wendelbow, 3444 (E).
Iran, Kerman, Mt. Lalehzar, 3180 m, Jamzad et al. 80006
(TARI)
Iran, cultivated at RBG Kew, Chase 10031 (K).
Iran, Khorassan, 9 km from Kashmar towards Neyshabour,
1300 m, Assadi and Mozaffarian 35559 (TARI).
Iran, Bakhtiari, Gandoman, Comunication Station, 2100 m,
Jamzad et al. 79955 (TARI).
Cultivated in RBG.Edinburgh, no: 1947010A, Chase 10100
(K).
Afghanistan, East of Augardan, pass on road from Panjao to
Bisut, rocky slopes, 2750-3150 m, Hedge and Wendelbow, W
5011 (E).
Iran, Lorestan, cultivated in R.B.G.Kew, Chase, 10030 (K).
N. racemosa Lam.
N. rechingeri Hedge
N. rivularis Bornm.
N. saccharata Bunge
N. satureioides Boiss.
N. schiraziana Boiss.
N. sibirica Bunge
N. spathulifera Benth.
N. straussii Hausskn. & Bornm.
a
Herbaria: E ⫽ RBG Edinburgh, K ⫽ RBG Kew, TARI ⫽ Tehran, Iran.
was made using an autosampler. Retention times and UV spectra of the flavonoids
in the extracts were compared with those of standards.
2.4. HPLC with Atmospheric Pressure Chemical Ionisation Mass Spectrometry
(LC-APCI-MS)
Some extracts were further analysed by LC-APCI-MS. Chromatography was performed in a similar manner to analytical HPLC except that the concentration of
HOAc in solvents A and B was 1%. Mass spectra were recorded using a quadruple
ion trap mass spectrometer (Finnigan LCQ) with the sample being ionised by an
APCI source operated in positive mode and using a vaporiser temperature of 550
°C, sheath and auxiliary nitrogen flow pressure of 80 and 20 psi, respectively, a
capillary temperature of 150 °C and a needle current of 5 µA. The mass spectrometer
was controlled by Xcalibur 1.1 software (Finnigan) and programmed to record survey
scans in the range m/z 125–500, followed by collision-induced dissociation spectra
(CID) of the most intense ions in each survey scan. CID spectra were obtained by
prior isolation of the parent ion in the trap (isolation width 5 amu) and then applying
a collision energy of 45% without wideband activation. Dynamic exclusion allowed
the CID spectra of co-eluting compounds to be recorded automatically by the instrument without prior knowledge of their molecular masses.
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Z. Jamzad et al. / Biochemical Systematics and Ecology 31 (2003) 587–600
Table 2
Chromatographic and spectral data of surface flavonoids found in species of Nepeta and related genera
(n.d. ⫽ no data, as the compounds were present in too low concentrations in the extracts to give good
LC-MS data)
Compound no. Flavonoid
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Luteolin (5,7,3⬘,4⬘-tetrahydroxyflavone)
8-Hydroxycirsiliol (5,8,3⬘,4⬘tetrahydroxy-6,7-dimethoxyflavone)
Thymusin (5,6,4⬘-trihydroxy-7,8dimethoxyflavone)
Chrysoeriol (5,7,4⬘-trihydroxy-3⬘methoxyflavone) or Diosmetin (5,7,3⬘trihydroxy-4⬘-methoxy flavone)?
Cirsiliol (5,3⬘,4⬘-trihydroxy-6,7dimethoxyflavone)
8-Hydroxycirsimaritin or isothymusin
(5,8,4⬘-trihydroxy-6,7dimethoxyflavone)
Apigenin (5,7,4⬘-trihydroxyflavone)
Kaempferol 3-methyl ether (5,7,4⬘trihydroxyflavonol 3-methyl ether)
Cirsimaritin (5,4⬘dihydroxy-6,7dimethoxyflavone)
Ladanein (5,6-dihydroxy-7,4⬘dimethoxyflavone)
Xanthomicrol (5,4⬘-dihydroxy-6,7,8methoxyflavone)
8-Hydroxysalvigenin (5,8-dihydroxy6,7,4⬘-trimethoxyflavone)
Acacetin (5,7-dihydroxy-4⬘methoxyflavone)
Genkwanin (5,4⬘-dihydroxy-7methoxyflavone)
Kaempferol 3,7-dimethyl ether? (5,4⬘dihydroxy-3,7-dimethoxyflavonol)
Salvigenin (5-hydroxy-6,7,4⬘trimethoxyflavone)
λmax (nm)
MH+ (m/z)
17.6
17.8
255, 267sh, 348
285, 295sh, 345
287
347
18.7
295, 336
331
18.8
253, 267sh, 346
301
19.0
273, 348
331
19.1
304, 333
331
19.2
19.7
267, 338
266, 352
271
301
20.4
276, 336
315
21.4
284, 334
n.d.
21.8
280, 290sh, 333
345
21.9
304, 333
n.d.
22.5
266, 333
n.d.
22.9
267, 338
285
23.2
266, 347
315
23.3
276, 333
329
Rt (min)
3. Results and discussion
3.1. Identification of the flavonoids
A total of 14 flavones and two flavonols were detected in the surface extracts of
species of Nepeta, Agastache, Dracocephalum and Lallemantia (Table 2). Compounds 1, 3, 5–7, 9–14 and 16 could be identified by comparing their HPLC retention
times, UV spectra and mass spectra (especially the CID spectra) with the data
obtained from standards in in-house libraries. The remaining flavonoids, 2, 4, 8 and
15, could only be identified provisionally.
Table 3
Distribution of surface flavonoids in species of Nepeta and related genera (the flavones are grouped according to their A-ring substitution patterns)a
Species
5,7-diOH flavones
F
1
H
+
++
+
∗
∗
∗
∗
4
7
+
±
±
∗
∗
∗
+
+
∗
∗
∗
∗
∗
∗
∗
∗
+
±
∗∗
±
±
∗
±
∗
∗
∗
13
14
10
5,65-OH-6,7-diOMe
diOH- flavones
7,8diOMe
flavones
3
5
9
16
+
±
++
±
±
±
±
t
+
±
5,8-diOH-6,7diOMe flavones
5-OH- Flavonol 36,7,8Me ethers
triOMe
flavones
2
11
8
15
++
++
+
+
+
++
+
+
+
∗∗
∗
∗
∗
∗
∗
5-OH- 5,67-OMe diOH flavones 7-OMe
flavones
++
±
+
+
++
±
++
+
+
±
±
++
+
+
±
++
+
±
++
±
++
±
6
12
+
++
+
++
±
+
++
+
t
++
+
+
±
+
t
±
t
±
++
±
±
±
±
±
±
t
±
Z. Jamzad et al. / Biochemical Systematics and Ecology 31 (2003) 587–600
Agastache barberi
Dracocephalum grandiflorum
D. kotschyii
Lallemantia peltata
N. amoena
N. assurgens
N. asterotricha
N. bornmulleri
N. bucharica
N. cataria
N. cephalotes
N. congesta var. cryptantha
N. crassifolia
N. crispa
N. daenensis
N. denudata
N. dschuparensis
N. fissa
N. gloeocephala
N. glomerulosa
N. grandiflora
N. isaurica
N. ispahanica
N. kurdica
N. menthoides
N. microsiphon
Plant
material
usedb
++
593
(continued on next page)
594
Table 3 (continued)
N.
N.
N.
N.
N.
N.
N.
N.
N.
N.
N.
N.
N.
N.
N.
N.
mirzayanii
mussinii
nuda
oxyodonta
persica
pogonosperma
pungens
racemosa
rechingeri
rivularis
saccharata
saturejoides
schiraziana
sibirica
spathulifera
strausii
Plant
material
usedb
5,7-diOH flavones
F
1
H
∗
4
7
5-OH- 5,67-OMe diOH flavones 7-OMe
flavones
13
±
∗
∗
∗
∗
∗
14
±
±
±
+
∗
∗
±
∗
++
±
∗
∗
∗
∗
∗
±
t
∗
∗
±
t
+
±
t
10
5,65-OH-6,7-diOMe
diOH- flavones
7,8diOMe
flavones
3
5
9
16
t
±
+
+
+
++
±
±
+
±
±
±
++
±
±
±
+
±
±
+
5,8-diOH-6,7diOMe flavones
5-OH- Flavonol 36,7,8Me ethers
triOMe
flavones
2
11
±
±
±
++
+
±
6
t
±
+
±
+
++
±
++
±
±
12
+
8
15
+
±
++
+
++
±
±
a
Relative amounts of compounds according to the UV absorbance on the HPLC chromatograms: t ⫽ trace; ± 0.005–0.05 absorbance units; ⫹ 0.05–0.5 absorbance
units; ++ more than 0.5 absorbance units.
b
Plant material used for extraction: F ⫽ fresh; H ⫽ herbarium: ∗∗ recently dried herbarium material; ∗ old herbarium material.
Z. Jamzad et al. / Biochemical Systematics and Ecology 31 (2003) 587–600
Species
Z. Jamzad et al. / Biochemical Systematics and Ecology 31 (2003) 587–600
595
The molecular mass determined for flavonoid 2 was 346 Da, which is consistent
with a tetrahydroxy-dimethoxy-flavone. The λmax of the long wave UV band was
345 nm, suggesting that the 3⬘- and 4⬘-carbons of the B-ring are oxygenated. The
remaining four hydroxyl and methoxyl groups should be in the A-ring (positions 5,
6, 7 and 8). In a previous study it was found that the A-ring hydroxy/methoxy substitution pattern of flavones oxygenated in the 5, 6, and 7-positions or in the 5, 6, 7
and 8-positions can be predicted from the product ions generated and their relative
abundance during CID when using APCI-MS in the positive mode (Grayer et al.,
2001). The CID spectrum of 2 indicated that the A-ring of this flavone has a 5,8dihydroxy-6,7-dimethoxy substitution pattern (product ions at [MH-15]+ and [MH33]+ with relative abundances of 100% and 40%, respectively, very similar to those
found in 5,8,4⬘-trihydroxy-6,7-dimethoxyflavone and 5,8-dihydroxy-6,7,4⬘-trimethoxyflavone (Grayer et al., 2001). Therefore compound 2 was tentatively identified
as 5,8,3⬘,4⬘-tetrahydroxy-6,7-dimethoxyflavone. This is a new plant compound, but
Horie et al. (1995) have synthesised it and described the spectra of this flavone. The
λmax of the UV spectrum of the compound given by these authors are similar to
those determined for flavone 2 (see Table 2). We propose the name 8-hydroxycirsiliol
for this flavone, because cirsiliol is the likely biogenetic precursor of 2 and also
because it accentuates the fact that the compound has a free 8-hydroxyl group, which
is rare in external flavones.
Horie et al. (1995) also synthesised the 8-hydroxyflavones isothymusin (5,8,4⬘trihydroxy-6,7-dimethoxyflavone) and its 4⬘-methyl ether (5,8-dihydroxy-6,7,4⬘trimethoxyflavone). The latter compound was first described by La Duke (1982)
from Tithonia pedunculata and called pedunculin, but appears to have been wrongly
identified. Horie et al. (1995) showed that the structure of the flavone from this plant
is in fact 5,7-dihydroxy-6,8,4⬘-trimethoxyflavone (nevadensin), and that therefore the
name pedunculin is a synonym of nevadensin and should no longer be used. The
compound reported as pedunculin from species of Ocimum (Grayer et al., 2001) is
really 5,8-dihydroxy-6,7,4⬘-trimethoxyflavone (it has a CID spectrum in accordance
with this substitution pattern and the same UV spectrum as that given by Horie et
al., 1995), so that it could be called either isothymusin 4⬘-methyl ether, to show the
A-ring substitution relationship of this flavone with isothymusin, or 8-hydroxysalvigenin, to show its biogenetic relationship and free 8-hydroxyl group. We propose the
latter option and also propose to change the name isothymusin (compound 6) to 8hydroxycirsimaritin, for the same reasons and because the name isothymusin often
gets confused with that of its isomer thymusin (5,6,4⬘-trihydoxy-6,7dimethoxyflavone). Isothymusin was originally obtained as an acid-catalysed
interconversion product (Wessely-Moser rearrangement) of thymusin from Thymus
membranaceous (Ferreres et al., 1985), but it was subsequently detected in Becium
grandiflorum Pic. Serm. ( ⫽ Ocimum grandiflorum Lam.) (Grayer and Veitch, 1998)
and other species of Ocimum (Grayer et al., 2001).
Flavonoid 4 has the same molecular mass and a similar UV spectrum as chrysoeriol and diosmetin, the 3⬘- and 4⬘-monomethyl ethers of luteolin, respectively, which
are difficult to distinguish by HPLC procedures only. It also could be the 7-methyl
ether of luteolin, which has the same molecular mass and a similar UV spectrum,
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but not the 5-methyl ether, because this has a quite different UV spectrum. To establish the position of the methyl group, this compound should be isolated and the UV
spectra determined using shift reagents (Mabry et al., 1970), but this could not be
carried out because lack of sufficient plant material for isolation work.
The UV spectrum of flavonoid 8 was that of a kaempferol derivative with a substituted 3-hydroxy group. As the molecular mass (300 Da) was consistent with a monomethyl ether of kaempferol, compound 8 was tentatively identified as the flavonol,
kaempferol 3-methyl ether. The UV spectrum of flavonol 15 was very similar to that
of 8, but the molecular mass was 14 Da more (consistent with a methyl group),
suggesting that 15 is a dimethyl ether of kaempferol. Therefore 15 was provisionally
identified as kaempferol 3,7-dimethyl ether, which has a similar UV spectrum as the
3-methyl ether. However, it also could be the 3,4⬘-dimethyl ether of kaempferol.
Isolation of 15 and UV spectroscopy using shift reagents are needed to ascertain the
position of the second methyl group.
3.2. Distribution of the surface flavonoids in species of Nepeta and related
genera
Table 3 shows the distribution of surface flavonoids in the species of Nepeta,
Agastache, Dracocephalum and Lallemantia examined. The flavones have been
arranged in this Table according to their A-ring substitution patterns, as these have
been found to have phylogenetic importance in some taxa of the Lamiaceae. For
example, flavones with a 5-hydroxy-6,7-dimethoxy A-ring are widespread in the family Lamiaceae as a whole, whereas flavones with a 5,6-dihydroxy-7,8-dimethoxy Aring have only been found in genera of the tribe Mentheae, subfamily Nepetoideae
(Tomás-Barberán and Gil, 1992). Furthermore, the 5,7,8-trihydroxy-6-methoxy and
5,7-dihydroxy-6,8-dimethoxy A-ring patterns are unique in the Lamiaceae for the
genus Ocimum (Grayer et al., 2001).
Surface flavonoids were found in all species of Agastache, Dracocephalum and
Lallemantia studied and in 36 out of 38 species of Nepeta investigated. Most species
of Nepeta have remarkably similar surface flavonoid profiles with cirsimaritin (9)
being the major surface flavone and this was sometimes accompanied by its 4⬘methyl ether, salvigenin (16). Two other flavones frequently present in Nepeta species were 8-hydroxycirsimaritin (6), detected in 30 of the species, and genkwanin
(14), detected in 20 species. 8-Hydroxycirsimaritin (or isothymusin) is a very
unstable compound and often disintegrates during drying (Grayer and Veitch, 1998).
Therefore the absence of this compound from extracts of N. amoena, N. macrosiphon
and N. pungens (see Table 3), all prepared from old herbarium material, does not
necessarily mean that these species do not produce this flavone; analysis of fresh
material of these species may reveal that the compound is present. Cirsimaritin is a
common surface flavonoid in the Lamiaceae (Tomás-Barberán and Gil, 1992), but
8-hydroxycirsimaritin is very rare having only been found before in a few species
of Ocimum (including Becium) (Grayer and Veitch, 1998; Grayer et al., 2001). The
equally rare 4⬘-methyl ether of 8-hydroxycirsimaritin, 8-hydroxysalvigenin (5,8-dihydroxy-6,7,4⬘-trimethoxyflavone, compound 12), which also has the 5,8-dihydroxy-
Z. Jamzad et al. / Biochemical Systematics and Ecology 31 (2003) 587–600
597
6,7-dimethoxy A-ring substitution pattern, was detected in four species of Nepeta,
including N. crispa, in which 8-hydroxycirsimaritin itself could not be detected. The
new compound 8-hydroxycirsiliol (2), which also has the same A-ring substitution
pattern, was present in three species. Therefore, the presence of surface flavones with
a 5,8-dihydroxy-6,7-dimethoxy A-ring substitution pattern appears to be a feature of
the genus Nepeta. This is an other example that A-ring substitution patterns in surface
flavones can be useful characters at a higher level of classification as discussed above.
The third relatively common compound, detected in 20 of the Nepeta species
examined, is genkwanin (apigenin 7-methyl ether, 14). Genkwanin is likely to be a
precursor in the biosynthesis of cirsimaritin and 8-hydroxycirsimaritin. Apigenin (7)
was present in eight species of Nepeta, one of Dracocephalum and one of Lallemantia. In herbarium material this compound may have been derived from hydrolysed
vacuolar apigenin glycosides. The 8-methyl ether of 8-hydroxycirsimaritin, xanthomicrol (11), was found in four species of Nepeta and one of Dracocephalum (D.
kotschyii). Each of the remaining flavonoids in Nepeta only occurred in a few species
and seem to be distributed at random. The presence of the surface flavone thymusin
(3) in just one species of Nepeta is of interest. This compound has an A-ring substitution pattern (5,6-dihydroxy-7,8-dimethoxy) which is characteristic of several other
genera of the tribe Mentheae, e.g. Thymus and Mentha (Tomás-Barberán and Gil,
1992), but it is not a characteristic chemical feature of Nepeta.
In Agastache barberi the main methoxylated flavonoids were cirsimaritin (9) and
8-hydroxycirsimaritin (6) like in many species of Nepeta, but genkwanin
( ⫽ apigenin 7-methyl ether, 14) was not detected. Instead, apigenin 4⬘-methyl ether,
acacetin (13) was found. This appears to be a characteristic compound for the genus,
as several authors have found acacetin in all the Agastache species they investigated.
These are A. micrantha (Sanders et al., 1980), A. aurantia (Exner et al., 1981), A.
urticifolia, A. occidentalis, A. parviflora, A. cusicki, A. rugosa, A. foeniculum, A.
nepetoides and A. scrophulariifolia (Vogelman, 1984). Sanders et al. (1980) and
Exner et al. (1981) also found flavones with a 5-hydroxy-6,7-dimethoxy A-ring,
closely related to cirsimaritin, in A. aurantia and A. micrantha, but not their 8hydroxy derivatives. However, these authors studied the surface flavonoids by isolation rather than HPLC, and since 8-hydroxylated flavones tend to disintegrate during isolation, this may be the reason why they were not detected in these Agastache
species. The unidentified flavone that Vogelman (1984) found in many of the Agastache species he studied may be cirsimaritin or a related compound.
The two species investigated for Dracocephalum produced cirsimaritin, but 8hydroxycirsimaritin and genkwanin were not detected. Luteolin was also found in
both species, but the remainder of the flavonoids differed between the two species.
D. kotschyii produced two flavonols, kaempferol 3,7-dimethyl ether (15) and
kaempferol 3-methyl ether (8), which were not encountered in D. grandiflorum,
nor in species of Nepeta or Agastache. Previous work on the surface flavonoids of
Dracocephalum species gave similar results to our research. Shamyrina et al. (1979)
found luteolin and apigenin in D. nutans and Oganesyan et al. (1989) isolated
cirsimaritin, xanthomicrol, gardenin B (the 4⬘-methyl ether of xanthomicrol) and
two methoxylated kaempferol derivatives, penduletin (5,4⬘-dihydroxy-3,6,7-
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trimethoxyflavone) and calycopterin (5-hydroxy-3,6,7,8,4⬘-pentamethoxyflavone)
from D. multicaule. Thus the presence of methoxylated flavonols and the frequent
occurrence of flavones with the 5-hydroxy-6,7,8-trimethoxy A-ring substitution, as
found in xanthomicrol and gardenin B, may be chemical features which distinguish
the genus Dracocephalum from Nepeta.
The species investigated for Lallemantia showed a completely different surface
flavonoid profile, because only 5,7-dihydroxy flavones were found and no flavones
with 6- and/or 8-oxygenation were present. Lallemantia is a small genus with five
species that has been treated as one of the sections of the genus Dracocephalum
(Budantsev, 1993). However, there seem to be significant differences between the
flavonoid profiles of these genera. These differences are supported by the molecular
data obtained from comparison of DNA sequences of the internal transcribed spacers
region of nuclear ribosomal DNA (Jamzad, 2001; Jamzad et al., submitted) and their
essential oil composition (Jamzad, 2001). It is necessary to examine more species
of the genera Dracocephalum and Lallemantia to see whether the presence of flavonols and the absence of the 8-hydroxycirsimaritin in Dracocephalum and the presence of only 5,7-dihydroxyflavones in Lallemantia can be considered as diagnostic
characters that distiguish them from each other and from Nepeta.
4. Conclusions
The great majority of species of Nepeta contain lipophilic flavonoids of the flavone
group on the surface of their leaves; the synthesis of compounds of this class is a
characteristic feature of many other genera within Lamiaceae. Therefore the synthesis
of these compounds within Nepeta can be phylogenetically referred to as a plesiomorphic state (Kitching et al., 1998), and their absence from some of the members
of the family as a derived character (apomorphy). The 5,6-dihydroxy-7,8-dimethoxy
A-ring substitution pattern as found in the compound thymusin, and which is a
characteristic feature of several genera in the tribe Mentheae, was detected in only
one species of Nepeta (N. assurgens). However, the 5-hydroxy-6,7-dimethoxy Aring pattern as found in cirsimaritin (9) and salvigenin (16), and the unusual 5,8dihydroxy-6,7-dimethoxy flavone A-ring substitution pattern, as found in 8-hydroxycirsimaritin (6), 8-hydroxysalvigenin (12) and 8-hydroxycirsiliol (2), is present in
the majority of Nepeta species. Cirsimaritin and salvigenin are also common in many
other Lamiaceae genera (Gil-Muñoz, 1993). Therefore the presence of these flavonoids in Nepeta should be considered as a plesiomorphic state. On the other hand, 8hydroxycirsimaritin and 8-hydroxysalvigenin are rare in the Lamiaceae and therefore
the production of these 8-hydroxylated flavones should be considered as synapomorphies for Nepeta species. The distribution of the other identified flavones is variable and does not reflect any pattern of infrageneric classifications, but in a few
cases, seem autoapomorphies of some Nepeta species and therefore may be useful
to identify those species, e.g. the presence of thymusin (3) in N. assurgens. The
presence of acacetin (13) in Agastache and of methoxylated flavonols in Dracocephalum may be recognized as taxonomic markers at the generic level as they are
absent from Nepeta.
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599
Acknowledgements
We acknowledge the receipt of a grant from the Islamic Republic of Iran to Ziba
Jamzad for her Ph.D. studies. We wish to thank the authorities of the Research
Institute of Forests and Rangelands for the use of their herbaria and research facilities. The authors are grateful to Dr. F.A. Tomás-Barberán for an authentic sample
of thymusin.
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