Phytochemistry 57 (2001) 733–736 www.elsevier.com/locate/phytochem Chemical variability in the essential oil of Hyptis suaveolens Neucı́rio R. Azevedoa, Irani F.P. Camposb, Heleno D. Ferreirab, Tomás A. Portesb, Suzana C. Santosa, José C. Seraphinc, José R. Paulad, Pedro H. Ferria,* a Laboratório de Bioatividade Molecular, Instituto de Quı´mica, Universidade Federal de Goiás, C.P. 131, 74001-970 Goiânia, GO, Brazil b Departamento de Biologia Geral-ICB, Universidade Federal de Goiás, C.P. 131, 74001-970 Goiânia, GO, Brazil c Núcleo de Estatı´stica Aplicada, Instituto de Matemática e Estatı´stica, Universidade Federal de Goiás, C.P. 131, 74001-970 Goiânia, GO, Brazil d Laboratório de Farmacognosia, Faculdade de Farmácia, Universidade Federal de Goiás, C.P. 131, 74605-050 Goiânia, GO, Brazil Received 2 October 2000; received in revised form 29 December 2000 Abstract The essential oils of Hyptis suaveolens plants collected from 11 localities of the Brazilian Cerrado region were investigated by GC–MS. Sabinene, limonene, biclyclogermacrene, b-phellandrene and 1,8-cineole were the principal constituents. The results were submitted to principal component and chemometric cluster analysis which allowed three groups of essential oils to be distinguished with respect to the content of p-mentha-2,4(8)-diene, limonene/b-phellandrene/g-terpinene and germacrene D/bicyclogermacrene. In patterns of geographic variation in essential oil composition indicated that the sesquiterpenes are mainly produced in the samples grown at lower latitudes. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Hyptis suaveolens; Lamiaceae; Essential oils; Chemical variability; Multivariate analysis 1. Introduction Hyptis Jacq., with well over 300 species, exhibits a major morphological diversity in the Brazilian Cerrado region (Harley, 1988). Its species are quite aromatic and are frequently used for the treatment of gastrointestinal infections, cramps, and pain, as well as in the treatment of skin infections (Corrêa, 1931). The aggressive annual weedy species, Hyptis suaveolens (L.) Poit. is distributed in the tropics and subtropics, and it is not commonly found over 500 m. The plant is normally restricted to places where soils have been profoundly disturbed, and may be considered as a ruderal species (Wulff, 1973). Some work dealing with the composition and antifungal (Pandey et al., 1982; Singh et al., 1992; Zollo-Amvam et al., 1998), antibacterial (Iwu et al., 1990; Asekun et al., 1999), and anticonvulsant (Akah and Nwambie, 1993) activities of H. suaveolens leaf oil have been previously reported: these plants were obtained from various origins (Brazil, India, Malaysia, Nigeria, USA, Aruba). * Corresponding author. Tel.: 55-62-521-1008; fax: 55-62-521-1008. E-mail address: pedro@quimica.ufg.br (P.H. Ferri). Although relatively few samples have been analysed, it appears from reported data that leaf or aerial parts oils of H. suaveolens exhibited several compositions characterised by the occurrence, as major components, of bcaryophyllene (Malaysia and Nigeria), 1,8-cineole and sabinene (USA, India and Aruba), 1,8-cineole and bpinene (Brazilian Amazonian and Brazilian Northeast) (Craveiro et al., 1981; Gottlieb et al., 1981; Luz et al., 1984; Din et al., 1988; Iwu et al., 1990; Mallavarapu et al., 1993; Ahmed et al., 1994; Singh and Upadhyay, 1994; Asekun and Ekundayo, 2000). As part of our ongoing work on the characterisation of essential oils of aromatic plants growing wild in the Brazilian Cerrado region (Costa et al., 2000), we now report on the results obtained for the essential oil composition and variability of H. suaveolens. For that purpose, 11 different sampling sites (Fig. 1) were chosen, and the qualitative and quantitative analysis of the volatile oil of a representative population sample of each was performed by GC–MS. To study the chemical variability, the chemical constituents from the essential oil of samples from each locality were submitted to principal component and chemometric cluster analysis, in order to detect dome pattern distribution of samples and to identify which 0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00128-5 734 N.R. Azevedo et al. / Phytochemistry 57 (2001) 733–736 constituents can differentiate between the groups of individuals. 2. Results and discussion The essential oil of H. suaveolens plants collected from the eleven different sampling sites of Brazilian Cerrado (Fig. 1) gave an average yield of 0.7% (wt/wt). In total, 40 compounds were identified, accounting for 91– 97% of the volatile constituents. Monoterpene hydrocarbons were the main group of constituents in most of the populations (36.89–73.40%). Nevertheless, important differences in the amounts of the major constituents were found, mainly of sabinene (2.93–31.13%) which had the highest percentage in population D, p-mentha-2,4(8)diene (0.19–20.87%) as the principal constituent of populations E and G, b-phellandrene (0.92–18.17%), limonene (3.60–17.56%), g-terpinene (0.52–17.33%) as the major components of population C. Populations A Fig. 1. Map of Brazilian Cerrado region showing the locations where Hyptis suaveolens plants were collected and to which cluster it belongs: I (*), II (+), and III (*). Goiás State (GO): (A) Caldas Novas (17
410 2600 S/48
420 2300 W), at an altitude of 763 m, 13 January 2000; (B) Piracanjuba (17
130 5600 S/49
40 4000 W), 734 m, 13 January 2000; (E) Itumbiara (18
220 4800 S/49
130 800 W), 448 m, 29 January 2000; (F) Itumbiara (18
220 3000 S/49
130 1200 W), 448 m, 29 January 2000; (G) Goiatuba (18
10 2100 S/49
210 3900 W), 774 m, 14 January 2000; (H) Goiânia (16
340 2400 S/49
170 3200 W), 760 m, 15 January 2000; (I) Uruaçu (14
300 3200 S/49
70 2200 W), 524 m, 12 March 2000; (J) Uruaçu (14
300 2800 S/49
70 1900 W), 524 m, 12 March 2000; (K) Uruaçu (14
300 3500 S/49
70 2700 W), 523 m, 12 March 2000. Minas Gerais State (MG): (C) Frutal (20
10 3300 S/48
560 1700 W), 549 m, 29 January 2000; (D) Frutal (20
10 2700 S/48
560 1400 W), 549 m, 29 January 2000. and B had the highest percentage of 1,8-cineole (0– 15.08%), and populations H, J and K had bicyclogermacrene (2.38–12.68%) as the main constituent. The results showed some important differences between the populations, indicating the existence of chemical polymorphism. Results obtained from nearest neighbour complete linkage cluster analysis using Ward’s technique (Ward, 1963) and principal component analysis (PCA) showed the existence of a highly chemical variability within the essential oil H. suaveolens (Fig. 2). First principal component separates monoterpene hydrocarbons (mean content 71.01%, S.D.=2.39; populations C and D) from sesquiterpene samples (20.70%, S.D.= 11.40; populations B, H and I–K), and the second principal component distinguishes the populations A, E, F and G for the content of p-mentha-2,4(8)-diene. In Fig. 2, the strong negative correlation between sesquiterpenes and monoterpene hydrocarbons is clear. Additionally, there is a correlation between essential oil composition and geographic variation (Table 1). Sesquiterpenes were mainly produced at lower latitudes, whereas monoterpenes at higher ones. Therefore, three main types of essential oils were found: Cluster I was characterised by a high percentage of p-mentha-2,4(8)diene (11.73%, S.D. 8.4); Cluster II had limonene (15.19%, S.D.=3.35), b-phellandrene (16.48%, S.D.= 2.39) and g-terpinene (10.65%, S.D.=9.45) as the major compounds; and Cluster III showed substantial percentages of germacrene D (6.54%, S.D.=2.43) and bicyclogermacrene (11.49%, S.D.=1.22). The chemical composition of the oils from USA (Ahmed et al., 1994), India (Mallavarapu et al., 1993), Aruba (Fun and Svendsen, 1990) and Brazilian Amazonian (Gottlieb et al., 1981) are closed to the composition determined for Fig. 2. Principal component analysis (PCA) ordination of Hyptis suaveolens plants collected from eleven populations (A-K) of Brazilian Cerrado. aAxes refer to the ordination scores obtained from the samples. bAxes refer to the ordination scores obtained for the discriminant oil components, which are represented as vectors from origin. Axis Ia accounts for 38.5% of the total variance and Axes IIa accounts for a further 17.6% of the total variance. N.R. Azevedo et al. / Phytochemistry 57 (2001) 733–736 Table 1 Correlation coefficients between essential oil components and latitude (south) of sampling sites of Hyptis suaveolensa Oil component Latitude Tricyclene Myrcene d-2-Carene cis-p-Menth-2-en-1-ol Limonene b-Phellandrene b-Boubonene b-Elemene (E)-Caryophyllene cis-Thujopsene Germacrene D Bicyclogermacrene Globulol epi-a-Muurolol d-Elemene 0.84b 0.73 0.86b 0.61 0.75b 0.90b 0.83b 0.93b 0.69 0.64 0.89b 0.84b 0.77b 0.62 0.82b a All correlations have a significant level, P < 0.05 (Student’s ttest). b P < 0.01 (Student’s t-test). sample of cluster I. Otherwise, the content of the bpinene in oil from Brazilian Amazonian is much higher than that of our samples. The chemical composition of Clusters II and III are original. The chemical variability presented in this paper corresponds well with the results from both polymorphism in seed germination and differential rate of growth in plantlets of Hyptis suaveolens (Wulff, 1973), which may be important factors in the colonising ability of this species. 735 ane) connected to an ion trap detector operating in EI mode at 70 eV; carrier gas: He (1 ml min1); injector and ion-source temperatures were 250
C and a split ratio of 1:5. Injection volume was 1 ml (20% in CH2Cl2) and the oven temperature was programmed from 60
C (isothermal for 2 min), with an increase of 3
C min1, to 240
C, then 10
C min1 to 280
C, ending with a 10 min isothermal at 280
C. Individual components were identified by comparing their RRt and mass spectra with those of the authentic samples, literature (Adams, 1995) and a computerized MS-data base using NIST libraries. 3.3. Chemical variability Principal component analysis (PCA), using SPAD.N software package (Lebart et al., 1994), was applied to examine the interrelationships between different populations and its chemical constituents. Cluster analysis was also applied to the study of similarity of samples on the basis of constituent distribution. Nearest neighbour complete linkage technique by Benzécri algorithm (Benzécri, 1980) was used as an index of similarity and hierarchical clustering was performed according to the Ward́s variance minimising method (Ward, 1963). Acknowledgements We thank Dr. A.J. Marsaioli (IQ-Unicamp) for supplying the authentic samples. Thanks are also due to CNPq, PADCT III/QEQ (Grant No. 620166/97-5) and Fundação de Apoio à Pesquisa-FUNAPE/UFG for their financial support. 3. Experimental 3.1. Plant material References H. suaveolens plants were collected at the vegetative stage in January and March 2000 and identified by two of the authors (I.F.P.C. and H.D.F.); 3–5 individuals, representing the local population, were collected from the each locality (Fig. 1). Voucher specimens of each sample are deposited in the Herbarium of the Universidade Federal de Goiás, Goiás State, Brazil. The aerial parts were air-dried at room temp. for 7 days. Then, the total plant material from each population was pulverized and submitted to hydrodistillation (6 h) using a Clevenger apparatus. Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured, Illinois. Ahmed, M., Scora, R.W., Ting, I.P., 1994. Composition of leof oil of Hyptis suaveolens (L.) Poit. J. Essent. Oil Res. 6, 571–575. Akah, P.A., Nwambie, A.I., 1993. Nigerian plants with anti-convulsant property. Fitoterapia 64, 42–44. Asekun, O.T., Ekundayo, O., 2000. Essential oil constituents of Hyptis suaveolens (L.) Poit. (bush tea) leaves from Nigeria. J. Essent. Oil Res. 12, 227–230. Asekun, O.T., Ekundayo, O., Adenivi, B.A., 1999. Antimicrobial activity of the essential oil of Hyptis suaveolens leaves. Fitoterapia 70, 440–442. Benzécri, J.P., 1980. L’Analyse des Données: la Taxinomie, vol. 1. Dunod, Paris. Corrêa, M.P., 1931. Dicionário das Plantas Úteis e das Exóticas Cultivadas. Imprensa Nacional, Rio de Janeiro. Costa, R.T., Fernandes, O.F.L., Santos, S.C., Oliveira, C.M.A., Lião, L.M., Ferri, P.H., Paula, J.R., Ferreira, H.D., Sales, B.H.N., Silva, M. R. R., 2000. Antifungal activity of volative constituents of Eugenia dysenterica leaf oil. J. Ethnopharmacol. 72, 113–119. Craveiro, A.A., Fernandes, A.G., Andrade, C.H.S., Matos, F.J.A., Alencar, J.W., Machado, M.I.L., 1981. Óleos Essenciais do Nordeste. Edições UFC, Fortaleza (68). 3.2. Analysis of essential oils Oil samples analysis were performed on a GC–MS Shimadzu QP5050A instrument employing the following conditions: Column: CBP-5 (Shimadzu) fused silica capillary column (50 m long  0.25 mm i.d.  0.25 mm film thickness composed of 5% phenylmethylpolysilox- 736 N.R. Azevedo et al. / Phytochemistry 57 (2001) 733–736 Din, L.B., Zakaria, Z., Sandudin, M.W., Brophy, J., Toia, R.F., 1988. Composition of the steam volatile oil from Hyptis suaveolens. Pertanika 11, 239–247. Fun, C.E., Svendsen, A.B., 1990. The essential oil of Hypis suaveolens Poit. grown in Aruba. Flav. Frag. J. 5, 161–163. Gottlieb, O.R., Koketsu, M., Magalhães, M.T., Maia, J.G.S., Mendes, P.H., Rocha, A.I., Silva, M.L., Wilberg, V.C., 1981. Essential oils of Amazonia. VII. Acta Amazonica 11, 143–148. Harley, R.M., 1988. Revision of generic limits in Hyptis Jacq. and its allies. Bot. J. Linn. Soc. 98, 87–95. Iwu, M.M., Ezeugwu, C.O., Okunji, C.O., Sanson, D.R., Tempesta, M.S., 1990. Antimicrobial activity and terpenoids of the essential oil of Hyptis suaveolens. Int. J. Crude Drug Res. 28, 73–76. Lebart, L., Morineau, A., Lambert, T., Pleuvret, P., 1994. SAPD.N Versión 2.5. Sistema Compatible para el Análisis de Datos. Centre International de Statistique et d’Informatique Appliqueés, Saint Mandé, France. Luz, A.I.R., Zoghbi, M.G.B., Ramos, L.S., Maia, J.G.S., Da Silva, M.L., 1984. Essential oils of some Amazonian Labiatae, 1. Genus Hyptis. J. Nat. Prod. 47, 745–747. Mallavarapu, G.R., Ramesh, S., Kaul, P.N., Bhattacharya, A.K., Rao, B.R.R., 1993. The essential oil of Hyptis suaveolens (L.) Poit. J. Essent. Oil Res. 5, 321–323. Pandey, D.K., Tripathi, N.N., Tripathi, R.D., Dixit, S.N., 1982. Fungitoxic and phytotoxic properties of the essential oil of Hyptis suaveolens. J. Plant Dis. Prot. 89, 344–349. Singh, G., Upadhyay, R.K., 1994. Sesquiterpene alcohols from the essential oil of Hyptis suaveolens. Fitoterapia 65, 186–187. Singh, G., Upadhyay, R.K., Rao, G.P., 1992. Fungitoxic activity of the volatile oil of Hyptis suaveolens. Fitoterapia 63, 462–465. Ward, J.H., 1963. Hierarchical grouping to optimize an objective function. J. Am. Stat. Assoc. 58, 238–244. Wulff, R., 1973. Intrapopulational variation in the germination of seeds in Hyptis suaveolens. Ecology 54, 646–649. Zollo-Amvam, P.H., Biviti, L., Tchoumbougnang, F., Menut, C., Lamaty, G., Bouchet, P., 1998. Aromatic plants of tropical Central Africa. Part XXXII. Chemical composition and antifungal activity of thirteen essential oils from aromatic plants of Cameroon. Flav. Flag. J. 13, 107–114.