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Essential oil composition of four Croton species from Madagascar and their chemotaxonomy
Biochemical Systematics and Ecology 34 (2006) 648e653 www.elsevier.com/locate/biochemsyseco
Essential oil composition of four Croton species from Madagascar and their chemotaxonomy Niko Radulovic´ a,b, Emilienne Mananjarasoa c, Liva Harinantenaina a, Asakawa Yoshinori a,* a
Tokushima Bunri University, Faculty of Pharmaceutical Sciences, Yamashiro-cho, Tokushima 770-8514, Japan b University of Nisˇ, Faculty of Sciences and Mathematics, Department of Chemistry, Visˇegradska 33, 18000 Nisˇ, Serbia and Montenegro c Centre National de recherche Environnementale, Tsimbazaza, 101 Antananarivo, Madagascar Received 6 October 2005; accepted 25 February 2006
Keywords: Croton antanosiensis Leandri; Croton decaryi Leandri; Croton geayi Leandri; Croton sakamaliensis Leandri; Euphorbiaceae; Essential oil; Chemotaxonomy
1. Subject and source Croton (Euphorbiaceae) is one of the largest genera of flowering plants, with nearly 1300 species of herbs, shrubs, and trees that are ecologically prominent and often important elements of secondary vegetation in the tropics and subtropics worldwide (Webster, 1993). Madagascar with ca. 200 species (150 endemic species) is one of the world hotspots for diversity of the genus (Radcliffe-Smith, 1988, 2001). Aerial parts of Croton antanosiensis Leandri, Croton decaryi Leandri, Croton geayi Leandri, Croton sakamaliensis Leandri (Euphorbiaceae), commonly known as andriambolafotsy, senjeno, zanapoly vavy, and zanapoly lahy, respectively, were collected at Ifotaka, Fort-Dauphin Madagascar on July 2003. The species were identified by Dr. R. Vonjy, Faculty of Sciences (Antananarivo University, Madagascar) and voucher specimens deposited in the Laboratoire de Microbiologie du Centre National de Recherche Environnementale, Tsimbazaza, Antananarivo, Madagascar. 2. Previous work To the best of our knowledge, no phytochemical studies have previously been reported on C. antanosiensis, C. decaryi, and C. sakamaliensis. From the wood of C. geayi (Palazzino et al., 1997) four 3,4-seco-diterpenes were * Corresponding author. Tel.: þ81 88 622 9611; fax: þ81 88 655 3051. E-mail address: [email protected] (A. Yoshinori). 0305-1978/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2006.02.005
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isolated, two belonging to the ent-kaur-16-ene series (geayine and 7-deoxogeayine) and two to the ent-8,13-epoxylabd-14-ene series (geayinine and isogeayinine). 3. Present study 3.1. Isolation of the essential oil The aboveground parts (leaves and stems) of the species were used for the analysis of the essential oil composition. Dried ground plant material (leaves and stems separate where possible) was subjected to hydrodistillation for 2.5 h using a Clevenger-type apparatus. The obtained essential oils were dried over anhydrous sodium sulphate and the yield calculated (w/w %): C. antanosiensis (0.25%); C. decaryi Leandri (stems: 0.19%/leaves: 0.29%); C. geayi Leandri (0.32%); C. sakamaliensis (stems: 0.15%/leaves: 0.38%). 3.2. Gas chromatography/mass spectrometry The GC/MS analysis of the oil was performed using a Hewlett-Packard 5890 series II gas chromatograph equipped with an SPB 1 capillary column (30 m 0.25 mm, 0.25 mm film thickness) directly coupled to a mass selective detector MSD 5971A of the same company which was operated in EI mode (70 eV). Helium was the carrier gas at a flow rate of 1 ml/min. The injector was operated at 250 C and the oven temperature was programmed as follows: isothermal 50 C for 3 min, then gradually increased to 250 C at 5 C/min and finally isothermal at 250 C for 15 min. 3.3. Identification procedure The linear retention indices for all of the compounds were determined by co-injection of the sample with a solution containing the homologous series of C8eC25 n-alkanes. Individual identification of components was based on comparison of their mass spectra with those of Wiley 275 MS library and those described by Adams (1995), as well as on comparison of their retention indices (Van Der Dool and Kratz, 1963) with literature values (Adams, 1995; Cavaleiro et al., 2002). The area percentage was obtained electronically from the GCeFID (that was conducted under identical conditions as GC/MS analysis) response without the use of an internal standard or correction factors. The obtained results are presented in Table 1. 4. Chemotaxonomical significance All the investigated Croton taxa contain essential oils that range from 0.15 to 0.38% based on dry weight. The highest oil content was found in the leaves of C. sakamaliensis. As it is shown in Table 1, essential oils of each species are relatively complex mixtures and contain more than 20 compounds. Contributions of the main constituents never exceed 40% of the total. Among them the sesquiterpenes make the main portion of the oils of C. geayi (45.74%) and leaf oils of C. decaryi (61.31%) and C. sakamaliensis (70.69%), while the oil of C. antanosiensis (73.07%) and stem oils of C. decaryi (74.72%) and C. sakamaliensis (96.25%) are mainly constituted of monoterpenes. Apart from the mono- and sesquiterpenes all species have rather low amounts of aliphatic compounds of nonterpenic origin, and contain no trace of phenylpropanoids. Phenylpropanoids have been reported to constitute the oil of Croton zehntneri, sect. Cascarilla (Craveiro et al., 1981), Cascarilla sarcopetalus, sect. Cyclostigma (de Heluani et al., 2000), Cyclostigma hieronymi, sect. Cascarilla (de Heluani et al., 2005), Cascarilla cuneatus, sect. Luntia subsect. Cuneati, and Cuneati malambo, sect. Tiglium (Suarez et al., 2005). It is interesting to note that phenylpropanoids are found in root or bark oils of the mentioned species. Generally, monoterpene hydrocarbons presented a much more significant fraction (Table 1) of the oils than their oxygenated counterparts except in the case of the stem oil of C. sakamaliensis, where the quantities were approximately equal, and the oil of C. geayi where no hydrocarbons were detected. Apart from the oil of C. antanosiensis, the same predominance of hydrocarbons over oxygenated derivatives stands for the sesquiterpene fraction. Among the major constituents of all of the oils were b-caryophyllene and/or a/b-pinene. Bracho and Crowley (1966) proposed that the co-occurrence of a/b-pinene might be a characteristic of the genus Croton, however, in the light of a larger number of thus far studied taxa,
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Table 1 Constituents (in %) of the essential oils of Croton antanosiensis, Croton decaryi Leandri, Croton geayi Leandri, Croton sakamaliensis from Madagascar Compounds
Nonane Tricyclene a-Thujene a-Pinene Camphene b-Pinene b-Myrcene a-Phellandrene a-Terpinene D3-Carene p-Cymene b-Phellandrene 1,8-Cineol Limonene g-Terpinene Linalool Borneol 4-Terpineol Isoborneol a-Terpineol Decanal Neral Geraniol Bornyl acetate 2-Undecanone a-Cubebene Geranyl acetate a-Copaene b-Bourbonene b-Elemene Decyl acetate Isocaryophyllene a-Gurjunene b-Caryophyllene b-Gurjunene trans-a-Bergamotene a-Humulene Valencene Germacrene D Bicyclogermacrene g-Cadinene d-Cadinene a-Elemene a-Elemol Germacrene B trans-Nerolidol trans-a-Bisabolene Caryophyllene oxide Spathulenol Humulene oxide II Viridiflorol T-Cadinol T-Muurolol b-Eudesmol
RIa
900 923 931 940 956 990 1013 1022 1033 1036 1040 1043 1046 1058 1083 1119 1162 1173 1174 1183 1191 1218 1246 1260 1302 1337 1360 1366 1375 1381 1395 1396 1402 1410 1425 1430 1441 1449 1465 1478 1493 1499 1505 1531 1534 1555 1557 1570 1571 1600 1603 1643 1645 1655
Croton antanosiensis Leandri sect. Monguia
Croton decaryi Leandri sect. Anisophyllum Leaves
Stems
Croton geayi Leandri sect. Argyrocroton
e tr tr 32.76 1.10 16.44 2.66 e e e 0.50 e e 6.06 e e tr 1.08 e 2.44 0.96 3.51 4.49 0.59 e e 1.44 e e 0.94 0.74 e e 2.02 e e 0.84 0.79 e 1.40 tr 0.76 tr tr e 5.64 e e 4.42 e 1.26 e 3.05 e
0.26 0.09 1.27 21.18 0.91 6.72 e e e 0.09 e e 0.17 0.52 tr e tr e e e e e e e 0.84 0.24 e 0.22 0.08 0.23 e e 0.15 26.65 e 1.48 19.03 e 1.20 2.12 e 2.10 e e 0.41 e e 5.11 e 2.29 e e e e
e 0.73 4.21 26.06 11.47 7.34 e e e 0.24 1.22 e 3.83 3.06 tr 0.51 13.27 e 1.85 e e e e 0.93 e e e e e e e e e 8.55 e e 3.35 e e e e e 1.60 e e e e 6.20 e e e e e 1.29
e e e e e e e e e e e e 15.69 e e e 2.59 4.50 e 14.09 e e e e e 0.65 e tr 0.25 2.26 e e tr 14.75 e 1.54 2.10 0.98 e 0.26 7.05 1.66 e 3.44 e e e 4.24 e e tr e 6.56 e
Croton sakamaliensis Leandri Leaves
Stems
e e 0.35 7.75 0.19 7.05 0.07 0.29 e e e tr 9.40 tr tr e e e e e e e e e e 0.20 e 2.44 0.32 0.44 e 0.14 0.50 28.23 1.09 1.54 4.12 2.70 2.77 1.31 2.55 2.36 e e e e 0.97 12.54 e 0.19
e e 3.04 12.05 0.48 4.00 0.60 1.35 0.01 0.35 1.22 14.69 37.95 11.62 e 8.89 e e e e e e e e e 0.22 e e e e e e e 0.95 e e tr e e tr e e e e e e e 0.51 e tr e e e e
4.18 1.89
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Table 1 (continued ) Compounds
a-Eudesmol Methyl palmitate Cembrene A Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others Total identified
RIa
1668 1909 1956
Croton antanosiensis Leandri sect. Monguia
Croton decaryi Leandri sect. Anisophyllum Stems
Croton geayi Leandri sect. Argyrocroton
Leaves
e e e 59.52 13.55 6.75 14.37 1.70 95.89
e e e 30.78 0.17 53.91 7.40 1.10 93.36
Croton sakamaliensis Leandri Leaves
Stems
e e e 54.33 20.39 13.50 7.49 0.00 95.71
e e e 0.00 36.87 31.50 14.24 0.00 82.61
e 0.21 0.80 15.70 9.40 51.68 18.80 1.01 96.59
0.03 e e 49.41 46.84 1.17 0.54 0.00 97.96
Percentages higher than 5% are bolded. a Retention index on SPB1; e, not detected; tr, trace (<0.01%).
b-caryophyllene and linalool seem to be equally frequent major constituents of many Croton species (relevant data concerning such species are summarised in Table 2). The oil of C. antanosiensis (sect. Morguia) differs from the other three oils since it contains trans-nerolidol (5.64%), spathulenol (4.42%) and T-muurolol (3.05%) instead of b-caryophyllene, a-humulene, and caryophyllene oxide as the major contributors of the sesquiterpene fraction. Similar patterns of spathulenol (6.0%) and T-muurolol (2.3%) are reported for Croton ovalifolius, sect. Micranthis, although this oil (Meccia et al., 2000) also contains significant amounts of b-caryophyllene (20.8%), a-humulene (3.5%), and caryophyllene oxide (5.2%). trans-Nerolidol was previously found to be one of the major constituents of the oil of Croton cajucara, sect. Cleodora (Lopes et al., 2000). The aerial parts of C. antanosiensis as well as the stems of C. sakamaliensis have a high concentration of limonene in the oils, 6.06% and 11.62%, respectively. Limonene (38%) was reported as a maker of the oil obtained from the flowering tops of Croton zambesicus, sect. Argyrocroton, from Saudi Arabia (Mekkawi, 1985). The relatively high content of camphene (11.47%) in the stem oil differentiates C. decaryi (sect. Anisophyllum) from the other members of the genus Croton since there are no published data on a Croton species containing this monoterpene hydrocarbon as one of the major constituents. Further more the stem oil contained borneol (13.27%) reported in the above mentioned C. zambesicus oil with 5e10.1% (Mekkawi, 1985; Fekam et al., 2002) as well as in C. hieronymi root oil with 19.0% (de Heluani et al., 2005). Section Anisophyllum includes about 10e15 species native only to Madagascar (Webster, 1993) and hence the dissimilarity with the species of other sections may be rationalized, however, C. decaryi still belongs to the characteristic caryophyllene/pinene group (Table 1) of Croton taxa (Table 2). The composition of C. geayi (sect. Argyrocroton) oil was marked by the presence of 1,8-cineol (15.69%), a-terpineol (14.09%) and the complete absence of pinenes. Other major constituents were b-caryophyllene (14.75%), g-cadinene (7.05%) and T-muurolol (6.56%). There is a similarity, in respect of 1,8-cineol (37.5%) and b-caryophyllene (23.0%) content between Croton nepetaefolius, sect. Ocalia (Craveiro et al., 1981) and C. geayi, also it can be noted that the absence of pinenes distinguishes C. geayi from C. zambesicus (Fekam et al., 2002) although they belong to the same section Argyrocroton. The leaf and stem oils of C. sakamaliensis were found to contain 1,8-cineol (9.40/37.95%), limonene (e/11.62%), b-phellandrene (e/14.69%) and linalool (e/8.89%) along with high amounts of a- and b-pinene, b-caryophyllene and caryophyllene oxide. C. sakamaliensis is not yet placed in either of the sections of the genus Croton (Webster, 1993). The composition of the oil suggests that it should be placed in Argyrocroton section of the genus Croton due to the greatest similarity (high amounts of 1,8-cineol, limonene, linalool, as well as the omnipresence of b-caryophyllene and/or a/b-pinene) between the Madagascar species C. geayi, here investigated, and the Saudi Arabian or Central African species C. zambesicus (Mekkawi, 1985; Fekam et al., 2002) which are the members of that section. From the above discussed it can be concluded that the oil compositions of Croton species vary greatly with respect to the plant organ that the oil was extracted from, show dependence of the geographical proximity (different species collected in the same region have similar composition) and are generally marked with the presence of b-caryophyllene and/or a/b-pinene as major constituents.
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Table 2 Previously investigated species of the genus Croton with b-caryophyllene, a/b-pinene, or linalool as major constituents of their essential oils Country
Sectiona
Species
Plant part
Major constituents
(%)
Reference
Brazil
Barhamia
C. adenocalyx A. DC.
leaves
Argyroglossum
C. argyrophylloides
aerial
30.9 13.4 12.6 27.4
Craveiro et al., 1990
Brazil
a-Pinene b-Pinene b-Caryophyllene a-Pinene
Central African Republic Brazil
Lamprocroton
C. aubrevillei J. Leonard
bark
Linalool b-Caryophyllene
34.6 11.9
Cleodora
C. cajucara Benth.
leaves
Venezuela
e
C. deserticolus
Brazil
Barhamia
C. essequiboensis
bark and wood aerial
Curacao
Cascarilla
C. flavens L. (Welensali)
b-Caryophyllene Linalool a-Pinene b-Pinene a-Pinene b-Caryophyllene a-Pinene
6.9 41.2 14.0 11.0 18.4 19.6 15e24
Lopes et al., 2000 Bracho and Crowley, 1966 Craveiro et al., 1981 Woerdenbag et al., 2000
Brazil
Geiseleria
C. glandulosus
b-Caryophyllene
42.3
Venezuela
Cyclostigma subsect. Cyclostigma Cascarilla
C. gossypiifolius Vahl.
bark
a-Pinene
11.0
Andrade-Neto et al., 1994 Bracho and Crowley, 1966
C. jimenezii
leaves
b-Caryophyllene
10.4e12.9
Brazil
Luntia subsect. Matourenses
C. lanjouwensis Jablonski
Leaves fruit inflorescence
Brazil
Podostachys
C. lundianus
bark aerial
a-Pinene b-Caryophyllene a-Pinene Linalool a-Pinene b-Caryophyllene
26.6 19.0 28.4 26.7 72.2 20.5
Brazil
Argyroglossum
C. micans
aerial
b-Caryophyllene
19.0
Brazil
Ocalia
C. nepetaefolius
aerial
b-Caryophyllene
23.0
Venezuela Venezuela
Micranthis e
20.8 12.0
Lasiogyne
leaves bark and wood leaves
b-Caryophyllene a-Pinene
Brazil Brazil S. Tome´ and Principe Venezuela
Lasiogyne e e
C. ovalifolius Vahl. C. populifolius Lam. C. sacaquinha Benth. C. sonderianus C. stellulifer Hutch. C. turumiquirensis Stey.
bark
b-Caryophyllene Linalool b-Caryophyllene a-Pinene Linalool a-Pinene
5.7 5.8 24.4 8.1e9.1 12.0e12.6 12.0
Venezuela
e
C. xanthochloros Croiz.
bark
a-Pinene
19.0
Cameroon
Argyrocroton
C. zambesicus Muell. Arg.
leaves/roots/ stem barks
a-Pinene b-Pinene Linaool b-Caryophyllene b-Caryophyllene Linalool a-Pinene Linalool
8.7/3.7/2.3 18.9/0.5/7.7 5.2/0.3/33.8 15.8/9.4/13.9 9.9 9.9 10 5
Costa Rica
aerial and underground parts aerial
aerial aerial
Tchad
leaves
Saudi Arabia
flowering tops
a
According to Webster (1993).
Craveiro et al., 1981 Menut et al., 1995
Ciccio and Segnini, 2002 Leao et al., 1998
Andrade-Neto et al., 1994 Craveiro et al., 1981 Craveiro et al., 1981 Meccia et al., 2000 Bracho and Crowley, 1966 Lopes et al., 2003 Craveiro et al., 1981 Martins et al., 2000 Bracho and Crowley, 1966 Bracho and Crowley, 1966 Fekam et al., 2002
Menut et al., 1995 Mekkawi, 1985
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Acknowledgements The author from Serbia and Montenegro acknowledges the Ministry of Science and Environmental Protection of the Republic of Serbia, for financial support. References Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography and Mass Spectroscopy. Allured, Carol Stream, IL. Andrade-Neto, M., Wilson de Alencar, J., Cunha, A.N., Silveira, E.R., 1994. J. Essent. Oil Res. 6, 191. Bracho, R., Crowley, K.J., 1966. Phytochemistry 5, 921. Cavaleiro, C., Selgueiro, L., Barroso, J.G., Figueiredo, A.C., Pedro, L.G., Fontihna, S.S., Bighelli, A., Casanova, J., Looman, A., Schreffer, J.J.C., 2002. Flavour Fragrance J. 17, 111. Ciccio, J.F., Segnini, M., 2002. J. Essent. Oil Res. 14, 357. Craveiro, A.A., Alencar, J.W., Matos, F.J.A., Machado, M.I.L., 1990. J. Essent. Oil Res. 2, 145. Craveiro, A.A.C., Rodrigues, A.S., Andrade, C.H.S., Matos, F.J.A., Alencar, J.W., Machado, M.I.L., 1981. J. Nat. Prod. 44, 602. Fekam, B.F., Keumedjio, F., Jazet, D.P.M., Ngadjui, B.T., Amvam Zollo, P.H., Menut, C., Bessiere, J.M., 2002. Flavour Fragrance J. 17, 215. de Heluani, C.S., Catalan, C.A.N., Hernandez, L.R., Burgueno-Tapia, E., Joseph-Nathan, P., 2000. J. Nat. Prod. 63, 222. de Heluani, C.S., de Lampasona, M.P., Vega, M.I., Catalan, C.A.N., 2005. J. Essent. Oil Res. 17, 351. Leao, I.M.S., Andrade, C.H.S., Pinheiro, M.L.B., Da Rocha, A.F.I., Machado, M.I.L., Craveiro, A.A., Alencar, J.W., Matos, F.J.A., 1998. J. Essent. Oil Res. 10, 643. Lopes, D., Bizzo, H.R., Sa Sobrinho, A.F., Pereira, M.V.G., 2000. J. Essent. Oil Res. 12, 705. Lopes, D., Bizzo, H.R., Sa Sobrinho, A.F., Pereira, M.V.G., 2003. J. Essent. Oil Res. 15, 48. Martins, A.P., Salgueiro, L.R., Goncalves, M.J., Vila, R., Tomi, F., Adzet, T., Da Cunha, A.P., Canigueral, S., Casanova, J., 2000. Planta Med. 66, 647. Meccia, G., Rojas, L.B., Rosquete, C., San Feliciano, A., 2000. Flavour Fragrance J. 15, 144. Mekkawi, A.G., 1985. Fitoterapia 56, 181. Menut, C., Lamaty, G., Bessiere, J.M., Seuleiman, A.M., Fendero, P., Maidou, E., Denamganai, J., 1995. J. Essent. Oil Res. 7, 419. Palazzino, G., Federici, E., Rasoanaivo, P., Galeffi, C., Delle Monache, F., 1997. Gazz. Chim. Ital. 127, 311. Radcliffe-Smith, A., 1988. Croton. In: Polhill, R.M. (Ed.), Flora of Tropical East Africa, Euphorbiaceae. Part 1, pp. 135e160 (London). Radcliffe-Smith, A., 2001. Genera Euphorbiacearum. Royal Botanic Gardens, Kew. Suarez, A.I., Vasquez, L.J., Manzano, M.A., Compagnone, R.S., 2005. Flavour Fragrance J. 20, 611. Van Der Dool, H., Kratz, P.D., 1963. J. Chromatogr. 11, 463. Webster, G.L., 1993. Taxon 42, 793. Woerdenbag, H.J., Bos, R., van Meeteren, H.E., Baarslag, J.J.J., de Jong-van den Berg, L.T.W., Pras, N., do Rego Kuster, G., Petronia, R.R.L., Vos, G.I., 2000. J. Essent. Oil Res. 12, 667.
Essential oil composition of four Croton species from Madagascar and their chemotaxonomy Niko Radulovic´ a,b, Emilienne Mananjarasoa c, Liva Harinantenaina a, Asakawa Yoshinori a,* a
Tokushima Bunri University, Faculty of Pharmaceutical Sciences, Yamashiro-cho, Tokushima 770-8514, Japan b University of Nisˇ, Faculty of Sciences and Mathematics, Department of Chemistry, Visˇegradska 33, 18000 Nisˇ, Serbia and Montenegro c Centre National de recherche Environnementale, Tsimbazaza, 101 Antananarivo, Madagascar Received 6 October 2005; accepted 25 February 2006
Keywords: Croton antanosiensis Leandri; Croton decaryi Leandri; Croton geayi Leandri; Croton sakamaliensis Leandri; Euphorbiaceae; Essential oil; Chemotaxonomy
1. Subject and source Croton (Euphorbiaceae) is one of the largest genera of flowering plants, with nearly 1300 species of herbs, shrubs, and trees that are ecologically prominent and often important elements of secondary vegetation in the tropics and subtropics worldwide (Webster, 1993). Madagascar with ca. 200 species (150 endemic species) is one of the world hotspots for diversity of the genus (Radcliffe-Smith, 1988, 2001). Aerial parts of Croton antanosiensis Leandri, Croton decaryi Leandri, Croton geayi Leandri, Croton sakamaliensis Leandri (Euphorbiaceae), commonly known as andriambolafotsy, senjeno, zanapoly vavy, and zanapoly lahy, respectively, were collected at Ifotaka, Fort-Dauphin Madagascar on July 2003. The species were identified by Dr. R. Vonjy, Faculty of Sciences (Antananarivo University, Madagascar) and voucher specimens deposited in the Laboratoire de Microbiologie du Centre National de Recherche Environnementale, Tsimbazaza, Antananarivo, Madagascar. 2. Previous work To the best of our knowledge, no phytochemical studies have previously been reported on C. antanosiensis, C. decaryi, and C. sakamaliensis. From the wood of C. geayi (Palazzino et al., 1997) four 3,4-seco-diterpenes were * Corresponding author. Tel.: þ81 88 622 9611; fax: þ81 88 655 3051. E-mail address: [email protected] (A. Yoshinori). 0305-1978/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2006.02.005
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isolated, two belonging to the ent-kaur-16-ene series (geayine and 7-deoxogeayine) and two to the ent-8,13-epoxylabd-14-ene series (geayinine and isogeayinine). 3. Present study 3.1. Isolation of the essential oil The aboveground parts (leaves and stems) of the species were used for the analysis of the essential oil composition. Dried ground plant material (leaves and stems separate where possible) was subjected to hydrodistillation for 2.5 h using a Clevenger-type apparatus. The obtained essential oils were dried over anhydrous sodium sulphate and the yield calculated (w/w %): C. antanosiensis (0.25%); C. decaryi Leandri (stems: 0.19%/leaves: 0.29%); C. geayi Leandri (0.32%); C. sakamaliensis (stems: 0.15%/leaves: 0.38%). 3.2. Gas chromatography/mass spectrometry The GC/MS analysis of the oil was performed using a Hewlett-Packard 5890 series II gas chromatograph equipped with an SPB 1 capillary column (30 m 0.25 mm, 0.25 mm film thickness) directly coupled to a mass selective detector MSD 5971A of the same company which was operated in EI mode (70 eV). Helium was the carrier gas at a flow rate of 1 ml/min. The injector was operated at 250 C and the oven temperature was programmed as follows: isothermal 50 C for 3 min, then gradually increased to 250 C at 5 C/min and finally isothermal at 250 C for 15 min. 3.3. Identification procedure The linear retention indices for all of the compounds were determined by co-injection of the sample with a solution containing the homologous series of C8eC25 n-alkanes. Individual identification of components was based on comparison of their mass spectra with those of Wiley 275 MS library and those described by Adams (1995), as well as on comparison of their retention indices (Van Der Dool and Kratz, 1963) with literature values (Adams, 1995; Cavaleiro et al., 2002). The area percentage was obtained electronically from the GCeFID (that was conducted under identical conditions as GC/MS analysis) response without the use of an internal standard or correction factors. The obtained results are presented in Table 1. 4. Chemotaxonomical significance All the investigated Croton taxa contain essential oils that range from 0.15 to 0.38% based on dry weight. The highest oil content was found in the leaves of C. sakamaliensis. As it is shown in Table 1, essential oils of each species are relatively complex mixtures and contain more than 20 compounds. Contributions of the main constituents never exceed 40% of the total. Among them the sesquiterpenes make the main portion of the oils of C. geayi (45.74%) and leaf oils of C. decaryi (61.31%) and C. sakamaliensis (70.69%), while the oil of C. antanosiensis (73.07%) and stem oils of C. decaryi (74.72%) and C. sakamaliensis (96.25%) are mainly constituted of monoterpenes. Apart from the mono- and sesquiterpenes all species have rather low amounts of aliphatic compounds of nonterpenic origin, and contain no trace of phenylpropanoids. Phenylpropanoids have been reported to constitute the oil of Croton zehntneri, sect. Cascarilla (Craveiro et al., 1981), Cascarilla sarcopetalus, sect. Cyclostigma (de Heluani et al., 2000), Cyclostigma hieronymi, sect. Cascarilla (de Heluani et al., 2005), Cascarilla cuneatus, sect. Luntia subsect. Cuneati, and Cuneati malambo, sect. Tiglium (Suarez et al., 2005). It is interesting to note that phenylpropanoids are found in root or bark oils of the mentioned species. Generally, monoterpene hydrocarbons presented a much more significant fraction (Table 1) of the oils than their oxygenated counterparts except in the case of the stem oil of C. sakamaliensis, where the quantities were approximately equal, and the oil of C. geayi where no hydrocarbons were detected. Apart from the oil of C. antanosiensis, the same predominance of hydrocarbons over oxygenated derivatives stands for the sesquiterpene fraction. Among the major constituents of all of the oils were b-caryophyllene and/or a/b-pinene. Bracho and Crowley (1966) proposed that the co-occurrence of a/b-pinene might be a characteristic of the genus Croton, however, in the light of a larger number of thus far studied taxa,
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Table 1 Constituents (in %) of the essential oils of Croton antanosiensis, Croton decaryi Leandri, Croton geayi Leandri, Croton sakamaliensis from Madagascar Compounds
Nonane Tricyclene a-Thujene a-Pinene Camphene b-Pinene b-Myrcene a-Phellandrene a-Terpinene D3-Carene p-Cymene b-Phellandrene 1,8-Cineol Limonene g-Terpinene Linalool Borneol 4-Terpineol Isoborneol a-Terpineol Decanal Neral Geraniol Bornyl acetate 2-Undecanone a-Cubebene Geranyl acetate a-Copaene b-Bourbonene b-Elemene Decyl acetate Isocaryophyllene a-Gurjunene b-Caryophyllene b-Gurjunene trans-a-Bergamotene a-Humulene Valencene Germacrene D Bicyclogermacrene g-Cadinene d-Cadinene a-Elemene a-Elemol Germacrene B trans-Nerolidol trans-a-Bisabolene Caryophyllene oxide Spathulenol Humulene oxide II Viridiflorol T-Cadinol T-Muurolol b-Eudesmol
RIa
900 923 931 940 956 990 1013 1022 1033 1036 1040 1043 1046 1058 1083 1119 1162 1173 1174 1183 1191 1218 1246 1260 1302 1337 1360 1366 1375 1381 1395 1396 1402 1410 1425 1430 1441 1449 1465 1478 1493 1499 1505 1531 1534 1555 1557 1570 1571 1600 1603 1643 1645 1655
Croton antanosiensis Leandri sect. Monguia
Croton decaryi Leandri sect. Anisophyllum Leaves
Stems
Croton geayi Leandri sect. Argyrocroton
e tr tr 32.76 1.10 16.44 2.66 e e e 0.50 e e 6.06 e e tr 1.08 e 2.44 0.96 3.51 4.49 0.59 e e 1.44 e e 0.94 0.74 e e 2.02 e e 0.84 0.79 e 1.40 tr 0.76 tr tr e 5.64 e e 4.42 e 1.26 e 3.05 e
0.26 0.09 1.27 21.18 0.91 6.72 e e e 0.09 e e 0.17 0.52 tr e tr e e e e e e e 0.84 0.24 e 0.22 0.08 0.23 e e 0.15 26.65 e 1.48 19.03 e 1.20 2.12 e 2.10 e e 0.41 e e 5.11 e 2.29 e e e e
e 0.73 4.21 26.06 11.47 7.34 e e e 0.24 1.22 e 3.83 3.06 tr 0.51 13.27 e 1.85 e e e e 0.93 e e e e e e e e e 8.55 e e 3.35 e e e e e 1.60 e e e e 6.20 e e e e e 1.29
e e e e e e e e e e e e 15.69 e e e 2.59 4.50 e 14.09 e e e e e 0.65 e tr 0.25 2.26 e e tr 14.75 e 1.54 2.10 0.98 e 0.26 7.05 1.66 e 3.44 e e e 4.24 e e tr e 6.56 e
Croton sakamaliensis Leandri Leaves
Stems
e e 0.35 7.75 0.19 7.05 0.07 0.29 e e e tr 9.40 tr tr e e e e e e e e e e 0.20 e 2.44 0.32 0.44 e 0.14 0.50 28.23 1.09 1.54 4.12 2.70 2.77 1.31 2.55 2.36 e e e e 0.97 12.54 e 0.19
e e 3.04 12.05 0.48 4.00 0.60 1.35 0.01 0.35 1.22 14.69 37.95 11.62 e 8.89 e e e e e e e e e 0.22 e e e e e e e 0.95 e e tr e e tr e e e e e e e 0.51 e tr e e e e
4.18 1.89
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Table 1 (continued ) Compounds
a-Eudesmol Methyl palmitate Cembrene A Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others Total identified
RIa
1668 1909 1956
Croton antanosiensis Leandri sect. Monguia
Croton decaryi Leandri sect. Anisophyllum Stems
Croton geayi Leandri sect. Argyrocroton
Leaves
e e e 59.52 13.55 6.75 14.37 1.70 95.89
e e e 30.78 0.17 53.91 7.40 1.10 93.36
Croton sakamaliensis Leandri Leaves
Stems
e e e 54.33 20.39 13.50 7.49 0.00 95.71
e e e 0.00 36.87 31.50 14.24 0.00 82.61
e 0.21 0.80 15.70 9.40 51.68 18.80 1.01 96.59
0.03 e e 49.41 46.84 1.17 0.54 0.00 97.96
Percentages higher than 5% are bolded. a Retention index on SPB1; e, not detected; tr, trace (<0.01%).
b-caryophyllene and linalool seem to be equally frequent major constituents of many Croton species (relevant data concerning such species are summarised in Table 2). The oil of C. antanosiensis (sect. Morguia) differs from the other three oils since it contains trans-nerolidol (5.64%), spathulenol (4.42%) and T-muurolol (3.05%) instead of b-caryophyllene, a-humulene, and caryophyllene oxide as the major contributors of the sesquiterpene fraction. Similar patterns of spathulenol (6.0%) and T-muurolol (2.3%) are reported for Croton ovalifolius, sect. Micranthis, although this oil (Meccia et al., 2000) also contains significant amounts of b-caryophyllene (20.8%), a-humulene (3.5%), and caryophyllene oxide (5.2%). trans-Nerolidol was previously found to be one of the major constituents of the oil of Croton cajucara, sect. Cleodora (Lopes et al., 2000). The aerial parts of C. antanosiensis as well as the stems of C. sakamaliensis have a high concentration of limonene in the oils, 6.06% and 11.62%, respectively. Limonene (38%) was reported as a maker of the oil obtained from the flowering tops of Croton zambesicus, sect. Argyrocroton, from Saudi Arabia (Mekkawi, 1985). The relatively high content of camphene (11.47%) in the stem oil differentiates C. decaryi (sect. Anisophyllum) from the other members of the genus Croton since there are no published data on a Croton species containing this monoterpene hydrocarbon as one of the major constituents. Further more the stem oil contained borneol (13.27%) reported in the above mentioned C. zambesicus oil with 5e10.1% (Mekkawi, 1985; Fekam et al., 2002) as well as in C. hieronymi root oil with 19.0% (de Heluani et al., 2005). Section Anisophyllum includes about 10e15 species native only to Madagascar (Webster, 1993) and hence the dissimilarity with the species of other sections may be rationalized, however, C. decaryi still belongs to the characteristic caryophyllene/pinene group (Table 1) of Croton taxa (Table 2). The composition of C. geayi (sect. Argyrocroton) oil was marked by the presence of 1,8-cineol (15.69%), a-terpineol (14.09%) and the complete absence of pinenes. Other major constituents were b-caryophyllene (14.75%), g-cadinene (7.05%) and T-muurolol (6.56%). There is a similarity, in respect of 1,8-cineol (37.5%) and b-caryophyllene (23.0%) content between Croton nepetaefolius, sect. Ocalia (Craveiro et al., 1981) and C. geayi, also it can be noted that the absence of pinenes distinguishes C. geayi from C. zambesicus (Fekam et al., 2002) although they belong to the same section Argyrocroton. The leaf and stem oils of C. sakamaliensis were found to contain 1,8-cineol (9.40/37.95%), limonene (e/11.62%), b-phellandrene (e/14.69%) and linalool (e/8.89%) along with high amounts of a- and b-pinene, b-caryophyllene and caryophyllene oxide. C. sakamaliensis is not yet placed in either of the sections of the genus Croton (Webster, 1993). The composition of the oil suggests that it should be placed in Argyrocroton section of the genus Croton due to the greatest similarity (high amounts of 1,8-cineol, limonene, linalool, as well as the omnipresence of b-caryophyllene and/or a/b-pinene) between the Madagascar species C. geayi, here investigated, and the Saudi Arabian or Central African species C. zambesicus (Mekkawi, 1985; Fekam et al., 2002) which are the members of that section. From the above discussed it can be concluded that the oil compositions of Croton species vary greatly with respect to the plant organ that the oil was extracted from, show dependence of the geographical proximity (different species collected in the same region have similar composition) and are generally marked with the presence of b-caryophyllene and/or a/b-pinene as major constituents.
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Table 2 Previously investigated species of the genus Croton with b-caryophyllene, a/b-pinene, or linalool as major constituents of their essential oils Country
Sectiona
Species
Plant part
Major constituents
(%)
Reference
Brazil
Barhamia
C. adenocalyx A. DC.
leaves
Argyroglossum
C. argyrophylloides
aerial
30.9 13.4 12.6 27.4
Craveiro et al., 1990
Brazil
a-Pinene b-Pinene b-Caryophyllene a-Pinene
Central African Republic Brazil
Lamprocroton
C. aubrevillei J. Leonard
bark
Linalool b-Caryophyllene
34.6 11.9
Cleodora
C. cajucara Benth.
leaves
Venezuela
e
C. deserticolus
Brazil
Barhamia
C. essequiboensis
bark and wood aerial
Curacao
Cascarilla
C. flavens L. (Welensali)
b-Caryophyllene Linalool a-Pinene b-Pinene a-Pinene b-Caryophyllene a-Pinene
6.9 41.2 14.0 11.0 18.4 19.6 15e24
Lopes et al., 2000 Bracho and Crowley, 1966 Craveiro et al., 1981 Woerdenbag et al., 2000
Brazil
Geiseleria
C. glandulosus
b-Caryophyllene
42.3
Venezuela
Cyclostigma subsect. Cyclostigma Cascarilla
C. gossypiifolius Vahl.
bark
a-Pinene
11.0
Andrade-Neto et al., 1994 Bracho and Crowley, 1966
C. jimenezii
leaves
b-Caryophyllene
10.4e12.9
Brazil
Luntia subsect. Matourenses
C. lanjouwensis Jablonski
Leaves fruit inflorescence
Brazil
Podostachys
C. lundianus
bark aerial
a-Pinene b-Caryophyllene a-Pinene Linalool a-Pinene b-Caryophyllene
26.6 19.0 28.4 26.7 72.2 20.5
Brazil
Argyroglossum
C. micans
aerial
b-Caryophyllene
19.0
Brazil
Ocalia
C. nepetaefolius
aerial
b-Caryophyllene
23.0
Venezuela Venezuela
Micranthis e
20.8 12.0
Lasiogyne
leaves bark and wood leaves
b-Caryophyllene a-Pinene
Brazil Brazil S. Tome´ and Principe Venezuela
Lasiogyne e e
C. ovalifolius Vahl. C. populifolius Lam. C. sacaquinha Benth. C. sonderianus C. stellulifer Hutch. C. turumiquirensis Stey.
bark
b-Caryophyllene Linalool b-Caryophyllene a-Pinene Linalool a-Pinene
5.7 5.8 24.4 8.1e9.1 12.0e12.6 12.0
Venezuela
e
C. xanthochloros Croiz.
bark
a-Pinene
19.0
Cameroon
Argyrocroton
C. zambesicus Muell. Arg.
leaves/roots/ stem barks
a-Pinene b-Pinene Linaool b-Caryophyllene b-Caryophyllene Linalool a-Pinene Linalool
8.7/3.7/2.3 18.9/0.5/7.7 5.2/0.3/33.8 15.8/9.4/13.9 9.9 9.9 10 5
Costa Rica
aerial and underground parts aerial
aerial aerial
Tchad
leaves
Saudi Arabia
flowering tops
a
According to Webster (1993).
Craveiro et al., 1981 Menut et al., 1995
Ciccio and Segnini, 2002 Leao et al., 1998
Andrade-Neto et al., 1994 Craveiro et al., 1981 Craveiro et al., 1981 Meccia et al., 2000 Bracho and Crowley, 1966 Lopes et al., 2003 Craveiro et al., 1981 Martins et al., 2000 Bracho and Crowley, 1966 Bracho and Crowley, 1966 Fekam et al., 2002
Menut et al., 1995 Mekkawi, 1985
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Acknowledgements The author from Serbia and Montenegro acknowledges the Ministry of Science and Environmental Protection of the Republic of Serbia, for financial support. References Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography and Mass Spectroscopy. Allured, Carol Stream, IL. Andrade-Neto, M., Wilson de Alencar, J., Cunha, A.N., Silveira, E.R., 1994. J. Essent. Oil Res. 6, 191. Bracho, R., Crowley, K.J., 1966. Phytochemistry 5, 921. Cavaleiro, C., Selgueiro, L., Barroso, J.G., Figueiredo, A.C., Pedro, L.G., Fontihna, S.S., Bighelli, A., Casanova, J., Looman, A., Schreffer, J.J.C., 2002. Flavour Fragrance J. 17, 111. Ciccio, J.F., Segnini, M., 2002. J. Essent. Oil Res. 14, 357. Craveiro, A.A., Alencar, J.W., Matos, F.J.A., Machado, M.I.L., 1990. J. Essent. Oil Res. 2, 145. Craveiro, A.A.C., Rodrigues, A.S., Andrade, C.H.S., Matos, F.J.A., Alencar, J.W., Machado, M.I.L., 1981. J. Nat. Prod. 44, 602. Fekam, B.F., Keumedjio, F., Jazet, D.P.M., Ngadjui, B.T., Amvam Zollo, P.H., Menut, C., Bessiere, J.M., 2002. Flavour Fragrance J. 17, 215. de Heluani, C.S., Catalan, C.A.N., Hernandez, L.R., Burgueno-Tapia, E., Joseph-Nathan, P., 2000. J. Nat. Prod. 63, 222. de Heluani, C.S., de Lampasona, M.P., Vega, M.I., Catalan, C.A.N., 2005. J. Essent. Oil Res. 17, 351. Leao, I.M.S., Andrade, C.H.S., Pinheiro, M.L.B., Da Rocha, A.F.I., Machado, M.I.L., Craveiro, A.A., Alencar, J.W., Matos, F.J.A., 1998. J. Essent. Oil Res. 10, 643. Lopes, D., Bizzo, H.R., Sa Sobrinho, A.F., Pereira, M.V.G., 2000. J. Essent. Oil Res. 12, 705. Lopes, D., Bizzo, H.R., Sa Sobrinho, A.F., Pereira, M.V.G., 2003. J. Essent. Oil Res. 15, 48. Martins, A.P., Salgueiro, L.R., Goncalves, M.J., Vila, R., Tomi, F., Adzet, T., Da Cunha, A.P., Canigueral, S., Casanova, J., 2000. Planta Med. 66, 647. Meccia, G., Rojas, L.B., Rosquete, C., San Feliciano, A., 2000. Flavour Fragrance J. 15, 144. Mekkawi, A.G., 1985. Fitoterapia 56, 181. Menut, C., Lamaty, G., Bessiere, J.M., Seuleiman, A.M., Fendero, P., Maidou, E., Denamganai, J., 1995. J. Essent. Oil Res. 7, 419. Palazzino, G., Federici, E., Rasoanaivo, P., Galeffi, C., Delle Monache, F., 1997. Gazz. Chim. Ital. 127, 311. Radcliffe-Smith, A., 1988. Croton. In: Polhill, R.M. (Ed.), Flora of Tropical East Africa, Euphorbiaceae. Part 1, pp. 135e160 (London). Radcliffe-Smith, A., 2001. Genera Euphorbiacearum. Royal Botanic Gardens, Kew. Suarez, A.I., Vasquez, L.J., Manzano, M.A., Compagnone, R.S., 2005. Flavour Fragrance J. 20, 611. Van Der Dool, H., Kratz, P.D., 1963. J. Chromatogr. 11, 463. Webster, G.L., 1993. Taxon 42, 793. Woerdenbag, H.J., Bos, R., van Meeteren, H.E., Baarslag, J.J.J., de Jong-van den Berg, L.T.W., Pras, N., do Rego Kuster, G., Petronia, R.R.L., Vos, G.I., 2000. J. Essent. Oil Res. 12, 667.