Essential oil variability in Baccharis uncinella DC and. Baccharis dracunculifolia DC growing wild in southern Brazil, Bolivia and Uruguay

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1 FLAVOUR AND FRAGRANCE JOURNAL Flavour Fragr. J. 2008; 23: Published online in Wiley InterScience ( Essential oil variability in Baccharis uncinella DC and John Wiley & Sons, Ltd. Baccharis dracunculifolia DC growing wild in southern Brazil, Bolivia and Uruguay Caren D. Frizzo, 1 Luciana Atti-Serafini, 2 Sergio Echeverrigaray Laguna, 2 Eduardo Cassel, 3 Daniel Lorenzo 4 and Eduardo Dellacassa 4 * 1 Aripê Citrus Ltda., RS 124 km 1.2, Montenegro, RS, Brazil 2 Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS, Brazil 3 Facultad de Ingeniería, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil 4 Cátedra de Farmacognosia y Productos Naturales, Facultad de Química, General Flores 2124, Montevideo, Uruguay Received 1 April 2007; Revised 13 November 2007; Accepted 6 January 2008 ABSTRACT: Essential oils of Baccharis uncinella DC and B. dracunculifolia DC collected from different wild populations growing in Bolivia, southern Brazil and Uruguay were analysed using gas chromatography and gas chromatography mass spectrometry techniques, to check for chemical variability. Cluster analysis led to the identi¼cation of two chemotypes for B. dracunculifolia, related to aromadendrane- or cadinane-derived components. As the timing of sampling of the plant material may have an in½uence on the composition of the essential oil isolated, the seasonal in½uence at the moment of harvesting was studied for two populations of each species. The results suggested that the ½owering season was optimal both species and this supported the practice of harvesting at the full bloom stage. Copyright 2008 John Wiley & Sons, Ltd. KEY WORDS: Baccharis uncinella DC; Baccharis dracunculifolia DC; essential oils; GC; GC MS; sesquiterpene alcohols; statistical analysis; chemotypes; seasonal variation Introduction Baccharis (Compositae Asteraceae) is a large (500 species) genus of plants distributed from the USA to Argentina, of which 90% are located in South America. In recent decades species of the genus have been studied, given their importance as sources of novel active components with possible applications in the pharmaceutical and chemical industries. 1 3 This interest is still ongoing, and in the present study two species of the genus (B. dracunculifolia and B. uncinella) were selected. Their essential oils were analysed by GC and GC MS, and the results submitted to statistical analysis in order to establish more precisely their intraspeci¼c chemical variability, as well as the in½uence of different factors, such as geographical distribution and harvesting season, affecting their composition. Baccharis uncinella is a shrub with spontaneous occurrence in the southern and south-eastern regions of Brazil. 4 The only previous phytochemical study reported for this species was published by Frizzo et al. 5 By contrast, * Correspondence to: E. Dellacassa, Cátedra de Farmacognosia y Productos Naturales, Facultad de Química, Universidad de la República, Avda. General Flores 2124, CP Montevideo, Uruguay. edellac@fq.edu.uy the essential oil of B. dracunculifolia, commercially known as vassoura oil, has been studied by several authors This oil has importance in the perfumery industry, mainly due to the presence of the sesquiterpene alcohol (E)-nerolidol, responsible for the herbaceous and green notes of the oil. The differences observed in the composition of this oil for populations of Brazil, Bolivia and Uruguay suggest the existence of chemotypes. 10,11,13 In this paper we evaluate the existence of possible chemotypes in B. dracunculifolia populations located in different geographic areas of Bolivia, Uruguay and southern/southeastern Brazil. The chemical variability of the essential oils, related to their geographic origin, was studied for two populations of B. uncinella and nine of B. dracunculifolia. The chemical composition of essential oils from two populations of each species was monitored for a year to establish their chemical response to seasonal variations. Materials and Methods Plant Material and Extraction of the Essential Oil Aerial parts of B. dracunculifolia and B. uncinella were collected from 11 populations (two from B. uncinella Copyright 2008 John Wiley & Sons, Ltd.

2 100 C. D. FRIZZO ET AL. Table 1. Identification of Baccharis samples studied Species Sample Population Location Features of collect regions B. dracunculifolia 1 Capão do Leão Southern Brazil Altitude 21 m Latitude S Longitude W 2 Campestre da Serra Southern Brazil Altitude 770 m Latitude S Longitude W 3 Caxias do Sul Southern Brazil Altitude 817 m Latitude S Longitude W 4 Limeira South East Brazil Altitude 588 m Latitude S Longitude W 5 Santa Rosa Southern Brazil Altitude 277 m Latitude S Longitude W 6 Las Brujas Uruguay Altitude 43 m Latitude S Longitude W 7 Cuchilla Alta Uruguay Altitude 15 m Latitude Longitude W 8 La Floresta Uruguay Altitude 12 m Latitude S Longitude W 9 Cochabamba Bolivia Altitude m Latitude S Longitude W B. uncinella 10 Campestre da Serra Southern Brazil Altitude 770 m Latitude S Longitude W 11 Caxias do Sul Southern Brazil Altitude 817 m Latitude S Longitude W and nine from B. dracunculifolia), as speci¼ed in the Table 1. To study the possible variation in the chemical compositions due to seasonal factors, ¼ve samples of fresh leaves and stems, representing two populations of each species, were collected randomly in southern Brazil (B. uncinella in Caxias do Sul and Campestre da Serra; B. dracunculifolia in Campestre da Serra and Capão do Leão). The samples were representative of the different species and of their geographic distribution, and were chosen in order to be representative of the same parameters involved in the interaction between atmospheric climate and soil (pedoclimatic parameters) and collection conditions. The extraction conditions were identical for all samples. In this way the in½uence of environmental and technical parameters on the chemical compositions of the essential oils were annulled. The plants were identi¼ed and voucher specimens were deposited at the Herbarium of the Facultad de Química in Montevideo (MVFA 26336). The plants were air-dried at room temperature (<28 C, 2 days, relative humidity(rh) <55%, ¼nal water content 35 45%). The essential oils were obtained by hydrodistillation of ca. 300 g samples, using a Clevenger system. 19 The oils were dried by passing them through a cartridge (1 ml capacity) ¼lled with anhydrous sodium sulphate (0.3 g), the weight determined for yield calculation, and then kept in sealed ½asks under N 2 atmosphere at 17 C until GC analysis. GC and GC MS analysis The GC analyses of the oils were carried out on a Hewlett-Packard 6890 gas chromatograph equipped with a ½ame ionization detector, using two capillary columns of different polarity: HP-5 and HP-Innowax cross-linked fused-silica capillary columns (both 30 m 0.32 mm i.d., 0.25 μm ¼lm thickness). GC MS analyses were conducted using a Hewlett-Packard 6890 equipped with a MSD 5973 mass selective detector and reference libraries, 20,21 using two capillary columns: HP-5MS and HP- Innowax cross-linked fused-silica capillary columns (both 30 m 0.25 mm i.d., 0.25 μm phase thickness). The analytical conditions in both the GC and GC MS analyses were as previously described. 5 Identification and Quantification The components of the oils were identi¼ed by comparison of their linear retention indices (LRIs) on the two

3 ESSENTIAL OILS OF BACCHARIS UNCINELLA AND B. DRACUNCULIFOLIA 101 columns, determined in relation to a homologous series of n-alkanes (C 9 C 26 ), by comparison with those of pure standards or data reported in literature. 20,22,23 Comparison of fragmentation patterns in the mass spectra with those stored on the GC MS and published was also performed. 20,21,24 The percentages of each component were reported as raw percentages without standardization. Repeatability of the measuring system showed variation coef¼cients under 5% for all the components reported in Table 2. Statistical Analysis The essential oils chemical variability between species, and of populations of the same species, were determined by hierarchical cluster analysis (HCA), using Euclidean distance measurements and principal components analysis (PCA). The results of integration areas obtained from GC analysis were used to create the data matrix of independent variables. 25 The matrix data (11 samples of essential oils, 18 components with average values 1.0% at least for one species) were submitted to the software SPSS, Version (1999). This matrix was extracted from Table 2 and the components were selected considering that components with low values show higher variances and have a major relative error. The seasonal variability was determined using the same statistical tools, HCA and PCA. the matrix data submitted to statistical analysis was built using average values of integration areas resulting from GC analysis (10 essential oil samples of each species from two different populations and ¼ve stages of harvesting). The components were selected between those present in concentrations 1.0%, resulting in 17 components for B. uncinella and 16 for B. dracunculifolia. Results and Discussion The yields of essential oils from B. uncinella and B. dracunculifolia were in the range % w/w. The components identi¼ed in the aerial parts of the 11 populations studied, their retention indices and their percentage composition are listed in Table 2. The constituents are arranged in order of their elution on the HP-5 column. The oils obtained from two of the populations of B. uncinella (samples 10 and 11) show similar compositions, with 68 identi¼ed components, representing % of the total composition. The monoterpene hydrocarbons were the predominant fraction ( %), followed by oxygenated sesquiterpenes ( %). Oxygenated monoterpenes constitute a minor fraction. A literature search showed that components such as β- caryophyllene, bicyclogermacrene and germacrene D are commonly identi¼ed in different species of the genus Baccharis, such as B. dracunculifolia, B. latifolia, B. salicifolia and B. crispa. 11,12,14,17,26 Samples of B. uncinella essential oils here analysed showed the same major components, relating to mono- and sesquiterpene fractions, Table 2. Qualitative and quantitative composition of volatile compounds found in Baccharis uncinella (samples 1 to 9) and B. dracunculifolia (samples 10, 11) Components a LRI a (HP-5) LRI b (Innowax) Baccharis dracunculifolia Baccharis uncinella α-thujene d b tr c 0.1 tr tr tr tr α-pinene Camphene tr tr tr tr tr tr tr Sabinene tr tr 0.5 tr β-pinene β-myrcene δ-3-carene tr tr tr tr tr α-phellandrene tr tr tr tr tr 0.1 tr tr tr tr α-terpinene tr tr tr tr p-cymene tr Limonene ,8-Cineole tr tr 0.2 tr tr (E)-β-Ocimene γ-terpinene tr 0.2 tr tr 0.1 tr cis-sabinene hydrate tr α-terpinolene tr tr 0.1 tr Linalool p-menth-2-en-1-ol tr 0.1 tr tr Terpinen-4-ol tr α-terpineol δ-elemene tr α-cubebene tr tr α-copaene tr tr

4 102 C. D. FRIZZO ET AL. Table 2. (Continued) Components a LRI a (HP-5) LRI b (Innowax) Baccharis dracunculifolia Baccharis uncinella β-bourbonene tr β-elemene tr tr α-gurjunene tr β-caryophyllene Aromadendrene tr β-humulene tr α-humulene tr tr allo-aromadendrene tr γ-gurjunene 1470 tr tr 0.3 tr tr γ-muurolene tr Germacrene-D α-amorphene* tr tr tr 0.5 tr tr α-curcumene tr tr 0.6 tr tr cis-β-guaiene tr tr Bicyclogermacrene Ledene* tr tr α-muurolene trans-β-guaiene 1504 tr tr tr Germacrene A tr 0.2 tr tr (E,E)-Farnesene γ-cadinene tr δ-cadinene trans-calamenene tr β-bisabolene tr 0.1 α-cadinene tr tr Elemol epi-longipinanol tr Ledol tr (E)-Nerolidol Spathulenol Globulol Viridi½orol Guaiol β-oplopenone ,10-di-epi-Cubenol tr epi-Cubenol γ-eudesmol T-Cadinol T-Muurolol* tr 0.3 α-muurolol α-cadinol β-bisabolol α-bisabolol epi-α-bisabolol tr 0.4 Farnesol tr tr tr 2.3 tr 0.2 Bisabolene tr tr Farnesyl acetate tr Total Grouped components Hydrocarbons Monoterpenes Sesquiterpenes Oxygenated components Monoterpenes Sesquiterpenes a The components are reported according their elution order on HP-5. b Relative proportions of the essential oil constituents were expressed as percentages obtained by peak-area normalization, all relative response factors being taken as one. Percentages were obtained on HP-5, except for compounds (*) which were obtained on Innowax. For each compound reported, the values were not signi¼cantly different between samples (p < 0.05). c tr, trace (<0.1%). d Peak identi¼cations are based on comparison of LRI values on two columns with those from pure standards or reported in the literature, and on comparison of MS with ¼le spectra.

5 ESSENTIAL OILS OF BACCHARIS UNCINELLA AND B. DRACUNCULIFOLIA 103 Table 3. Average relative percentages of essential oils from B. uncinella and B. dracunculifolia Components B. uncinella X 2 B. dracunculifolia X 9 X 11 Components B. uncinella X 2 B. dracunculifolia X 9 X 11 α-thujene δ-cadinene α-pinene (E)-Nerolidol β-pinene Spathulenol β-myrcene Globulol Limonene Viridi½orol β-caryophyllene Guaiol Germacrene D T-Cadinol Bicyclogermacrene α-cadinol γ-cadinene α-bisabolol X 2, average relative percentages for two populations of B. uncinella; X 9, average relative percentages for nine populations of B. dracunculifolia; X 11, average relative percentages for 11 populations of Baccharis; components presented average values 1.0% at least for one species and were selected from matrix data used in the statistical analysis; tr, traces (<0.1%). that had been previously found for representative samples of B. dracunculifolia from Brazil 10,13,18 and Uruguay 11 over their geographical area of distribution. Seventy components were identi¼ed in B. dracunculifolia oils (samples 1 9) from Brazil, Uruguay and Bolivia. The components identi¼ed accounted for % of the total oil compositions (Table 2). The essential oils of B. dracunculifolia from the different origins were quite different in quantitative and qualitative composition. The Brazilian oils were characterized by high contents of (E)-nerolidol, the Uruguayan oils showed a predominance of viridi½orol with absence of (E)-nerolidol, and Bolivian oils had higher contents of γ- cadinene, δ-cadinene, T-cadinol and α-cadinol. The presence and percentages of these dominant sesquiterpene compounds in the populations studied differs from the results published earlier for Bolivia 10,13,18 and Uruguay 11 for the essential oil compositions of B. dracunculifolia. To study the chemical variability of B. dracunculifolia, 11 samples of essential oils (nine samples for B. dracunculifolia and two for B. uncinella; 18 components with average values 1.0% at least for one species) were processed by cluster analysis. The matrix data are shown in Table 3, which was extracted from Table 2, considering that components with lower percentages will show higher variances and therefore higher relative errors. The 11 samples could be subdivided into four clusters, A, B, C and D, as shown in the Figure 1 dendrogram. The ¼rst group (cluster A) was formed by seven populations of B. dracunculifolia, and all came from Brazilian samples from the southern and south-eastern regions (samples 1 5) and the Uruguayan samples from the region near the Rio de La Plata (Cuchilla Alta and La Floresta) (samples 7 and 8). Cluster A was characterized by the predominance of high contents of aromadendranederived oxygenated sesquiterpenes, such as spathulenol ( %), globulol ( %) and viridi½orol (Brazilian samples, 1.1 5%; Uruguayan samples, %). In this cluster, the closer samples belonged to the Figure 1. Dendogram of 11 populations of Baccharis, obtained by hierarchical cluster analysis (HCA) with average linkage between groups (Euclidean distance measure), based on their essential oil composition. The 11 samples could be subdivided into four clusters, A, B, C and D Brazilian populations from Campestre da Serra and Santa Rosa (samples 2 and 5), from the regions of mountain ridge (770 m) and plateau (277 m), respectively. Cluster B was formed by one sample (6), which belonged to the Uruguayan population of Las Brujas, located at 40 km NW from Montevideo. The Bolivian sample (9) nearest from a high altitude region (2548 m) belonged to cluster C, which was the most remote population of B. dracunculifolia analysed. This cluster was characterized by high contents of components derived from cadinane, such as γ-cadinene, δ-cadinene and their oxygenated derivatives T-cadinol and α-cadinol. Finally, cluster D was formed by the populations of B. uncinella, the other species studied, and included Brazilian samples 10 and 11. To assess the adequacy of this classi¼cation, the clustering obtained was con¼rmed by PCA. Results are given in Figure 2, where the samples are divided into four groups, I, II, III and IV.

6 104 C. D. FRIZZO ET AL. Figure 2. Principal components analysis (PCA) of 11 populations of Baccharis, based on their essential oil composition. The samples are divided into four groups, I, II, III and IV The multivariate analysis performed on the essential oil compositions of nine populations of B. dracunculifolia reveals the existence of two chemotypes: 1. Aromadendrane-derived components, characterized by high percentages of alcohols derived from aromadendrane: spathulenol, globulol and viridi½orol. This chemotype includes Brazilian samples 1 5 (spathulenol, %; globulol, %; viridi½orol, %) and Uruguayan samples 6 8 (spathulenol, %; globulol, %; viridi½orol, %). 2. Cadinane-derived components, represented by the Bolivian sample (9), characterized by higher concentrations of components derived from cadinane (γcadinene, 5.5%; δ-cadinene, 12.1%; T-cadinol, 11.6%; α-cadinol, 6.7%). B. dracunculifolia essential oils showed clear chemical differences between populations from Brazil and Bolivia. Uruguayan samples had compositions similar to those from Brazil. The in½uence of external factors, such as altitude and latitude, was clear in the Bolivian samples with a clearly different chemical composition from those from Brazil and Uruguay. The higher concentrations of viridi½orol in Uruguayan samples and the absence of (E)-nerolidol, the main sesquiterpene alcohol reported for Brazilian samples, did not constitute factors relevant enough to result in the separation of Uruguayan samples in the statistical analysis, annulling the possibility of the existence of a third chemotype. The dendrogram showed the greatest similarity in the compositions of the essential oils from B. uncinella populations, with no signi¼cant differences as a result of geographic origin. This can be explained by the limited dispersion of this species. B. uncinella oils showed similar compositions to some B. dracunculifolia oils, but the relationship of spathulenol and (E)-nerolidol concentrations showed an inverse ratio for the two species. The chemical variability of the oils has been discussed so far as regards the geographical origin of both Baccharis species, but the compositions of essential oils from aromatic plants is also related to their developmental stage. It is well known that the essential oil composition is controlled by genetic information However, changes in the biosynthesis can be attributed to environmental factors, which may strongly in½uence the composition of an essential oil, as is the case of the timing of sampling the plant material. 29,30 In this section the parameter selected for evaluation of the seasonal variability of essential oils was the oxygenated compounds content for both species, in particularly the oxygenated sesquiterpenes, as they have commercial interest. The statistical analysis matrix data used for B. uncinella is shown in Table 4. Cluster analysis resulted in a dendogram (Figure 3) with samples grouped into two clusters, A and B. Cluster A grouped populations of similar compositions harvested in April May (sample 2) and October November (sample 5), corresponding to autumn and spring seasons, respectively, in the Southern Hemisphere, the blooming period for this species. Cluster B grouped populations collected at the end of summer (sample 1) and in winter (samples 3 and 4). This behaviour was also con¼rmed by PCA (Figure 4), where populations were divided into two groups, I and II. These results suggest that the suitable period for industrial processing B. uncinella corresponds to the ½owering season (April May, sample 2; October November, sample 5), when the plants produce lower concentrations of monoterpene hydrocarbons α-thujene, α-pinene, β-pinene and limonene, while keeping stable concentrations of valuable oxygenated sesquiterpenes. In the case of B. dracunculifolia, the HCA resulted in a dendogram (Figure 5) where the samples grouped into Figure 3. Dendogram of two populations of B. uncinella (Campestre da Serra and Caxias do Sul) for five stages of harvesting, obtained by HCA with average linkage between groups (Euclidean distance measure). Samples were grouped into two clusters, A and B

7 ESSENTIAL OILS OF BACCHARIS UNCINELLA AND B. DRACUNCULIFOLIA 105 Table 4. Average relative percentages of essential oils of B. uncinella for populations of Campestre da Serra and Caxias do Sul for 5 stages of harvesting Components* Campestre da Serra Caxias do Sul Components Campestre da Serra Caxias do Sul α-thujene Bicyclogermacrene α-pinene δ-cadinene Sabinene (E)-Nerolidol β-pinene Spathulenol β-myrcene Globulol Limonene Viridi½orol Terpinen-4-ol T-Cadinol β-caryophyllene α-cadinol Germacrene D * The components have average values 1.0% at least for one species and were selected to matrix data used in the statistical analysis. Table 5. Average relative percentages of essential oils of B. dracunculifolia for populations of Campestre da Serra and Capão do Leão for 5 stages of harvesting Components* Campestre da Serra Capão do Leão Components Campestre da Serra Capão do Leão α-pinene (E)-Nerolidol β-pinene Spathulenol β-myrcene Globulol Limonene Viridi½orol β-caryophyllene Guaiol Germacrene-D T-Cadinol Bicyclogermacrene α-cadinol δ-cadinene α-bisabolol * The components have average values 1.0% at least for one species and were selected to matrix data used in the statistical analysis. Figure 5. Dendogram of two populations of B. dracunculifolia (Campestre da Serra and Capão do Leão) for five stages of harvesting, obtained by HCA with average linkage between groups (Euclidean distance measure). The samples grouped into clusters A and B Figure 4. PCA of two populations of B. uncinella (Campestre da Serra and Caxias do Sul) for five stages of harvesting, based on their essential oil composition. Populations were divided into two groups, I and II clusters A and B. The matrix data is shown in Table 5. Cluster A was represented by samples from October November (5) and February March (1), while cluster B by samples from June July (3), August September (4) and April May (2). PCA (Figure 6) con¼rmed this clustering with the samples divided in two groups, I and II. In conclusion, we report the chemical variability of the essential oils from two species of genus Baccharis, B. dracunculifolia and B. uncinella. The in½uence of external factors such as altitude and latitude clearly affected B. dracunculifolia populations, and the chemical compositions established two chemotypes, de¼ned by aromadendrane- or cadinane-derived components. The chemical compositions of the analysed essential oils appear to show quantitative variations due to the in½uence of season of harvest. It is quite probable that a wide variability in essential oil compositions has ecological advantages, protecting plants against pathogens and/or climatic conditions. These facts indicate that the genus possesses

8 106 C. D. FRIZZO ET AL. Figure 6. PCA of two populations of B. dracunculifolia (Campestre da Serra and Capão do Leão) for five stages of harvesting, based on their essential oil composition. Samples can be divided in two groups, I and II a rich genetic diversity to biosynthesize sesquiterpenoids. Consequently, breeding and selection of cultivars from the wild species producing potentially valuable oils appears a promising research area. These ¼ndings have ecological and economic signi¼cance for utilization of the species in the pharmaceutical, cosmetic and chemical industries. Acknowledgements The authors would like to thank Professor Patrick Moyna, PhD, FRCS, for the language revision of this manuscript. Author C.D.F. is grateful to the Conselho Nacional de Desenvolvimento Cientí¼co e Tecnológico (CNPq) for ¼nancial support. References 1. Fukuda M, Ohkoshi E, Makino M et al. Chem. Pharm. Bull. 2006; 54: de Silva Filho AA, Bueno PC, Gregorio LE et al. J. Pharm. Pharmacol. 2004; 56: Boldt PE. Baccharis (Asteraceae). A Review of Its Taxonomy, Phytochemistry, Ecology, Economic Status, Natural Enemies and Potential for Its Biological Control in the United States. Texas A&M University System: College Station, TX, Barroso GM. Rodriguesia 1976; 18: Frizzo CD, Sera¼ni LA, Dellacassa E et al. Flavour Fragr. J. 2001; 16: Ribeiro dos Santos S, Mollan TRM, D Andrea Pinto AJ et al. Riv. Ital. EPPOS 1966; 98: Bauer AGLS, Siqueira NCS, Bacha CTM et al. Revist. Centro Ciencias Saude 1978; 6: Bohlmann F, Zdero C, Genz M et al. Phytochemistry 1981; 20: Motl O, Trka A. Parfum. Kosm. 1983; 64: Queiroga CL, Fukai A, Marsaioli A. J. Braz. Chem. Soc. 1990; 1: Loayza I, Collin G, Gagnon M et al. Riv. Ital. EPPOS 1993 (special edn): Loayza I, Abujder D, Aranda R et al. Phytochemistry 1995; 38: Ferracini VL, Paraiba LC, Leitao Filho HF et al. J. Essent. Oil Res. 1995; 7: Weyerstahl P, Marschall-Weyerstahl H, Christiansen C. Planta Med. 1990; 56: Weyerstahl P, Christiansen C, Marschall H. Liebigs Ann. Chem. 1992; 12: Weyerstahl P, Marschall H, Schneider K. Liebigs Ann. Chem. 1995; 2: Weyerstahl P, Christiansen C, Marschall H. Liebigs Ann. Chem. 1995; 6: Weyerstahl P, Christiansen C, Marschall H. Flavour Fragr. J. 1996; 11: Mechkovski A, Akerele CO. Quality Control Methods for Medicinal Plant Materials. WHO/PHARM/92.559/rev.1. World Health Organization: Geneva, Adams RP. Identi¼cation of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured: Carol Stream, IL, McLafferty FW, Stauffer DB. The Wiley/NBS Registry of Mass Spectral Data, 5th edn. Wiley: New York, Jennings W, Shibamoto T. Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography. Academic Press: New York, Davies NW. J. Chromatogr. A 1990; 503: Stenhagen E, Abrahamsson S, MacLafferty FW. Registry of Mass Spectral Data. Wiley: New York, Cruz CD, Regazzi JA. Modelos Biométricos Aplicados ao Melhoramento Genético, 2nd edn. Editora Universidade Federal de Viçosa: Minas Gerais, Zunino MP, Novillo-Newton M, Maestri DM et al. Flavour Fragr. J. 1997; 12: Takeda O, Miki E, Terabayashi S et al. Planta Med. 1996; 62: Slavkovska V, Jancic R, Milosavljevic S et al. J. Essent. Oil Res. 1997; 9: Egerton-Warburton LM, Ghisalberti EL, Considine JA. Biochem. Syst. Ecol. 1998; 26: Boira H, Blanquer A. Biochem. Syst. Ecol. 1998; 26: Plummer JA, Wann JM, Spadek ZE. Ann. Bot. 1999; 83: