Accepted Article
Article Type: Full Paper
Chemical composition, enantiomeric analysis, AEDA sensorial
evaluation and antifungal activity of the essential oil from the
Ecuadorian plant Lepechinia mutica Benth (Lamiaceae).
Jorge Ramírez*a,d, Gianluca Gilardonia, Miriam Jácomea, José Montesinosa, Marinella Rodolfib,
Maria Lidia Guglielminettib, Cecila Caglieroc, Carlo Bicchic and Giovanni Vidarid
aDepartamento
bDepartment
of Earth and Environmental Sciences, Lab. Mycology, University of Pavia, Via San
Epifanio 14, 27100, Pavia, Italy
cDipartimento
dDipartimento
de Química y Ciencias Exactas, Universidad Técnica Particular de Loja, San
Cayetano Alto s/n, Loja, Ecuador. jyramirez@utpl.edu.ec
di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via Pietro
Giuria 9, 10125, Torino, Italy.
di Chimica and CEMEC, Università degli Studi di Pavia, Viale Taramelli 12, 27100,
Pavia, Italy.
This study describes the GC-FID, GC-MS, GC-O, and enantioselective GC analysis of the essential
oil hydrodistilled from leaves of Lepechinica mutica (Lamiaceae), collected in Ecuador. GC-FID
and GC-MS analyses allowed the characterization and quantification of 79 components,
representing 97.3% of the total sample. Sesquiterpene hydrocarbons (38.50%) and
monoterpene hydrocarbons (30.59%) were found to be the most abundant volatiles, while
oxygenated sesquiterpenes (16.20%) and oxygenated monoterpenes (2.10%) were the minor
components. In order to better characterize the oil aroma, the most important odorants, from
the sensorial point of view, were identified by Aroma Extract Dilution Analysis (AEDA) GC-O.
They were α-pinene, β-phellandrene, and dauca-5,8-diene, exhibiting the characteristic woody,
herbaceus and earthy odors, respectively. Enantioselective GC analysis of L. mutica essential oil
revealed the presence of twelve couples and two enantiomerically pure chiral monoterpenoids.
Their enantiomeric excesses were from a few percent units to 100%. Moreover, the essential oil
exhibited moderate in vitro activity against five fungal strains, being especially effective against
M. canis, which is a severe zoophilic dermatophyte causal agent of pet and human infections.
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201700292
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Keywords: Lepechinia mutica • Ecuador • Enantiomeric analysis • Gas chromatographyolfactometry • Antifungal activity.
Introduction
The genus Lepechinia belongs to the family Lamiaceae and comprises about 43 species
distributed from South-West USA to Chile. Sesquiterpenes, diterpenes, triterpenes and
flavonoids have been isolated from different species of this genus. Some species are used for
their antitumor and insulin-mimetic properties, to treat uterine infections and stomach pains [1].
Regarding the essential oil components, 15 species of the genus Lepechinia have been studied so
far, including L. conferta [1], L. floribunda [2–5], L. graveolens [3], L. caulescens [6], L. paniculata [7], L.
betonicifolia [8], L. meyeni [3,9], L. salviaefolia [10], L. bullata [11], L. calycina [12], L. schiedeana [13–15], L.
urbanii [16], L. chamaedryoides [17], L. radula [18]. In table 1, we have reported the major
compounds from some Lepechinia species.
However, due to the heterogeneity of compounds identified in Lepechinia spp, it has not been
possible to establish a typical metabolic pattern. The essential oils of seven different Ecuadorian
plants of the family Lamiaceae have been studied so far, including two species belonging to the
genus Lepechinia [7, 19]. In 2002, Malagón et al. identified 54 compounds in the essential oil from
L. mutica, collected at “Cerro el Villonaco” (Loja, Ecuador), on the basis of their retention indices,
referred to a series of homologous fatty acid methyl esters [19], and the comparison of the mass
spectra with an early collection of reference spectral data [20].
The gas chromatography-olfactometry (GC-O) technique couples traditional gas
chromatographic analysis with sensory detection. Thus GC-O may be considered a biological
detection method for the characterization of the odor of compounds separated and quantified
by GC chromatography [21]. Indeed, the correlation of the eluted peaks with specific odors
affords to establish accurate retention indices or retention times for the odor active
components, while peak areas in the GC-FID chromatogram are indicative of the relative
abundance of the components. Moreover, GC-O in combination with mass spectrometry enables
the identification of odor compounds with the aid of mass spectral information and leads to a
partial correlation between the chemical nature of an odorant and its perceived smell [21]. To
collect and process GC-O data and to estimate the relative odor potency of each aroma-active
compound and thus its sensory contribution to the total odor of the oil, we used the dilution to
threshold quantitative procedure known as the “Aroma Extract Dilution Analysis” (AEDA) [22-24].
Another important point, related to the sensorial properties of the volatile fraction, is that many
common constituents of essential oils are chiral and thus they may be present as one or both
enantiomeric forms. Since the odor properties as well as the biological activity of the two
stereoisomers may be greatly different, a complete characterization of odor-active components
of an essential oil requires enantiomer recognition and enantiomeric excess (EE) and/or ratio
(ER) determination [25, 26]. These separations are usually achieved by enantioselective gas
chromatography, using a capillary column endowed with a chiral stationary phase, usually
cyclodextrin derivatives, as chiral selectors [25]. Therefore, we decided to repeat the analysis of
the oil by GC-FID and GC-MS techniques, and identified their components by comparison of their
linear retention indices and mass spectra with an updated and more complete reference data
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Accepted Article
collection [27]. Moreover, the essential oil was submitted, for the first time with this genus, to GCO and enantioselective GC analyses to characterize better the pleasant minty smell of L. mutica
essential oil and to identify odor active compounds.
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ccepted Articl
Table 1. Major components in the essential oils of genus Lepechinia.
Major compounds
[1] L.
conferta
[2-5] L.
floribunda
[3] L.
graveolens
[6] L.
caulescens
[7] L.
paniculata
[8] L.
betonicifolia
[3-9] L.
meyeni
(-)-palustrol
[10] L.
salviaefolia
[13-15] L.
schiedeana
[16] L.
urbanii
x
1,8-cineole
x
x
Aromadendrene
x
Borneol
x
Bornyl acetate
x
Camphene
x
Camphor
Ledyl acetate
[12] L.
calycina
x
(-)-spirolepechinene
Ledol
[11] L.
bullata
x
x
x
x
x
x
x
Limonene
x
m-cymene
x
o-cymene
x
p-cymene
x
Viridiflorene
α-copaene
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x
x
[17] L.
chamaedryoides
[18] L.
radula
ccepted Articl
α-humulene
x
α-pinene
x
β-caryophyllene
β-phellandrene
x
x
x
x
x
x
x
β-pinene
x
x
β-selinene
x
x
γ-cadinene
x
δ-cadinene
Δ3-carene
x
x
x
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x
x
x
x
x
x
Results and Discussion
Accepted Article
GC-MS and GC-FID analyses.
The average and standard deviation of each oil component were calculated by six consecutive
GC-FID analyses. The results of GC-MS and GC-FID analyses are reported in Table 2.
Table 2. Components of the essential oil from L. mutica.
No.
Componentsa
RTb
FID
Calculated linear
retention indexc
Linear retention
index from
reference [27]
RI
% Area
1
Tricyclene
4.07
921
926
5
Trd
-
2
-Thujene
4.17
924
928
4
0.09
0.06
3
-Pinene
4.38
932
934
2
1.23
0.89
4
Camphene
4.88
946
949
3
0.75
0.80
5
Sabinene
5.65
969
971
2
0.24
0.15
6
-Pinene
5.82
974
976
2
3.78
1.76
7
Oct-3-en-1-ol
6.08
974
983
9
0.07
0.07
8
Myrcene
6.27
988
989
1
0.52
0.28
9
p-Mentha-1(7),8-diene
6.73
1003
1002
-1
0.16
0.13
10
-phellandrene
6.82
1005
1003
-2
3.80
1.70
11
3-Carene
6.93
1008
1008
0
8.69
4.24
12
-Terpinene
7.19
1014
1015
1
0.11
0.07
13
p-Cymene
7.31
1020
1019
-1
0.10
0.06
14
Sylvestrene
7.45
1025
1023
-2
0.29
0.18
15
o-Cymene
7.48
1022
1024
2
16
Limonene
7.64
1024
1028
4
3.79
2.18
17
-Phellandrene
7.69
1025
1030
5
18
-Terpinene
8.64
1054
1057
3
0.23
0.12
19
cis-Sabinene hydrate
9.12
1065
1071
6
0.05
0.03
20
p-Mentha-2,4(8)-diene
9.43
1085
1080
-5
0.35
0.18
21
Terpinolene
9.56
1086
1083
-3
0.60
0.33
22
trans-Linalool oxide
9.67
1084
1086
2
Trd
-
23
Linalool
10.20
1095
1102
7
0.20
0.09
24
Oct-1-en-3-yl acetate
10.44
1110
1109
-1
1.37
0.60
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25
Camphor
11.58
1141
1145
4
Trd
-
26
Borneol
12.46
1165
1172
7
0.25
0.05
27
4-Terpineol
12.71
1174
1180
6
0.14
0.02
28
-Terpineol
13.23
1186
1196
10
0.11
0.02
29
Isobornyl acetato
15.81
1283
1281
-2
2.20
1.04
30
-Elemene
17.41
1335
1328
-7
Trd
-
31
-Cubebene
17.81
1348
1340
-8
0.57
0.08
32
-Terpinyl acetate
17.89
1346
1342
-4
33
-Ylangene
18.57
1373
1361
-12
0.15
0.05
34
Isoledene
18.61
1374
1362
-12
35
-Copaene
18.81
1374
1367
-7
1.46
0.23
36
-Bourbonene
19.08
1387
1375
-12
0.47
0.25
37
-Cubebene
19.28
1387
1380
-7
0.15
0.04
38
-Gurjunene
19.97
1409
1400
-9
1.94
0.37
39
-Cedrene
20.26
1410
1407
-3
0.05
0.10
40
(E)-Caryophyllene
20.47
1417
1412
-5
4.55
2.16
41
Longifolene
20.74
1407
1418
11
0.15
0.07
42
-Copaene
20.86
1430
1421
-9
0.50
0.08
43
-Gurjunene
21.19
1431
1429
-2
1.47
0.78
44
cis-Muurola-3,5-diene
21.54
1448
1437
-11
0.45
0.36
45
-Humulene
21.89
1452
1445
-7
1.20
0.47
46
Aromadendrene
22.05
1439
1449
10
0.56
0.10
47
cis-Cadina-1(6),4-diene
22.19
1461
1452
-9
0.99
1.36
48
Amorpha-4,11-diene
22.36
1449
1456
7
0.15
0.07
49
Dauca-5,8-diene
22.64
1471
1463
-8
0.38
0.09
50
trans-Cadina-1(6),4-diene
22.79
1475
1466
-9
0.99
0.12
51
-Muurolene
22.98
1478
1471
-7
0.92
0.23
52
-Selinene
23.29
1492
1478
-14
0.81
0.08
53
cis--Guaiene
23.42
1492
1481
-11
0.71
0.11
54
Bicyclogermacrene
23.62
1500
1486
-14
55
epi-Cubebol
23.74
1493
1489
-4
4.62
0.58
56
-Zingiberene
23.75
1493
1489
-4
57
-Muurolene
23.83
1500
1491
-9
0.91
0.17
58
(E,E)--Farnesene
24.34
1505
1503
-2
0.83
0.25
59
-Cadinene
24.44
1513
1505
-8
2.86
0.37
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60
Cubebol
24.59
1514
1508
-6
0.36
0.21
61
-Cadinene
24.74
1522
1511
-11
6.96
0.99
62
trans-Calamenene
24.78
1521
1512
-9
0.15
0.04
63
trans-Cadina-1,4-diene
25.29
1533
1523
-10
0.37
0.10
64
-Cadinene
25.48
1537
1527
-10
0.39
0.12
65
Selina-3,7(11)-diene
25.61
1545
1530
-15
0.14
0.04
66
Germacrene B
26.33
1559
1545
-14
0.18
0.06
67
Germacrene D-4-ol
27.31
1574
1567
-7
1.46
0.40
68
Caryophyllene oxide
27.42
1582
1569
-13
0.29
0.24
69
Globulol
28.11
1590
1584
-6
5.91
2.61
70
Viridiflorol
28.51
1592
1592
0
1.29
0.45
71
1,10-di-epi-Cubenol
29.71
1618
1617
-1
0.27
0.11
72
10-epi--Eudesmol
29.96
1622
1622
0
0.54
0.15
73
Junenol
29.97
1618
1623
5
1.39
0.42
74
-Acorenol
30.14
1632
1626
-6
0.09
0.15
75
-Acorenol
30,78
1636
1639
3
0.47
0.81
76
-Eudesmol
31.00
1649
1644
-5
77
-Eudesmol
31.02
1652
1644
-8
4.47
1.93
78
-Cadinol
31.10
1652
1646
-6
79
Shyobunol
32.83
1688
1681
-7
10.80
5.91
Monoterpenes hydrocarbons
30.59
Oxygenated monoterpenes
2.07
Sesquiterpene hydrocarbons
38.54
Oxygenated sesquiterpenes
16.22
Others
9.88
Total identified
97.30
aCompound identification methods: LRI and comparison of the mass spectrum with Adams. bRT= Retention time (min). cCalculated linear
retention index on a DB5-MS column. dTr = trace (<0.05%).
Table 2 reports the list of the identified components. Indeed, seventy-eight compounds were
identified, representing 95.60% the total oil sample. Sesquiterpene hydrocarbons (38.54%) and
monoterpene hydrocarbons (28.89%) were the principal groups of compounds. Oxygenated
sesquiterpenes (16.22%) and oxygenated monoterpenes (2.07%) were the minor groups. The
most abundant components were shyobunol (10.80%), 3-carene (8.69%), -cadinene (6.96%),
globulol (5.91%), (E)-caryophyllene (4.55%), -pinene (3.78%) and -cadinene (2.86%). 3Carene has also been found as one of the three main components in the oils of L. conferta [1], L.
meyeni [3,9], L. calycina [12], L. schiedeana [13–15], L. urbanii [16], Sphacele chamaedryoides [17] and L.
radula [18].
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In the previous study by Malagon of the oil from L. mutica, monoterpene hydrocarbons were
found to be the main group of constituents (72%), among which -phellandrene (30%),
camphene (13%), limonene (8%), 3-carene (6%) and -pinene (3%) were the most abundant
ones [19]. The different chemical composition of the essential oil from L. mutica reported in the
previous [19] and present works, may depend on many factors, such as the phenological status of
the plant, different time and place of collection, distillation time, method of analysis. In fact, the
plant material studied by Malagón et.al. was collected in a different place and period (March
2000), giving a possible explication to the different composition.
AEDA GC-O
GC-O results were processed by means of AEDA technique, and is shown in Figure 2, and in
Table 3. In Figure 1 each (red) signal in the aromagram represents the perception of the
compound corresponding to the retention index reported in the underlying GC chromatogram;
the intensity of each signal is proportional to the dilution corresponding to the FD factor;
therefore, the greater is the signal intensity in the aromagram, the more important is the
contribution of the corresponding compound to the olfactory profile of the essential oil. Figure 2
depicts the AEDA aromagram of the essential oil from L. mutica resulting from the FD factors of
odor-active components from L. mutica.
Figure 1. Superposition of the gas chromatogram and the aromagram of the essential oil.
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The most important compounds from the olfactory point of view are reported in Table 3. Thus,
the main odor contributors, according to AEDA analysis, were -pinene, having a strong woody
odor, -phellandrene, endowed with a characteristic herbaceus tonality, and dauca-5,8-diene
possessing a typical earthy odor.
Figure 2. AEDA aromagram of the essential oil from L. mutica.
Table 3. Components of the olfactory profile of L. mutica essential oil.
Odour
AEDA (FD)
Compound
Calculated LRI
woody
8
-Pinene
934
vanilla
4
Camphene
949
woody
4
-Pinene
976
lemon
4
3-Carene
1008
herbaceous
8
-Phellandrene
1030
lavender
4
Oct-1-en-3-yl acetate
1109
woody
4
(E)-Caryophyllene
1412
earthy
8
Dauca-5,8-diene
1463
woody
2
-Zingiberene
1489
woody
2
-Cadinene
1511
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woody
4
-Cadinene
1527
herbaceous
4
Globulol
1584
woody
2
Shyobunol
1681
According to the AEDA analysis, the importance of odorous compounds does not correspond to
the component percentage in the oil. For example, shyobunol and 3-carene, which are among
the most abundant compounds, are not among the most powerful odorants; instead, the
opposite is true, for example, for -phellandrene and dauca-5,8-diene.
Enantioselective GC-MS analysis
Enantiomer components and enantiomeric excesses (EEs) of L. mutica essential oil were
determined as the mean value of two enantioselective GC-MS analyses [26,28], performed on
samples obtained from two different distillation processes. Since enantiomers have the same
chemical properties, the enantiomeric distribution (unlike the chemical composition) should
not be affected by the distillation process. For this reason, the enantioselective analysis can be
performed as the mean value of two replicates. Twelve couples and two enantiomerically pure
chiral monoterpenoids were detected (Table 4) and baseline separated. (+)-camphor and (-)borneol were detected as enantiomerically pure compounds, while (-)-camphene, (-)--pinene,
(-)--pinene were present in mixture with their enantiomers but with a very high E.E. In
contrast, the enantiomeric excesses of (-)-sabinene, (+)--phellandrene, (-)-limonene, (+)linalool and (-)--terpineol were only moderate, while terpinen-4-ol and trans-linalool oxide
were almost racemic. These results further confirm that secondary metabolites can be present
in plants as enantiomeric mixtures.
Table 4. Enantiomeric analysis of the components of L. mutica essential oil.
RT
Enantiomeric
distribution (%)
(1S,4R)-(-)-Camphene
9.57
98.48
(1R,4S)-(+)-Camphene
10.24
1.52
(1R)-(+)--Pinene
9.76
5.70
Enantiomers
E.E. (%)
96.96
88.61
(1S)-(-)--Pinene
9.82
94.31
(1R)-(+)--Pinene
10.88
1.88
96.25
(1S)-(-)--Pinene
11.29
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98.13
(1R,5R)-(+)-Sabinene
12.19
32.97
(1S,5S)-(-)-Sabinene
12.93
67.03
(R)-(-)--Phellandrene
14.38
6.85
(S)-(+)--Phellandrene
14.54
93.16
(R)-(-)--Phellandrene
15.90
9.12
(S)-(+)--Phellandrene
16.60
90.89
(S)-(-)-Limonene
16.44
70.94
(R)-(+)-Limonene
17.39
29.06
(1R)-(+)-Camphor
20.94
>99%
(+)-trans-Linalool oxide (furanoid)
18.56
52.89
(-)-trans-Linalool oxide (furanoid)
18.91
47.12
(R)-(-)-Linalool
23.13
27.83
(S)-(+)-Linalool
23.97
72.18
(2R)-(-)-Borneol
24.25
>99%
(S)-(+)-Terpinen-4-ol
27.21
44.72
(R)-(-)-4-Terpinen-4-ol
27.47
55.29
(S)-(-)--Terpineol
29.83
73.64
(R)-(+)--Terpineol
30.52
26.36
Accepted Article
34.06
86.31
81.77
41.88
100
5.77
44.35
100
10.57
47.28
Antifungal activity
Different biological properties have been attributed to the essential oils isolated from a few
Lepechinia species. They include the antimicrobial activity against Paenibacillus larvae for the
oil from L. floribunda) [2,4,5], in vitro anti-Vibro cholera activity for the oil from L. caulescens) [6],
repellent activity against Tribolium castaneum [8] and high total antioxidant activity (TAA) in the
DPPH assay for the oil from L. schiedeana [13–15], and insecticidal activity against Drosophila
melanogaster for the oil from L. chamaedryoides [29]. To the best of our knowledge, the biological
activity of L. mutica essential oil (Table 5) has not yet been investigated. We tested the
antifungal activity of the oil against three severe human fungal pathogens, Candida albicans,
Trichophytum rubrum and Microsporum canis, and two potent plant pathogens, Pyricularia
oryzae and Fusarium graminearum. Compared to the positive controls (amphotericin B and
voriconazole) the essential oil exhibited moderate activity against M. canis and T. rubrum,
having MIC values ranging between 2.2 and 4.5 mg/ml).
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Table 5. MIC (Minimum Inhibitory Concentration) and MFC (Minimum Fungicidal Concentration) values (mg/ml) of the essential oil hydrodistilled
from L. mutica.
Sample
Candida albicans
(human pathogen)
Trichophyton rubrum
(human pathogen)
Microsporum canis
(human pathogen)
Pyricularia
oryzae (plant
pathogen)
Fusarium
graminearum (plant
pathogen)
Oil
MIC > 9 mg/ml
2.2 <MIC ≤ 4.5 mg/ml
2.2 <MIC ≤ 4.5 mg/ml
MIC > 9 mg/ml
MIC > 9 mg/ml
MFC> 9 mg/ml
4.5 <MCF ≤ 9 mg/ml
Amphotericin Ba
MIC = 0.001 mg/ml
MIC = 0.5 mg/ml
MIC = 0.0005 mg/ml
n.d.
n.d.
Voriconazolea
MIC = 0.00006 mg/ml
MIC = 0.5 mg/ml
MIC = 0.00025 mg/ml
n.d.
n.d.
Flutriafol
PESTANAL®a
n.d.
n.d.
n.d.
MIC = 0.04
mg/ml
n.d.
aPositive
reference antifungal compound. n.d. = not determined
Conclusions
The chemical composition of the essential oil hydrodistilled from the leaves of L. mutica
determined in this work resulted to be quite different from that previously found by Malagón et
al in 2002 [19]. In this investigation we identified seventy-nine components of the essential oil,
which represented 97.30% the total sample. Sesquiterpene hydrocarbons (38.54%) and
monoterpenes hydrocarbons (30.59%) were the prevalent groups of compounds, while
oxygenated sesquiterpenoids (16.22%) and oxygenated monoterpenoids (2.07%) were present
in minor amounts. The most abundant components were shyobunol (10.80%), 3-carene
(8.69%), -cadinene (6.96%), globulol (5.91%), and (E)-caryophyllene (4.55%), while the most
sensorially important components, determined by AEDA GC-O, were -pinene, -phellandrene,
and dauca-5,8-diene, possessing typical woody, herbaceus and earthy odors, respectively.
Enantioselective GC-MS analysis of L. mutica essential oil revealed the presence of twelve
enantiomeric couples and two enantiomerically pure monoterpenes. Their enantiomeric
excesses varied from a few percent units to virtually 100%. Moreover, the essential oil exhibited
moderate in vitro activity against five fungal strains, being especially effective against M. canis,
which is a severe zoophilic dermatophyte causal agent of pet and human skin infections (tinea).
This oil might be used for an alternative antifungal treatment of infected men and animals, as
well as for the remediation of indoor environments contaminated by infected hair shedding,
which is a potential source of new infections.
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Experimental Section
Accepted Article
Plant material and preparation of the essential oil.
The collection of L. mutica leaves, authorized by the Ministry of Environment of Ecuador (MAE)
(authorization no. 001-IC-FLO-DBAP-VS-DRLZCH-MA), was performed in the Quilanga region of
the Loja Province, Ecuador, in November and December 2009. The plant was identified by
Bolivar Merino, “Herbarium of the Universidad Nacional Loja”. A voucher specimen, with the
number PPN-la-005, has been deposited in the Herbarium of the “Universidad Técnica
Particular de Loja”. The essential oil, d20 = 0.916 ± 0.026, n20 = 1.4867 ± 0.0009, []D20 = -5.8º
(neat), was obtained in 0.40 ± 0.12% yield (w/w) by steam distillation of fresh leaves
(approximately 10.36 kg) in a Clevenger-type apparatus for four hours. Subsequently, the
essential oil was tagged and stored in a brown vial at 4ºC until analysis.
GC-MS analysis
Qualitative analysis of the essential oil (six replicates) was performed by GC-MS using an Agilent
Chromatograph (model 6890N series), coupled to a mass spectrometer-detector (model Agilent
series 5973 inert). The spectrometer, controlled by the data system MSD-Chemstation D.01.00
SP1, operated at 70 eV; electron multiplier 1600 V; scan rate: 2 scan/s; mass range: 40-350 m/z.
A non-polar capillary column, DB-5ms 5%-phenyl-methylpolysiloxane, 30 m x 0.25 mm,
thickness 0.25 μm film, was used. Samples were dissolved in dichloromethane. An autosampler
(series 7673) was used. Helium was the carrier gas at a flow rate of 1.0 mL/min in constant flow
mode; the detector and injector temperatures were set at 250 ºC. The injector operated in split
mode (split ratio 20:1). The oven temperature was set at 60 ºC for 5 min, then increased to 110
ºC, with a gradient rate of 5 ºC/min, followed by an increase to 148 ºC with a gradient of 2
ºC/min. A third gradient rate of 20 ºC/min increased the temperature to 250 ºC, which was hold
for 2.4 min. The ion source temperature was 250 ºC. Chemical components of L. mutica essential
oil were identified by comparing their EI MS spectra with the spectra of compounds having
close retention indices (RI) reported in the Adam’s comprehensive work [27]. Retention indices
were determined, according to Van Den Dool and Kratz [30], on the basis of the retention times of
a homologous series of hydrocarbons C10-C25 (TPH-6RPM of CHEM SERVICE), which were
analyzed by GC under the same conditions.
GC-FID analysis
The analysis of the essential oil (six replicates) was carried out on an Agilent Technologies
chromatograph (model 6890N series), using a flame ionization detector (FID). The percentage
content of each oil component was computed from the corresponding GC-FID peak area without
applying any correction factor. The analytical parameters were the same as the GC-MS analysis.
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Accepted Article
GC-O analysis by incremental dilution technique (AEDA)
GC-O-MS analysis was performed using an Agilent Technologies chromatograph (model 6890N
series), and a Gerstel Olfactory Detection Port ODP 3. The same analytical parameters as those
used in the GC-MS analysis were applied. GC-O analysis was performed by injecting, at
corresponding to FD=1), the essential oil dissolved in CH2Cl2, while two trained panelists
signaled the perceived odors by pushing a button, without seeing the chromatogram in progress
[25]. Furthermore, the panelists were asked to describe the odor perceived at the sniffing port in
the two analyses of the oil. During AEDA, stepwise dilutions of the original oil were performed
and the diluted samples were then evaluated by GC-O to provide flavour dilution (FD) factors,
i.e. the highest oil dilution at which the odor of analyzed compound could distinctively be
detected. The overall results obtained with AEDA have been reported in an aromagram (Figure
2), showing the exponential (2x) FD values against the retention indices (RI).
Enantioselective GC analysis
Enantioselective GC-MS analysis was performed (two replicates) using a Shimadzu QP2010 GCMS system. The mass spectrometer operated in electron impact ionization mode at 70 eV, with a
mass range of m/z 35-350 full scan mode. The ion source temperature was set at 200 ºC. Helium
was the carrier gas at a flow rate of 1.0 mL/min. The injector operated in split mode (split ratio
20:1) at 200 ºC, with a transfer line at 230 ºC. The oven temperature was set at 50 ºC for 2 min,
and then increased to 220 ºC, with a gradient rate of 2 ºC/min, which was hold for 2.0 min. A
chiral capillary column, 30% 2,3-diethyl-6-tert-butyldimethylsilyl--CDX dissolved in Silicon PS
086 (25m x 0.25mm x 0.25m) from Mega (Legnano, MI, Italy), was used. Oil samples were
dissolved in cyclohexane. The enantiomer order in the enantioselective GC-MS analysis was
obtained by separated injections of enantiomerically pure standards.
Physical properties of the essential oil
The relative density, refractive index and optical activity of the essential oil were determined as
the means of three different experiments done at 20 ºC by using a pycnometer (5 mL) and an
analytical balance (METTLER AC100), a refractometer (model ABBE), and a Perkin-Elmer 241
polarimeter, respectively.
In vitro evaluation of antifungal activity
The in vitro antifungal activity of the oil was tested against different strains belonging to the
fungal collection deposited at the Laboratory of Mycology, University of Pavia, Candida albicans
(C.P. Robin) Berkhout, Microsporum canis E. Bodin ex Guég., Trichophyton rubrum (Castell.)
Sabour., Fusarium graminearum Schwabe and Pyricularia oryzae Cavara. The first three fungi
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Accepted Article
were isolated from human patients suffering of cutaneous mycoses, while the last two fungi
were isolated, respectively, from plants of rice (Oryza sativa L.) with blast disease and plants of
barley (Hordeum vulgare L.) infected by FHB (Fusarium Head Blight). All fungi were cultured
and maintained on Sabouraud agar (Oxoid, Basingstoke, UK) before performing the antifungal
tests.
The Minimum Inhibitory Concentration (MIC) of the essential oil was determined by brothmicrodilution method using 96 well flat-shaped microtitre plates (Sigma-Aldrich), according to
Gadd [31] and the Clinical and Laboratory Standards Institute [32, 33] procedures, with minor
modifications. The MIC is defined as the lowest drug concentration completely inhibiting
observable fungal growth compared to the control. The essential oil was added to the liquid
culture medium RPMI 1640 in micro-wells at a final concentration from 10 to 1 µl/mL. 0.002%
Tween 80 (v/v) was included to enhance oil solubility. Inoculum suspensions were prepared by
transferring fungi in 2 mL of sterile water with 0.85% NaCl (API BioMerieux) adjusted to 0.5
McFarland by nephelometric measurement. At first, the filamentous fungi were homogenously
disrupted by vortex in glass tubes containing sterile water and sterile broken cover glasses.
Total volume in each micro-well was 100 μL. Incubating temperature was 25°C, except for C.
albicans (cultured at 37°C). Test plates were examined after 24 and 48 h for C. albicans, and
after 5 days for T. rubrum, M. canis, P. oryzae, and F. graminearum. Amphotericin B, voriconazole
(ATB Fungus 3, BioMerieux), and flutriafol PESTANAL®, containing the antifungal compound
(R,S)-2,4-difluoro (1H-1,2,4-triazol-1-ylmethyl)benzhydryl (Sigma-Aldrich), were used as
positive controls. To determine the Minimum Fungicidal Concentration (MFC) by brothmicrodilution method, the initial inoculum was sub-cultured from microwell plates containing
the extract where no fungal growth was observed (100% inhibition) in fresh culture medium,
free of the essential oil, and Petri plates were examined for 10 days at 24 h interval [31–33]. The
MFC is defined as the sample lowest concentration causing total reduction of the initial
inoculum on culture medium. All bioassays were performed in triplicate.
Acknowledgements
This work has been supported by a grant (no 20110941) from the Secretaría Nacional de
Educación Superior, Ciencia y Tecnología (SENESCYT) del Ecuador.
.
Author Contribution Statement
J.R., G.G. and J.M. collected the plant material; M.J. and J.M. performed the olfactometric analysis;
J.R., C.C. and G.G. performed the chemical and enantiomeric analyses; J.R., G.V. and C.B. wrote
and revised the article; J.R. and G.G. elaborated the analytical data; M.R. and M.L.G. performed
the biological activity test.
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