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Phytochemical Diversity of the Essential Oils of Mexican Oregano (Lippia graveolens Kunth) Populations along an Edapho-Climatic Gradient by Luz Mara Calvo-Irabie´n* a ), Victor Parra-Tabla b ), Violeta Acosta-Arriola a ), Fabiola EscalanteErosa c ), Luciana Daz-Vera a ), Gabriel R. Dzib a ), and Luis Manuel Pen˜a-Rodrguez c ) a
) Unidad de Recursos Naturales, Centro de Investigacio´n Cientfica de Yucata´n, A. C. Calle 43 #130 Chuburna´ de Hidalgo, Me´rida, Yucata´n 97200, Me´xico (phone: þ 52-999-9428330; fax: þ 52-999-9813900; e-mail: [email protected]) b ) Universidad Auto´noma de Yucata´n, Campus de Ciencias Biolo´gicas y Agropecuarias, Departamento de Ecologa Tropical, Km. 15.5, Carretera Me´rida-Xmatkuil, 4-116, Me´rida, Yucata´n 97315, Me´xico c ) Unidad de Biotecnologa, Centro de Investigacio´n Cientfica de Yucata´n, A. C. Calle 43 #130 Chuburna´ de Hidalgo, Me´rida, Yucata´n 97200, Me´xico
Mexican oregano (Lippia graveolens) is an important aromatic plant, mainly used as flavoring and usually harvested from non-cultivated populations. Mexican oregano essential oil showed important variation in the essential-oil yield and composition. The composition of the essential oils extracted by hydrodistillation from 14 wild populations of L. graveolens growing along an edaphoclimatic gradient was evaluated. Characterization of the oils by GC-FID and GC/MS analyses allowed the identification of 70 components, which accounted for 89 – 99% of the total oil composition. Principal component and hierarchical cluster analyses divided the essential oils into three distinct groups with contrasting oil compositions, viz., two phenolic chemotypes, with either carvacrol (C) or thymol (T) as dominant compounds (contents > 75% of the total oil composition), and a non-phenolic chemotype (S) dominated by oxygenated sesquiterpenes. While Chemotype C was associated with semi-arid climate and shallower and rockier soils, Chemotype T was found for plants growing under less arid conditions and in deeper soils. The plants showing Chemotype S were more abundant in subhumid climate. High-oil-yield individuals ( > 3%) were identified, which additionally presented high percentages of either carvacrol or thymol; these individuals are of interest, as they could be used as parental material for scientific and commercial breeding programs.
Introduction. – Mexican oregano, Lippia graveolens Kunth (Verbenaceae), is an important aromatic plant mainly used as condiment or flavoring [1], but also used in the traditional medicine, because of the different biological activities reported for its essential oil, including antioxidant and antibiotic properties [2 – 6]. These characteristics, and their applications in a wide range of industries, confirm the economic potential of this phylogenetic resource. Mexico is the main exporting country of L. graveolens in the world [1], with ca. 4,000 t of Mexican oregano leaves exported annually, most of which are harvested from wild populations found in forests and secondary vegetation [7] [8]. The harvest pressure on this aromatic plant, combined with other factors such as habitat destruction and deforestation, have caused a significant decrease of its natural populations [9]. This is one of the reasons why, at the moment, Mexican oregano is considered a priority species for germplasm conservation [10] [11]. 2014 Verlag Helvetica Chimica Acta AG, Zrich
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L. graveolens is widely distributed throughout the southeast of the United States, Mexico, Guatemala, Nicaragua, and Honduras, growing under diverse ecological conditions in arid, semi-arid, and subhumid regions [12] [13]. A high morphological variability [11] and a significant variation in the essential-oil yield [14] and its chemical composition have been described [15 – 21]. However, most of these studies are restricted to a few individuals and locations. To the best of our knowledge and despite its economic importance, to date, there are no studies that evaluate the effect of environmental conditions on the variation of Mexican oreganos essential-oil composition within and among populations. As part of an ongoing research project directed towards establishing breeding and conservation programs for L. graveolens, and to improve our understanding of the factors responsible for the observed chemical variation, the essential-oil composition in 14 wild populations (see Table 1) of L. graveolens growing along an edaphoclimatic gradient was evaluated. In particular, two questions were addressed: i) Is the chemical variation observed in the essential-oil composition associated or correlated with the edaphoclimatic conditions? ii) Does this variation present a particular spatial pattern? The results obtained will contribute to our understanding of the relative importance of ecological factors on the production of essential oils in aromatic plants in general and, particularly, in Mexican oregano. Additionally, to ensure market demands of a homogeneous production in quantity and quality of this essential oil, the present results will provide valuable information related to the best suitable germplasm and environmental conditions for the management and cultivation of this important aromatic species.
Table 1. Location and Environmental Characteristics of Lippia graveolens Populations 1 – 14 from Southeast Mexico Region
Population
Longitude ( W) a )
Latitude ( N ) a)
Climate
Mean temperature [8]
Annual precipitation [mm]
I
1 2 3
89.5738 89.1158 89.8078
21.2538 21.3388 21.1638
Very warm semi-arid
25.5 25.7 25.7
538 684 614
II
4 5 6
88.1898 88.0848 88.7078
21.5618 21.5648 21.3418
Driest warm sub-humid
25.5 25.4 25.7
650 664 893
III
7 8 9 10 11 12
88.9838 89.6528 89.5998 90.1488 89.8578 90.0048
21.1408 20.6348 20.6018 20.6168 20.5398 20.5768
Warm sub-humid with summer rains
25.9 26.1 26.1 26.7 26.3 26.6
979 1007 1014 1021 1069 1099
IV
13 14
88.9098 88.9468
20.5008 20.5148
Intermediate warm sub-humid
26.1 26.2
1151 1156
a
) Longitude and latitude are expressed as decimals of degrees.
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Results and Discussion. – Edaphoclimatic Gradient. The studied area (Yucata´n Peninsula) provided an ideal scenario to assess the influence of edaphoclimatic variation on the intraspecific chemical variation found in L. graveolens essential oil. This area is a portion of flat land, characterized by an edaphoclimatic gradient with aridity being greatest in the northwestern part and presenting a more humid and colder climate in the southeastern part. Additionally, the soils in the NW of the Yucata´n Peninsula are rockier and younger than in the SE [22] [23]. The location and the environmental characteristics of the 14 sampled populations of L. graveolens, growing in four different regions with different climate types (Regions I – IV), are shown in Table 1. Chemotypes and Multivariate Analysis. The GC-FID and GC/MS analyses of the different samples of L. graveolens essential oil allowed the identification of 70 oil components, which accounted for 89 – 99% of the total oil composition (area of the chromatograms). The identified components are listed in Table 2, in the order of their elution from the Ultra 1 capillary column. The 15 components that represented more than 75% of the total area, mainly oxygenated monoterpenes and sesquiterpenes, are shown in bold type. The plot obtained by principal component analysis (PCA; Fig. 1) showed three distinct groups with contrasting essential-oil compositions and explained
Fig. 1. Plot showing the similarity among Lippia graveolens individuals from 14 natural populations (Populations 1 – 14, cf. Table 1) obtained by principal component analysis (PCA) of the mayor essentialoil constituents (p-cymene, g-terpinene, eucalyptol ( ¼ 1,8-cineole), carvacrol, thymol, b-caryophyllene, a-humulene, and caryophyllene oxide). Together, PCA Axis 1 and 2 explained 98% of the observed variation in the essential-oil compositions.
MH MH MH MH MH MH MH MO MH MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO
a-Thujene a-Pinene Camphene b-Pinene Myrcene d-Carene p-Cymene 1,8-Cineole g-Terpinene cis-Sabinene hydrate 6,7-Epoxymyrcene trans-Sabinene hydrate Linalool cis-p-Menth-2-enol trans-Sabinol Borneol Umbellulone Terpinen-4-ol a-Terpineol trans-Piperitol trans-Carveol cis-p-Mentha-1(7),8-dien-2-ol Thymol methyl ether Carvacrol methyl ether 2-Phenethyl acetate Thymol Carvacrol Eugenol 921 929 941 968 980 1005 1012 1019 1047 1051 1073 1079 1083 1104 1119 1145 1150 1158 1173 1187 1195 1202 1214 1223 1232 1279 1282 1326
Class a ) RI b )
Compound name and class
n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1.83 (0.13 – 5.14) n.d. n.d. n.d. 0.23 0.26 0.26 0.74 (0.26 – 1.70) 1.30 (0.17 – 2.19) n.d. 0.42 (0.11 – 0.95) 2.91 0.52 0.59 0.31 0.52 n.d. n.d. 4.16 (1.13 – 11.28) 1.24 (0.65 – 1.89) 0.35
0.03 (0.01 – 0.06) 0.03 (0.02 – 0.06) 0.01 0.02 0.08 (0.03 – 0.13) 0.02 0.96 (0.23 – 3.13) 0 – 59 (0.03 – 0.92) 0.04 (0.01 – 0.07) 0.04 (0.01 – 0.06) n.d. n.d. 0.72 (0.57 – 0.93) n.d. n.d. 0.13 (0.08 – 0.20) 0.08 (0.001 – 0.15) 0.82 (0.66 – 0.97) 0.13 (0.01 – 0.26) n.d. n.d. n.d. 0.83 (0.19 – 2.25) 0.09 (0.03 – 0.14) 0.14 6.18 (3.35 – 8.60) 78.50 (72.20 – 83.49) 0.13 (0.09 – 0.18)
0.02 0.04 (0.02 – 0.05) 0.01 0.01 0.13 (0.02 – 0.26) 0.01 1.28 (0.37 – 2.22) 0.43 (0.18 – 0.80) 0.19 (0.09 – 0.30) n.d 0.06 n.d. 0.59 (0.25 – 0.92) n.d. n.d. 0.07 0.14 (0.06 – 0.25) 0.76 (0.58 – 0.97) 0.14 (0.05 – 0.35) n.d. tr n.d. 2.07 (0.68 – 3.83) n.d. 0.07 88.47 (84.76 – 91.77) 0.44 (0.38 – 0.59) 0.134
0.8 1 0.2
0.6 0.2 0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.6 0.6
0.8
1 1 0.2 1 1 1
0.25 0.75 1 1
1 1 1 1
0.25 1 1 0.25
1
1
1
0.25
0.25 0.5 0.25 0.25 1 0.25 1 1 0.5
T f)
1
0.4 0.6 0.2 0.2 0.4 0.2 1 0.8 0.4 0.4
C f)
S f)
Chemotype T f )
Chemotype S f )
Chemotype C f )
Frequency d )
Relative content [%] c )
RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,
MS MS MS MS MS MS MS MS, CoI MS, CoI MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS, CoI MS, CoI MS
Identification e )
Table 2. Chemical Composition of the Essential Oils Obtained by Hydrodistillation of the Leaves of 14 Wild Lippia graveolens Populations Growing along an Edaphoclimatic Gradient in Southeastern Mexico. The 14 oils were divided into three chemotypes, a non-phenolic ( S ), a carvacrol (C ), and a thymol (T ) chemotype.
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MO MO SH SH SH SH SH SH SH OC SH SH SH SH SH SH SH SH SH SH SH SH SO SO SO SO SO SO SO SO
Thymol acetate Carvacrol acetate a-Copaene a-Cedrene b-Caryophyllene b-Copaene a-trans-Bergamotene a-Humulene Alloaromadendrene 3-(tert-Butyl)-4-hydroxyanisole g-Muurolene Germacrene D ar-Curcumene b-Selinene a-Selinene a-Muurolene g-Cadinene a-Alaskene trans-Calamene d-Cadinene cis-Calamene a-Calacorene cis-Sesquisabinene hydrate trans-Sesquisabinene hydrate trans-Nerolidol Spathulenol Caryophyllene oxide Viridiflorol Guaiol Humulene epoxide II
1327 1344 1367 1401 1407 1416 1428 1440 1447 1457 1461 1462 1465 1470 1480 1483 1496 1497 1500 1506 1510 1518 1534 1538 1545 1551 1556 1560 1575 1580
Class a ) RI b )
Compound name and class
Table 2 (cont.)
n.d. n.d. 0.28 (0.18 – 0.45) 0.20 2.45 (1.33 – 3.30) 0.20 (0.08 – 0.28) n.d. 1.48 (0.88 – 2.09) 0.26 (0.18 – 0.41) n.d. 0.53 (0.11 – 0.99) n.d. 0.35 0.68 (0.27 – 1.03) 0.46 (0.15 – 0.78) 0.28 (0.20 – 0.36) 0.27 (0.17 – 0.41) n.d. 0.47 (0.17 – 0.91) 0.33 (0.16 – 0.53) 0.43 n.d. 0.56 0.69 1.28 (0.82 – 2.36) 1.79 (0.28 – 4.00) 7.37 (4.94 – 11.34) 3.71 (1.01 – 12.63) 0.54 (0.25 – 1.08) 4.34 (3.04 – 5.99)
Chemotype S )
f
f
n.d. 0.07 (0.03 – 0.14) 0.09 (0.02 – 0.2) n.d. 1.45 (0.27 – 2.45) n.d. 0.14 (0.09 – 0.19) 0.75 (0.19 – 1.28) 0.04 (0.03 – 0.04) 3.32 (2.21 – 4.91) n.d. 0.10 (0.06 – 0.17) n.d. n.d. n.d. 0.03 (0.01 – 0.04) 0.06 (0.05 – 0.08) n.d. 0.07 (0.001 – 0.12) 0.13 (0.02 – 0.23) n.d. 0.01 n.d. n.d. 0.06 (0.03 – 0.09) n.d. 2.29 (0.40 – 3.15) n.d. 0.22 1.20 (0.23 – 1.64)
Chemotype C )
Relative content [%] c )
0.35 (0.07 – 0.83) 0.02 n.d. 0.12 1.21 (0.35 – 2.49) 0.55 (0.16 – 0.94) 0.05 (0.04 – 0.06) 0.83 (0.26 – 1.60) 0.06 0.76 (0.58 – 1.09) n.d. 0.02 0.34 0.01 n.d. 0.03 (0.01 – 0.05) 0.01 0.14 0.04 n.d. 0.07 (0.001 – 0.26) n.d. n.d. 0.02 0.06 n.d. 0.91 (0.48 – 1.28) n.d. n.d. 0.51 (0.33 – 0.71)
Chemotype T )
f
0.2 0.2 1 0.6 1 1 0.8 1
0.8 1 0.2
0.2 0.6 0.8 0.4 0.8
0.6
1 0.6
0.6 0.2 1 0.6
S f)
0.5 1
1
0.6 1 0.2 1
0.2
1
0.5 0.25 0.25 0.25
0.6 0.8 0.8 1
0.25 0.25 0.25
0.25 1 0.5 0.75 1 0.25 1
1 0.25
T f)
0.8
0.8 1 0.4 1
1
0.8 0.6
C f)
Frequency d )
RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,
MS MS MS MS MS, CoI MS MS MS, CoI MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS, CoI MS MS MS
Identification e )
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SO SO SO SO SO SO SO SO SO SH SO SO
1,10-Diepicubenol Muurola-4,10(14)-dien-1b-ol g-Eudesmol Caryophylla-4(12),8(13)-dien-5a-ol epi-a-Cadinol þ epi-a-Muurolol b-Eudesmol a-Eudesmol þ a-Cadinol cis-Calamen-10-ol Bulnesol Cadalene b-Epibisabolol a-Bisabolol 88.8 0.0 12.4 7.2 80.4 0.0
Monoterpene hydrocarbons ( MH) Oxygenated monoterpenes ( MO) Sesquiterpene hydrocarbons ( SH ) Oxygenated sesquiterpenes ( SO ) Other oxygenated compounds (OC)
1.2 88.2 3.1 4.3 3.3
99.4
1.6 93.2 2.5 2.0 0.8
98.97
n.d n.d. 0.08 (0.03 – 0.15) 0.08 (0.04 – 0.12) 0.26 n.d 0.16 (.005 – 0.26) n.d. n.d. n.d. 0.10 (0.05 – 0.17) 0.10
Chemotype T )
f
0.8
1 0.4 0.2 1 1 0.8 0.6 1 0.2
S)
f
0.2 1
0.6 0.8 0.8 0.6 0.6 0.4 0.8
C)
f
f
0.75 0.25
0.5
0.75 1 0.25
T)
Frequency d )
RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,
MS MS MS MS MS MS MS MS MS MS MS MS
Identification e )
) The abbreviations of the compound classes are given at the end of the table (first column, last five rows). b ) RI: Linear retention indices determined experimentally on the Ultra I column relative to a series of n-alkanes (C8 – C26 ) using a non-logarithmic scale. c ) Relative contents are given as mean GC-FID peak areas with minimal and maximal peak areas in parentheses and expressed as percentages; for the five most abundant compounds of each chemotype, the contents are given in bold type; n.d., not detected. d ) The frequency of appearance of the compounds in the oil samples was calculated as number of samples in which the compound was present/total number of samples. e ) Identification method: RI, tentative identification based on RI; MS, tentative identification based on mass spectra; CoI, coinjection with commercial standard. f ) Three chemotypes were identified for the L. graveolens oils of the 14 populations: Chemotype S, non-phenolic chemotype dominated by oxygenated sesquiterpenes (n ¼ 5); Chemotype C, carvacrol chemotype (n ¼ 5); Chemotype T, thymol chemotype (n ¼ 4).
a
Chemotype C )
f
2.70 (1.32 – 3.73) 0.04 (0.03 – 0.05) 2.42 (1.37 – 3.47) 0.05 (0.04 – 0.07) 11.68 0.22 (0.07 – 0.28) 19.89 (12.68 – 25.20) 0.15 (0.07 – 0.23) 7.67 (3.06 – 15.02) 0.09 (0.05 – 0.12) 9.56 (2.84 – 21.65) 0.09 (0.04 – 0.15) 12.84 (7.37 – 22.54) 0.15 (0.08 – 0.23) 4.06 (1.68 – 8.85) n.d. 0.42 0.03 n.d. 0.17 (0.06 – 0.28) 5.53 (4.54 – 7.55) n.d. n.d. n.d.
Chemotype S )
f
Relative content [%] c )
Total identified [%]
1590 1600 1605 1607 1615 1621 1626 1632 1640 1644 1646 1659
Class a ) RI b )
Compound name and class
Table 2 (cont.)
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98% of the observed variation in the essential-oil composition of L graveolens. The most important compounds for the discrimination were carvacrol, thymol, and caryophyllene oxide (Fig. 1). The UPGMA (unweighted pair-group method with arithmetic mean) cluster analysis (Fig. 2) confirmed the PCA results and, again, divided the individuals, according to the chemical composition of their essential oil, into three groups. These results indicated the existence of three different chemotypes in the populations of L. graveolens growing in southeastern Mexico, i.e., two phenolic chemotypes, with contents of either carvacrol (C) or thymol (T) representing more than 75% of the total oil composition, and one non-phenolic chemotype (S), dominated by oxygenated sesquiterpenes. For this latter chemotype, none of the individual components showed a concentration higher than 20% of the total composition (Table 2). Geographical Distribution. The Chi-squared test of independence showed a significantly different distribution of the individuals from these three chemotypes, among populations (c2 ¼ 170.75, df ¼ 26). Populations 1, 2, 6, and 7 presented only individuals of the Chemotype C, while Populations 3, 10, 13, and 14 included only individuals of the Chemotype S. Individuals of the Chemotype T were predominant in Populations 4, 5, and 8 (Fig. 3). L. graveolens essential oils with Chemotypes C and/or T are in agreement with previous reports found in the literature [15 – 21] [24], while a L.
Fig. 2. Dendrogram obtained by UPGMA (unweighted pair-group method with arithmetic mean) cluster analysis based on the essential-oil composition of Lippia graveolens individuals from 14 natural populations (Populations 1 – 14, cf. Table 1). Three chemotypes were observed, i.e., a carvacrol, a thymol, and a non-phenolic chemotype.
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graveolens oil with a Chemotype S has only been described once before, for a sample from Guatemala [25]. To the best of our knowledge, this is the first report of oils with a Chemotype S occurring in Mexico for this species. The populations with oils dominated by Chemotype S were more abundant in areas with deeper soils and colder and more humid climates, while the populations with oils of Chemotypes C and T were confined to areas with less developed and rockier soils and drier and warmer climates. Among these latter chemotypes, carvacrol-dominated oils were more abundant for individuals growing in very warm, semi-arid climates, while thymol-dominated oils occurred in individuals from less arid and colder areas (Fig. 3). Although it is well known that the essential-oil composition is under genetic control [26] [27], the distinct geographical distribution of the three chemotypes of L. graveolens can partly explain the observed association between particular climatic and edaphic conditions and the oil composition, which may confer ecological advantages to the individuals, as has been clearly demonstrated for Thymus vulgaris chemotypes [28] [29]. As a consequence of this differential spatial distribution of the chemotypes, the mean content (% area) of carvacrol, thymol, and sesquiterpenes (b-caryophyllene, a-humulene, and caryophyllene oxide) observed in the studied populations varied according to the edaphoclimatic gradient (Fig. 4). These findings are in agreement with those reported for Thymus
Fig. 3. Spatial distribution of the studied wild populations of Lippia graveolens (Populations 1 – 14, cf. Table 1) located along an edaphoclimatic gradient in southeastern Mexico. The pie charts show the percentage of individuals of the three described chemotypes in each population.
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Fig. 4. Variation in the contents (% total chromatogram area; mean 2 S. E.) of thymol, carvacrol, and sesquiterpenes (sum of b-caryophyllene, a-humulene, and caryophyllene oxide) in the essential oils of Lippia graveolens populations along an edaphoclimatic gradient. The number of studied individuals for Region I, II, III, and IV were 21, 24, 50, and 16 respectively (for details on the regions, cf. Table 1).
vulgaris, emphasizing the importance of chemical polymorphism for local adaptation both between and within chemotypes [28] [29]. Additionally, it has been shown that the yield and composition of the essential oil of other plants in the Lamiaceae family are related to the environmental conditions prevailing along their distribution range, with a high thymol and/or p-cymene content often associated with mesic conditions and a high carvacrol content associated with intense xerothermic conditions [30] [31]. Essential-Oil Yield. The essential-oil yield showed an important variation among the individuals of different chemotypes of L. graveolens. Individuals with oils of Chemotypes C (yield ¼ 1.95 0.15% (w/w); mean S. E.) and T (2.26 0.12%) had significantly higher yields (KruskalWallis test H(df 2) ¼ 80.95; p < 0.0001) than those with oils of the Chemotype S (0.31 0.03%), indicating a clear association between the chemotype and the production of essential oil. These findings coincide with those reported for Lippia origanoides [32], a closely related species with a similar essentialoil composition. Finally, taking into account that the standard essential-oil yield recommended for profitable commercial exploitation is 1% [32], the majority of the populations of L. graveolens analyzed in this study (65%) showed an interesting mean essential-oil yield ( 1%), with a high concentration of either carvacrol or thymol. Recent findings suggest an association between the genetic variation found in L. graveolens and both the oil production and quality [33]. Conclusions. – It was evidenced that L. graveolens is a quite variable taxon with respect to its essential-oil yield and composition. It was shown that the qualitative and quantitative characteristics of the essential oil of L. graveolens were affected by the prevailing edaphoclimatic conditions. To the best of our knowledge, this is the first comparative report on the chemical diversity of the essential oil of L. graveolens
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populations and its relation with climatic and soil conditions. The northern to central region of the Yucatan Peninsula, representing the drier areas of the studied gradient, is an important source of intra-specific chemical variability for this species. This area concentrates high-yield individuals ( > 3%) with high contents of either carvacrol or thymol. These individuals could be used as parental material for both scientific and commercial breeding programs, as well as for germplasm conservation in ex situ and in situ collections. The authors wish to thank Daniela Martnez-Natare´n and Karina Canul for field and laboratory work. This study was financially supported by a grant given to L. M. C.-I (CONACYT CB-2008-01, 106389).
Experimental Part Plant Material. Climatic data, such as mean temp. and annual precipitation, were generated for each population, using geographic coordinates and the software DIVA-GIS (Version 7.5) [34]. Fully expanded and undamaged leaves of at least five adult plants of Lippia graveolens Kunth were randomly selected from each of the 14 populations (in total 112 individuals); the leaves were collected during the rainy season (July to August) to minimize variability in the essential-oil content due to developmental or phenological differences. The leaves were placed in paper bags and taken to the laboratory, where they were air-dried in an air-flux drying oven (NOVATECH HS60) at 358 for 36 h, as described in [25], and then stored at 48 until extraction. Voucher specimens of representative individuals were deposited with the Herbarium of the Unidad de Recursos Naturales, CICY (L. M. Calvo-Irabien collection numbers: 240 – 266 and 331 – 333). Essential-Oil Extraction. Essential oils were extracted individually from dry leaves (5 g) by hydrodistillation in a Clevenger-type apparatus for 1.5 h with hexane (2 ml) as the collector solvent. The oilhexane mixture was dried (Na2SO4 ), and the solvent was evaporated under a flow of N2 for 30 min. The oil yield was estimated on the dry-weight basis (% w/w). The essential-oil samples were kept in sealed amber vials at 48 until chromatographic analyses. GC-FID and GC/MS Analyses. The GC/MS analyses were performed with an Agilent 6890N gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA ) connected with an Agilent 5975 mass-selective detector (MSD; electron impact ionization, 70 eV) and equipped with G1701DA GC/ MSD ChemStation software and a non-polar Agilent Technologies Ultra 1 cap. column (25 m 0.32 mm i.d., film thickness 0.52 mm). The oven temp. was programmed isothermal at 758 for 4 min, then linearly rising from 75 to 2008 at 58/min, and finally held isothermal at 2008 for 1 min; injector temp., 2808; detector temp., 2908; carrier gas, He (1.5 ml/min); injection volume, 1 ml; split ratio, 1 : 50. The quantification of individual components in the 112 individual samples was carried out by GCFID with an Agilent 6890N gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with FID. The cap. column and the chromatographic conditions were the same as those described above for the GC/MS analyses, except that H2 was used as carrier gas. Identification of Compounds. For the identification of the essential-oil components by GC/MS analysis, 14 chromatograms having contrasting profiles were selected. The linear retention index (RI) of each compound was determined rel. to a homologous series of n-alkanes, C8 – C20 (SigmaAldrich, St. Louis, MO, USA), directly injected into the GC/MS system under the above-described conditions. Whenever possible, the individual components were identified by coinjection with commercially available standards, i.e., carvacrol (98%), caryophyllene oxide (99%), trans-b-caryophyllene (98%), 1,8cineol (99%), p-cymene (97%), a-humulene (98%), thymol (99%), g-terpinene (98%; all obtained by SigmaAldrich, St. Louis, MO, USA). Otherwise, the peaks were tentatively identified by comparing their RI and mass spectra with those included in the NIST-05a and ADAMS libraries and/or reported in the literature [35]. The relative contents expressed in % of the essential-oil components were obtained by internal peak-area normalization without using response factors. The analyses were repeated three
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times for each sample. The percentage of the sum of identified compounds in each sample ranged from 89 to 99% of the total chromatographic area (Table 2). Statistical Analysis. The variation in the chemical composition of the L. graveolens essential oils was described using multivariate analyses. The eight terpenes that individually and regularly represented 10% or more of the total chromatographic area (contents 10%), i.e., p-cymene, g-terpinene, eucalyptol, carvacrol, thymol, b-caryophyllene, a-humulene, and caryophyllene oxide, were considered for the statistical analyses. The data matrix consisted of the corresponding contents for these eight terpenes in the 112 individuals; missing data were substituted by 0.01%. The multivariate analyses [36] included i) principal component analysis (PCA) based on an Euclidean covariance matrix, to check for partition among the individual essential-oil components and ii) hierarchical cluster analysis with unweighted pairgroup method with arithmetic mean (UPGMA) using Euclidean distances, to establish the different groups/chemotypes. To verify the presence of a non-random distribution of chemotypes among populations, a chi-squared test of independence [37] was used to compare the number of individuals from each of the three chemotypes in the 14 populations. Essential-oil yield comparison among chemotypes was performed using the nonparametric KruskalWallis test, considering that there was no data normality [37]. All statistical analyses were performed with the appropriate procedures of the software package STATISTICA 5.1 [38].
REFERENCES [1] S. E. Kintzios, Oregano: the Genera Origanum and Lippia, CRC Press, Boca Raton, FL, 2002. [2] T. Herna´ndez, M. Canales, J. G. Avila, A. Duran, J. Caballero, A. Romo de Vivar, R. Lira, J. Ethnopharmacol. 2003, 88, 181. [3] C. C. Arcila-Lozano, G. Loarca-Pin˜a, S. Lecona-Uribe, E. Gonza´lez de Meja, Arch. Latinoam. Nutr. 2004, 54, 100. [4] M. C. Portillo-Ruiz, S. Viramontes-Ramos, L. N. Mun˜oz-Castellanos, M. G. Gaste´lum-Franco, G. V. Neva´rez-Moorillo´n, J. Food Prot. 2005, 68, 2713. [5] N. E. Rocha-Guzma´n, J. A. Gallegos-Infante, R. F. Gonza´lez-Laredo, M. Ramos-Go´mez, M. E. Rodrguez-Mun˜oz, R. Reynoso-Camacho, A. Rocha-Uribe, M. R. Roque-Rosales, Food Chem. 2007, 102, 330. [6] R. Avila-Sosa, M. G. Gaste´lum-Franco, A. Camacho-Da´vila, J. V. Torres-Mun˜oz, G. V. Neva´rezMoorillo´n, Food Bioprocess. Technol. 2010, 3, 434. [7] C. M. Huerta, Biodiversitas 1997, 15, 8. [8] Market News Service (MNS), Essential Oils and Oleoresins, MNS Bulletin, International Trade Centre, Geneva, Switzerland, June 2009. [9] T. A. Osorno-Sa´nchez, R. Torres, R. Lindig-Cisneros, Afr. J. Agric. Res. 2012, 7, 100. [10] S. Padulosi, Oregano: Promoting the Conservation and Use of Underutilized and Neglected Crops, Proceedings of the IPGRI International Workshop on Oregano, Institute of Plant Genetics and Crop Plant Research, Gatersleben/International Plant Genetic Resources Institute, Valenzano, Italy, 1996. [11] M. A. Soto, F. Gonza´lez-Medrano, O. Sa´nchez, in Me´todo de Evaluacio´n del Riesgo de Extincio´n de las Especies Silvestres en Me´xico, Eds. O. Sa´nchez, R. Medelln, A. Aldama, B. Goettsch, J. Sobero´n-Mainero, M. Tambutti, SEMARNAT, INE, UNAM, CONABIO, Ciudad de Mexico, Me´xico, 2007, p. 91. [12] D. L. Nash, M. Nee, Flora de Veracruz, Fasc. 41, Instituto Nacional de Investigaciones sobre Recursos Bio´ticos, Xalapa, Me´xico, 1984. [13] A. Pool, R. Rueda, in Flora de Nicaragua, Angiospermas (Pandanaceae-Zigophillaceae), Eds. W. D. Stevens, C. Ulloa, A. Pool, O. M. Montiel, Missouri Botanical Garden Press, Missouri, MI, 2001, p. 2497. [14] D. A. Martnez-Natare´n, V. Parra-Tabla, G. Dzib, V. Acosta-Arriola, K. Canul-Puc, L. M. CalvoIrabie´n, J. Essent. Oil Bear. Plants 2012, 15, 589. [15] C. M. Compadre, R. A. Hussain, I. Leon, R. G. Enrquez, Planta Med. 1987, 53, 495.
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[16] J. Pino, A. Rosado, B. Baluja, P. Borges, Food/Nahrung 1989, 33, 289. [17] C. J. Uribe-Herna´ndez, J. B. Hurtado-Ramos, E. R. Olmedo-Arceaga, M. A. Martnez-Sosa, J. Essent. Oil Res. 1992, 4, 647. ´ ridas [18] L. Alaniz-Gutie´rrez, R. Castro-Franco, F. Go´mez-Lorence, Revista Chapingo Ser. Zonas A 2000, 1, 13. [19] F. Senatore, D. Rigano, Flavour Fragrance J. 2001, 16, 169. [20] G. Vernin, C. Lageot, E. M. Gaydou, C. Parkanyi, Flavour Fragrance J. 2001, 16, 219. [21] L. R. Salgueiro, C. Cavaleiro, M. J. GonÅalves, A. ProenÅa da Cunha, Planta Med. 2003, 69, 80. [22] R. Orellana, M. Balam, I. Ban˜uelos, E. Garca, J. A. Gonza´lez-Iturbe, F. Herrera, J. Vidal, in Atlas de Procesos Territoriales de Yucata´n, Eds. A. Garca de Fuentes, J. Cordoba-Ordo´n˜ez, P. Chico Ponce de Leo´n, Universidad Auto´noma de Yucata´n, Me´rida, Me´xico, 1999, p. 162. [23] F. Bautista, G. Palacio-Aponte, P. Quintana, J. A. Zinck, Geomorphology 2011, 135, 308. [24] L. M. Calvo-Irabien, J. A. Yam-Puc, G. Dzib, F. Escalante-Erosa, L. M. Pen˜a-Rodrguez, J. Herb Spices Med. Plants 2009, 15, 281. [25] C. Franz, J. Novak, in Handbook of Essential Oils: Science, Technology and Applications, Eds. K. H. C. Baser, G. Buchbauer, CRC Press, Boca Raton, FL, 2010, p. 39. [26] C. Franz, in Volatile Oil Crops, Eds. R. Hay, P. G. Waterman, Logman Scientific & Technical, Essex, England,1993, p. 63. [27] P. Vernet, P. H. Gouyon, G. Valdeyron, Genetica 1986, 6, 227. [28] J. D. Thompson, J. C. Chalchat, A. Michet, Y. B. Linhart, B. Ehlers, J. Chem. Ecol. 2003, 29, 859. [29] J. D. Thompson, P. Gauthier, J. Amiot, B. K. Ehlers, C. Collin, J. Fossat, V. Barrios, F. ArnaudMiramont, K. Keefover-Ring, Y. B. Linhart, Ecol. Monogr. 2007, 77, 421. [30] M. Skoula, J. B. Harborne, in Oregano: the genera Origanum and Lippia, Ed. S. E. Kintzios, CRC Press, Boca Raton, FL, 2002, p. 67. [31] R. D. Karousou, N. Koureas, S. Kokkini, Phytochemistry 2005, 66, 2668. [32] E. Stashenko, J. R. Martnez, C. A. Ruz, G. Arias, C. Dura´n, V. Salgar, M. Cala, J. Sep. Sci. 2010, 33, 93. [33] D. Martnez-Natare´n, V. Parra-Tabla, M. Ferrer-Ortega, L. M. Calvo-Irabie´n, Plant. Syst. Evol. 2014, 300, 535. [34] R. J. Hijmans, L. Guarino, P. Mathur, DIVA-GIS Version 7.5, 2012, http://www.diva-gis.org (accessed July 25, 2013). [35] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th edn., Allured Publishing Corporation, Carol Stream, IL, 2007. [36] K. Varmuza, P. Filzmoser, Introduction to Multivariate Analysis in Chemometrics, CRC Press, Boca Raton, FL, 2009. [37] R. R. Sokal, F. J. Rohlf, Biometry. The Principles and Practice of Statics in Biological Research, 3rd edn., W. H. Freeman and Company, New York, 1995. [38] STATSOFT STATISTICA (data analysis software system), version 5.1., StatSoft Inc., Tulsa, 1996, http://www.statsoft.com/. Received December 12, 2013
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Phytochemical Diversity of the Essential Oils of Mexican Oregano (Lippia graveolens Kunth) Populations along an Edapho-Climatic Gradient by Luz Mara Calvo-Irabie´n* a ), Victor Parra-Tabla b ), Violeta Acosta-Arriola a ), Fabiola EscalanteErosa c ), Luciana Daz-Vera a ), Gabriel R. Dzib a ), and Luis Manuel Pen˜a-Rodrguez c ) a
) Unidad de Recursos Naturales, Centro de Investigacio´n Cientfica de Yucata´n, A. C. Calle 43 #130 Chuburna´ de Hidalgo, Me´rida, Yucata´n 97200, Me´xico (phone: þ 52-999-9428330; fax: þ 52-999-9813900; e-mail: [email protected]) b ) Universidad Auto´noma de Yucata´n, Campus de Ciencias Biolo´gicas y Agropecuarias, Departamento de Ecologa Tropical, Km. 15.5, Carretera Me´rida-Xmatkuil, 4-116, Me´rida, Yucata´n 97315, Me´xico c ) Unidad de Biotecnologa, Centro de Investigacio´n Cientfica de Yucata´n, A. C. Calle 43 #130 Chuburna´ de Hidalgo, Me´rida, Yucata´n 97200, Me´xico
Mexican oregano (Lippia graveolens) is an important aromatic plant, mainly used as flavoring and usually harvested from non-cultivated populations. Mexican oregano essential oil showed important variation in the essential-oil yield and composition. The composition of the essential oils extracted by hydrodistillation from 14 wild populations of L. graveolens growing along an edaphoclimatic gradient was evaluated. Characterization of the oils by GC-FID and GC/MS analyses allowed the identification of 70 components, which accounted for 89 – 99% of the total oil composition. Principal component and hierarchical cluster analyses divided the essential oils into three distinct groups with contrasting oil compositions, viz., two phenolic chemotypes, with either carvacrol (C) or thymol (T) as dominant compounds (contents > 75% of the total oil composition), and a non-phenolic chemotype (S) dominated by oxygenated sesquiterpenes. While Chemotype C was associated with semi-arid climate and shallower and rockier soils, Chemotype T was found for plants growing under less arid conditions and in deeper soils. The plants showing Chemotype S were more abundant in subhumid climate. High-oil-yield individuals ( > 3%) were identified, which additionally presented high percentages of either carvacrol or thymol; these individuals are of interest, as they could be used as parental material for scientific and commercial breeding programs.
Introduction. – Mexican oregano, Lippia graveolens Kunth (Verbenaceae), is an important aromatic plant mainly used as condiment or flavoring [1], but also used in the traditional medicine, because of the different biological activities reported for its essential oil, including antioxidant and antibiotic properties [2 – 6]. These characteristics, and their applications in a wide range of industries, confirm the economic potential of this phylogenetic resource. Mexico is the main exporting country of L. graveolens in the world [1], with ca. 4,000 t of Mexican oregano leaves exported annually, most of which are harvested from wild populations found in forests and secondary vegetation [7] [8]. The harvest pressure on this aromatic plant, combined with other factors such as habitat destruction and deforestation, have caused a significant decrease of its natural populations [9]. This is one of the reasons why, at the moment, Mexican oregano is considered a priority species for germplasm conservation [10] [11]. 2014 Verlag Helvetica Chimica Acta AG, Zrich
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L. graveolens is widely distributed throughout the southeast of the United States, Mexico, Guatemala, Nicaragua, and Honduras, growing under diverse ecological conditions in arid, semi-arid, and subhumid regions [12] [13]. A high morphological variability [11] and a significant variation in the essential-oil yield [14] and its chemical composition have been described [15 – 21]. However, most of these studies are restricted to a few individuals and locations. To the best of our knowledge and despite its economic importance, to date, there are no studies that evaluate the effect of environmental conditions on the variation of Mexican oreganos essential-oil composition within and among populations. As part of an ongoing research project directed towards establishing breeding and conservation programs for L. graveolens, and to improve our understanding of the factors responsible for the observed chemical variation, the essential-oil composition in 14 wild populations (see Table 1) of L. graveolens growing along an edaphoclimatic gradient was evaluated. In particular, two questions were addressed: i) Is the chemical variation observed in the essential-oil composition associated or correlated with the edaphoclimatic conditions? ii) Does this variation present a particular spatial pattern? The results obtained will contribute to our understanding of the relative importance of ecological factors on the production of essential oils in aromatic plants in general and, particularly, in Mexican oregano. Additionally, to ensure market demands of a homogeneous production in quantity and quality of this essential oil, the present results will provide valuable information related to the best suitable germplasm and environmental conditions for the management and cultivation of this important aromatic species.
Table 1. Location and Environmental Characteristics of Lippia graveolens Populations 1 – 14 from Southeast Mexico Region
Population
Longitude ( W) a )
Latitude ( N ) a)
Climate
Mean temperature [8]
Annual precipitation [mm]
I
1 2 3
89.5738 89.1158 89.8078
21.2538 21.3388 21.1638
Very warm semi-arid
25.5 25.7 25.7
538 684 614
II
4 5 6
88.1898 88.0848 88.7078
21.5618 21.5648 21.3418
Driest warm sub-humid
25.5 25.4 25.7
650 664 893
III
7 8 9 10 11 12
88.9838 89.6528 89.5998 90.1488 89.8578 90.0048
21.1408 20.6348 20.6018 20.6168 20.5398 20.5768
Warm sub-humid with summer rains
25.9 26.1 26.1 26.7 26.3 26.6
979 1007 1014 1021 1069 1099
IV
13 14
88.9098 88.9468
20.5008 20.5148
Intermediate warm sub-humid
26.1 26.2
1151 1156
a
) Longitude and latitude are expressed as decimals of degrees.
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Results and Discussion. – Edaphoclimatic Gradient. The studied area (Yucata´n Peninsula) provided an ideal scenario to assess the influence of edaphoclimatic variation on the intraspecific chemical variation found in L. graveolens essential oil. This area is a portion of flat land, characterized by an edaphoclimatic gradient with aridity being greatest in the northwestern part and presenting a more humid and colder climate in the southeastern part. Additionally, the soils in the NW of the Yucata´n Peninsula are rockier and younger than in the SE [22] [23]. The location and the environmental characteristics of the 14 sampled populations of L. graveolens, growing in four different regions with different climate types (Regions I – IV), are shown in Table 1. Chemotypes and Multivariate Analysis. The GC-FID and GC/MS analyses of the different samples of L. graveolens essential oil allowed the identification of 70 oil components, which accounted for 89 – 99% of the total oil composition (area of the chromatograms). The identified components are listed in Table 2, in the order of their elution from the Ultra 1 capillary column. The 15 components that represented more than 75% of the total area, mainly oxygenated monoterpenes and sesquiterpenes, are shown in bold type. The plot obtained by principal component analysis (PCA; Fig. 1) showed three distinct groups with contrasting essential-oil compositions and explained
Fig. 1. Plot showing the similarity among Lippia graveolens individuals from 14 natural populations (Populations 1 – 14, cf. Table 1) obtained by principal component analysis (PCA) of the mayor essentialoil constituents (p-cymene, g-terpinene, eucalyptol ( ¼ 1,8-cineole), carvacrol, thymol, b-caryophyllene, a-humulene, and caryophyllene oxide). Together, PCA Axis 1 and 2 explained 98% of the observed variation in the essential-oil compositions.
MH MH MH MH MH MH MH MO MH MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO MO
a-Thujene a-Pinene Camphene b-Pinene Myrcene d-Carene p-Cymene 1,8-Cineole g-Terpinene cis-Sabinene hydrate 6,7-Epoxymyrcene trans-Sabinene hydrate Linalool cis-p-Menth-2-enol trans-Sabinol Borneol Umbellulone Terpinen-4-ol a-Terpineol trans-Piperitol trans-Carveol cis-p-Mentha-1(7),8-dien-2-ol Thymol methyl ether Carvacrol methyl ether 2-Phenethyl acetate Thymol Carvacrol Eugenol 921 929 941 968 980 1005 1012 1019 1047 1051 1073 1079 1083 1104 1119 1145 1150 1158 1173 1187 1195 1202 1214 1223 1232 1279 1282 1326
Class a ) RI b )
Compound name and class
n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1.83 (0.13 – 5.14) n.d. n.d. n.d. 0.23 0.26 0.26 0.74 (0.26 – 1.70) 1.30 (0.17 – 2.19) n.d. 0.42 (0.11 – 0.95) 2.91 0.52 0.59 0.31 0.52 n.d. n.d. 4.16 (1.13 – 11.28) 1.24 (0.65 – 1.89) 0.35
0.03 (0.01 – 0.06) 0.03 (0.02 – 0.06) 0.01 0.02 0.08 (0.03 – 0.13) 0.02 0.96 (0.23 – 3.13) 0 – 59 (0.03 – 0.92) 0.04 (0.01 – 0.07) 0.04 (0.01 – 0.06) n.d. n.d. 0.72 (0.57 – 0.93) n.d. n.d. 0.13 (0.08 – 0.20) 0.08 (0.001 – 0.15) 0.82 (0.66 – 0.97) 0.13 (0.01 – 0.26) n.d. n.d. n.d. 0.83 (0.19 – 2.25) 0.09 (0.03 – 0.14) 0.14 6.18 (3.35 – 8.60) 78.50 (72.20 – 83.49) 0.13 (0.09 – 0.18)
0.02 0.04 (0.02 – 0.05) 0.01 0.01 0.13 (0.02 – 0.26) 0.01 1.28 (0.37 – 2.22) 0.43 (0.18 – 0.80) 0.19 (0.09 – 0.30) n.d 0.06 n.d. 0.59 (0.25 – 0.92) n.d. n.d. 0.07 0.14 (0.06 – 0.25) 0.76 (0.58 – 0.97) 0.14 (0.05 – 0.35) n.d. tr n.d. 2.07 (0.68 – 3.83) n.d. 0.07 88.47 (84.76 – 91.77) 0.44 (0.38 – 0.59) 0.134
0.8 1 0.2
0.6 0.2 0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.6 0.6
0.8
1 1 0.2 1 1 1
0.25 0.75 1 1
1 1 1 1
0.25 1 1 0.25
1
1
1
0.25
0.25 0.5 0.25 0.25 1 0.25 1 1 0.5
T f)
1
0.4 0.6 0.2 0.2 0.4 0.2 1 0.8 0.4 0.4
C f)
S f)
Chemotype T f )
Chemotype S f )
Chemotype C f )
Frequency d )
Relative content [%] c )
RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,
MS MS MS MS MS MS MS MS, CoI MS, CoI MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS, CoI MS, CoI MS
Identification e )
Table 2. Chemical Composition of the Essential Oils Obtained by Hydrodistillation of the Leaves of 14 Wild Lippia graveolens Populations Growing along an Edaphoclimatic Gradient in Southeastern Mexico. The 14 oils were divided into three chemotypes, a non-phenolic ( S ), a carvacrol (C ), and a thymol (T ) chemotype.
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014) 1013
MO MO SH SH SH SH SH SH SH OC SH SH SH SH SH SH SH SH SH SH SH SH SO SO SO SO SO SO SO SO
Thymol acetate Carvacrol acetate a-Copaene a-Cedrene b-Caryophyllene b-Copaene a-trans-Bergamotene a-Humulene Alloaromadendrene 3-(tert-Butyl)-4-hydroxyanisole g-Muurolene Germacrene D ar-Curcumene b-Selinene a-Selinene a-Muurolene g-Cadinene a-Alaskene trans-Calamene d-Cadinene cis-Calamene a-Calacorene cis-Sesquisabinene hydrate trans-Sesquisabinene hydrate trans-Nerolidol Spathulenol Caryophyllene oxide Viridiflorol Guaiol Humulene epoxide II
1327 1344 1367 1401 1407 1416 1428 1440 1447 1457 1461 1462 1465 1470 1480 1483 1496 1497 1500 1506 1510 1518 1534 1538 1545 1551 1556 1560 1575 1580
Class a ) RI b )
Compound name and class
Table 2 (cont.)
n.d. n.d. 0.28 (0.18 – 0.45) 0.20 2.45 (1.33 – 3.30) 0.20 (0.08 – 0.28) n.d. 1.48 (0.88 – 2.09) 0.26 (0.18 – 0.41) n.d. 0.53 (0.11 – 0.99) n.d. 0.35 0.68 (0.27 – 1.03) 0.46 (0.15 – 0.78) 0.28 (0.20 – 0.36) 0.27 (0.17 – 0.41) n.d. 0.47 (0.17 – 0.91) 0.33 (0.16 – 0.53) 0.43 n.d. 0.56 0.69 1.28 (0.82 – 2.36) 1.79 (0.28 – 4.00) 7.37 (4.94 – 11.34) 3.71 (1.01 – 12.63) 0.54 (0.25 – 1.08) 4.34 (3.04 – 5.99)
Chemotype S )
f
f
n.d. 0.07 (0.03 – 0.14) 0.09 (0.02 – 0.2) n.d. 1.45 (0.27 – 2.45) n.d. 0.14 (0.09 – 0.19) 0.75 (0.19 – 1.28) 0.04 (0.03 – 0.04) 3.32 (2.21 – 4.91) n.d. 0.10 (0.06 – 0.17) n.d. n.d. n.d. 0.03 (0.01 – 0.04) 0.06 (0.05 – 0.08) n.d. 0.07 (0.001 – 0.12) 0.13 (0.02 – 0.23) n.d. 0.01 n.d. n.d. 0.06 (0.03 – 0.09) n.d. 2.29 (0.40 – 3.15) n.d. 0.22 1.20 (0.23 – 1.64)
Chemotype C )
Relative content [%] c )
0.35 (0.07 – 0.83) 0.02 n.d. 0.12 1.21 (0.35 – 2.49) 0.55 (0.16 – 0.94) 0.05 (0.04 – 0.06) 0.83 (0.26 – 1.60) 0.06 0.76 (0.58 – 1.09) n.d. 0.02 0.34 0.01 n.d. 0.03 (0.01 – 0.05) 0.01 0.14 0.04 n.d. 0.07 (0.001 – 0.26) n.d. n.d. 0.02 0.06 n.d. 0.91 (0.48 – 1.28) n.d. n.d. 0.51 (0.33 – 0.71)
Chemotype T )
f
0.2 0.2 1 0.6 1 1 0.8 1
0.8 1 0.2
0.2 0.6 0.8 0.4 0.8
0.6
1 0.6
0.6 0.2 1 0.6
S f)
0.5 1
1
0.6 1 0.2 1
0.2
1
0.5 0.25 0.25 0.25
0.6 0.8 0.8 1
0.25 0.25 0.25
0.25 1 0.5 0.75 1 0.25 1
1 0.25
T f)
0.8
0.8 1 0.4 1
1
0.8 0.6
C f)
Frequency d )
RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,
MS MS MS MS MS, CoI MS MS MS, CoI MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS, CoI MS MS MS
Identification e )
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SO SO SO SO SO SO SO SO SO SH SO SO
1,10-Diepicubenol Muurola-4,10(14)-dien-1b-ol g-Eudesmol Caryophylla-4(12),8(13)-dien-5a-ol epi-a-Cadinol þ epi-a-Muurolol b-Eudesmol a-Eudesmol þ a-Cadinol cis-Calamen-10-ol Bulnesol Cadalene b-Epibisabolol a-Bisabolol 88.8 0.0 12.4 7.2 80.4 0.0
Monoterpene hydrocarbons ( MH) Oxygenated monoterpenes ( MO) Sesquiterpene hydrocarbons ( SH ) Oxygenated sesquiterpenes ( SO ) Other oxygenated compounds (OC)
1.2 88.2 3.1 4.3 3.3
99.4
1.6 93.2 2.5 2.0 0.8
98.97
n.d n.d. 0.08 (0.03 – 0.15) 0.08 (0.04 – 0.12) 0.26 n.d 0.16 (.005 – 0.26) n.d. n.d. n.d. 0.10 (0.05 – 0.17) 0.10
Chemotype T )
f
0.8
1 0.4 0.2 1 1 0.8 0.6 1 0.2
S)
f
0.2 1
0.6 0.8 0.8 0.6 0.6 0.4 0.8
C)
f
f
0.75 0.25
0.5
0.75 1 0.25
T)
Frequency d )
RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,
MS MS MS MS MS MS MS MS MS MS MS MS
Identification e )
) The abbreviations of the compound classes are given at the end of the table (first column, last five rows). b ) RI: Linear retention indices determined experimentally on the Ultra I column relative to a series of n-alkanes (C8 – C26 ) using a non-logarithmic scale. c ) Relative contents are given as mean GC-FID peak areas with minimal and maximal peak areas in parentheses and expressed as percentages; for the five most abundant compounds of each chemotype, the contents are given in bold type; n.d., not detected. d ) The frequency of appearance of the compounds in the oil samples was calculated as number of samples in which the compound was present/total number of samples. e ) Identification method: RI, tentative identification based on RI; MS, tentative identification based on mass spectra; CoI, coinjection with commercial standard. f ) Three chemotypes were identified for the L. graveolens oils of the 14 populations: Chemotype S, non-phenolic chemotype dominated by oxygenated sesquiterpenes (n ¼ 5); Chemotype C, carvacrol chemotype (n ¼ 5); Chemotype T, thymol chemotype (n ¼ 4).
a
Chemotype C )
f
2.70 (1.32 – 3.73) 0.04 (0.03 – 0.05) 2.42 (1.37 – 3.47) 0.05 (0.04 – 0.07) 11.68 0.22 (0.07 – 0.28) 19.89 (12.68 – 25.20) 0.15 (0.07 – 0.23) 7.67 (3.06 – 15.02) 0.09 (0.05 – 0.12) 9.56 (2.84 – 21.65) 0.09 (0.04 – 0.15) 12.84 (7.37 – 22.54) 0.15 (0.08 – 0.23) 4.06 (1.68 – 8.85) n.d. 0.42 0.03 n.d. 0.17 (0.06 – 0.28) 5.53 (4.54 – 7.55) n.d. n.d. n.d.
Chemotype S )
f
Relative content [%] c )
Total identified [%]
1590 1600 1605 1607 1615 1621 1626 1632 1640 1644 1646 1659
Class a ) RI b )
Compound name and class
Table 2 (cont.)
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98% of the observed variation in the essential-oil composition of L graveolens. The most important compounds for the discrimination were carvacrol, thymol, and caryophyllene oxide (Fig. 1). The UPGMA (unweighted pair-group method with arithmetic mean) cluster analysis (Fig. 2) confirmed the PCA results and, again, divided the individuals, according to the chemical composition of their essential oil, into three groups. These results indicated the existence of three different chemotypes in the populations of L. graveolens growing in southeastern Mexico, i.e., two phenolic chemotypes, with contents of either carvacrol (C) or thymol (T) representing more than 75% of the total oil composition, and one non-phenolic chemotype (S), dominated by oxygenated sesquiterpenes. For this latter chemotype, none of the individual components showed a concentration higher than 20% of the total composition (Table 2). Geographical Distribution. The Chi-squared test of independence showed a significantly different distribution of the individuals from these three chemotypes, among populations (c2 ¼ 170.75, df ¼ 26). Populations 1, 2, 6, and 7 presented only individuals of the Chemotype C, while Populations 3, 10, 13, and 14 included only individuals of the Chemotype S. Individuals of the Chemotype T were predominant in Populations 4, 5, and 8 (Fig. 3). L. graveolens essential oils with Chemotypes C and/or T are in agreement with previous reports found in the literature [15 – 21] [24], while a L.
Fig. 2. Dendrogram obtained by UPGMA (unweighted pair-group method with arithmetic mean) cluster analysis based on the essential-oil composition of Lippia graveolens individuals from 14 natural populations (Populations 1 – 14, cf. Table 1). Three chemotypes were observed, i.e., a carvacrol, a thymol, and a non-phenolic chemotype.
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graveolens oil with a Chemotype S has only been described once before, for a sample from Guatemala [25]. To the best of our knowledge, this is the first report of oils with a Chemotype S occurring in Mexico for this species. The populations with oils dominated by Chemotype S were more abundant in areas with deeper soils and colder and more humid climates, while the populations with oils of Chemotypes C and T were confined to areas with less developed and rockier soils and drier and warmer climates. Among these latter chemotypes, carvacrol-dominated oils were more abundant for individuals growing in very warm, semi-arid climates, while thymol-dominated oils occurred in individuals from less arid and colder areas (Fig. 3). Although it is well known that the essential-oil composition is under genetic control [26] [27], the distinct geographical distribution of the three chemotypes of L. graveolens can partly explain the observed association between particular climatic and edaphic conditions and the oil composition, which may confer ecological advantages to the individuals, as has been clearly demonstrated for Thymus vulgaris chemotypes [28] [29]. As a consequence of this differential spatial distribution of the chemotypes, the mean content (% area) of carvacrol, thymol, and sesquiterpenes (b-caryophyllene, a-humulene, and caryophyllene oxide) observed in the studied populations varied according to the edaphoclimatic gradient (Fig. 4). These findings are in agreement with those reported for Thymus
Fig. 3. Spatial distribution of the studied wild populations of Lippia graveolens (Populations 1 – 14, cf. Table 1) located along an edaphoclimatic gradient in southeastern Mexico. The pie charts show the percentage of individuals of the three described chemotypes in each population.
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Fig. 4. Variation in the contents (% total chromatogram area; mean 2 S. E.) of thymol, carvacrol, and sesquiterpenes (sum of b-caryophyllene, a-humulene, and caryophyllene oxide) in the essential oils of Lippia graveolens populations along an edaphoclimatic gradient. The number of studied individuals for Region I, II, III, and IV were 21, 24, 50, and 16 respectively (for details on the regions, cf. Table 1).
vulgaris, emphasizing the importance of chemical polymorphism for local adaptation both between and within chemotypes [28] [29]. Additionally, it has been shown that the yield and composition of the essential oil of other plants in the Lamiaceae family are related to the environmental conditions prevailing along their distribution range, with a high thymol and/or p-cymene content often associated with mesic conditions and a high carvacrol content associated with intense xerothermic conditions [30] [31]. Essential-Oil Yield. The essential-oil yield showed an important variation among the individuals of different chemotypes of L. graveolens. Individuals with oils of Chemotypes C (yield ¼ 1.95 0.15% (w/w); mean S. E.) and T (2.26 0.12%) had significantly higher yields (KruskalWallis test H(df 2) ¼ 80.95; p < 0.0001) than those with oils of the Chemotype S (0.31 0.03%), indicating a clear association between the chemotype and the production of essential oil. These findings coincide with those reported for Lippia origanoides [32], a closely related species with a similar essentialoil composition. Finally, taking into account that the standard essential-oil yield recommended for profitable commercial exploitation is 1% [32], the majority of the populations of L. graveolens analyzed in this study (65%) showed an interesting mean essential-oil yield ( 1%), with a high concentration of either carvacrol or thymol. Recent findings suggest an association between the genetic variation found in L. graveolens and both the oil production and quality [33]. Conclusions. – It was evidenced that L. graveolens is a quite variable taxon with respect to its essential-oil yield and composition. It was shown that the qualitative and quantitative characteristics of the essential oil of L. graveolens were affected by the prevailing edaphoclimatic conditions. To the best of our knowledge, this is the first comparative report on the chemical diversity of the essential oil of L. graveolens
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populations and its relation with climatic and soil conditions. The northern to central region of the Yucatan Peninsula, representing the drier areas of the studied gradient, is an important source of intra-specific chemical variability for this species. This area concentrates high-yield individuals ( > 3%) with high contents of either carvacrol or thymol. These individuals could be used as parental material for both scientific and commercial breeding programs, as well as for germplasm conservation in ex situ and in situ collections. The authors wish to thank Daniela Martnez-Natare´n and Karina Canul for field and laboratory work. This study was financially supported by a grant given to L. M. C.-I (CONACYT CB-2008-01, 106389).
Experimental Part Plant Material. Climatic data, such as mean temp. and annual precipitation, were generated for each population, using geographic coordinates and the software DIVA-GIS (Version 7.5) [34]. Fully expanded and undamaged leaves of at least five adult plants of Lippia graveolens Kunth were randomly selected from each of the 14 populations (in total 112 individuals); the leaves were collected during the rainy season (July to August) to minimize variability in the essential-oil content due to developmental or phenological differences. The leaves were placed in paper bags and taken to the laboratory, where they were air-dried in an air-flux drying oven (NOVATECH HS60) at 358 for 36 h, as described in [25], and then stored at 48 until extraction. Voucher specimens of representative individuals were deposited with the Herbarium of the Unidad de Recursos Naturales, CICY (L. M. Calvo-Irabien collection numbers: 240 – 266 and 331 – 333). Essential-Oil Extraction. Essential oils were extracted individually from dry leaves (5 g) by hydrodistillation in a Clevenger-type apparatus for 1.5 h with hexane (2 ml) as the collector solvent. The oilhexane mixture was dried (Na2SO4 ), and the solvent was evaporated under a flow of N2 for 30 min. The oil yield was estimated on the dry-weight basis (% w/w). The essential-oil samples were kept in sealed amber vials at 48 until chromatographic analyses. GC-FID and GC/MS Analyses. The GC/MS analyses were performed with an Agilent 6890N gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA ) connected with an Agilent 5975 mass-selective detector (MSD; electron impact ionization, 70 eV) and equipped with G1701DA GC/ MSD ChemStation software and a non-polar Agilent Technologies Ultra 1 cap. column (25 m 0.32 mm i.d., film thickness 0.52 mm). The oven temp. was programmed isothermal at 758 for 4 min, then linearly rising from 75 to 2008 at 58/min, and finally held isothermal at 2008 for 1 min; injector temp., 2808; detector temp., 2908; carrier gas, He (1.5 ml/min); injection volume, 1 ml; split ratio, 1 : 50. The quantification of individual components in the 112 individual samples was carried out by GCFID with an Agilent 6890N gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with FID. The cap. column and the chromatographic conditions were the same as those described above for the GC/MS analyses, except that H2 was used as carrier gas. Identification of Compounds. For the identification of the essential-oil components by GC/MS analysis, 14 chromatograms having contrasting profiles were selected. The linear retention index (RI) of each compound was determined rel. to a homologous series of n-alkanes, C8 – C20 (SigmaAldrich, St. Louis, MO, USA), directly injected into the GC/MS system under the above-described conditions. Whenever possible, the individual components were identified by coinjection with commercially available standards, i.e., carvacrol (98%), caryophyllene oxide (99%), trans-b-caryophyllene (98%), 1,8cineol (99%), p-cymene (97%), a-humulene (98%), thymol (99%), g-terpinene (98%; all obtained by SigmaAldrich, St. Louis, MO, USA). Otherwise, the peaks were tentatively identified by comparing their RI and mass spectra with those included in the NIST-05a and ADAMS libraries and/or reported in the literature [35]. The relative contents expressed in % of the essential-oil components were obtained by internal peak-area normalization without using response factors. The analyses were repeated three
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times for each sample. The percentage of the sum of identified compounds in each sample ranged from 89 to 99% of the total chromatographic area (Table 2). Statistical Analysis. The variation in the chemical composition of the L. graveolens essential oils was described using multivariate analyses. The eight terpenes that individually and regularly represented 10% or more of the total chromatographic area (contents 10%), i.e., p-cymene, g-terpinene, eucalyptol, carvacrol, thymol, b-caryophyllene, a-humulene, and caryophyllene oxide, were considered for the statistical analyses. The data matrix consisted of the corresponding contents for these eight terpenes in the 112 individuals; missing data were substituted by 0.01%. The multivariate analyses [36] included i) principal component analysis (PCA) based on an Euclidean covariance matrix, to check for partition among the individual essential-oil components and ii) hierarchical cluster analysis with unweighted pairgroup method with arithmetic mean (UPGMA) using Euclidean distances, to establish the different groups/chemotypes. To verify the presence of a non-random distribution of chemotypes among populations, a chi-squared test of independence [37] was used to compare the number of individuals from each of the three chemotypes in the 14 populations. Essential-oil yield comparison among chemotypes was performed using the nonparametric KruskalWallis test, considering that there was no data normality [37]. All statistical analyses were performed with the appropriate procedures of the software package STATISTICA 5.1 [38].
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