Investigación

 

Bioactive Terpenoids from Roots and Leaves of Jatropha gaumeri

 

Roberto Can-Aké,¹ Gilda Erosa-Rejón,¹ Filogonio May-Pat,² Luis M. Peña-Rodríguez,¹ and Sergio R. Peraza-Sánchez¹*

 

¹ Grupo de Química Orgánica, Unidad de Biotecnología.

²Unidad de Recursos Naturales, Centro de Investigación Científica de Yucatán (CICY), Calle 43 No. 130, Col. Chuburná de Hidalgo, Mérida, Yucatán, México 97200. Fax: (+52-999) 981-3900. E-mail: speraza@cicy.mx

 

Recibido el 2 de octubre del 2003.
Aceptado el 20 de febrero del 2004.

 

Abstract

The methanolic extracts of roots and leaves of Jatropha gaumeri showed antimicrobial and antioxidant activity, respectively. The bioassay-guided purification of the root extract resulted in the isolation and identification of 2-epi-jatrogrossidione (1), a rhamnofolane diterpene with antimicrobial activity, and of 15-epi-4E-jatrogrossidentadione (2), a lathyrane diterpene without biological activity. Similarly, the bioassay-guided purification of the crude leaf extract allowed the identification of β-sitosterol and the triterpenes α-amyrin, β-amyrin and taraxasterol, as the metabolites responsible for the antioxidant activity. We wish to report here the isolation of the metabolites mentioned above and the complete assignment, not reported to date, of the ¹³C NMR signals for 1.

Key words: Antimicrobial activity, antioxidant activity, Jatropha gaumeri, Euphorbiaceae, Bacillus subtilis, 2-epi-jatrogrossidione, 15-epi-4E-jatrogrossidentadione, rhamnofolane, lathyrane, β-sitosterol, α-amyrin, β-amyrin, taraxasterol.

 

Resumen

Los extractos metanólicos de la raíz y hojas de Jatropha gaumeri mostraron actividad antimicrobiana y antioxidante, respectivamente. La purificación biodirigida del extracto de la raíz resultó en el aislamiento e identificación de 2-epi-jatrogrossidiona (1), un diterpeno tipo rhamnofolano con actividad antimicrobiana contra B. subtilis, y de 15-epi-4E-jatrogrossidentadiona (2), un diterpeno tipo lathyrano sin actividad en este ensayo. De manera similar, la purificación biodirigida del extracto crudo de las hojas permitió identificar al β-sitosterol y a los triterpenos α-amirina, β-amirina y taraxasterol como los responsables de la actividad antioxidante. En este trabajo se reporta el aislamiento de los productos mencionados y la asignación completa, no reportada hasta ahora, de las señales de RMN-¹³C de 1.

Palabras clave: Actividad antimicrobiana, actividad antioxidante, Jatropha gaumeri, Euphorbiaceae, Bacillus subtilis, 2-epi-jatrogrossidiona, 15-epi-4E-jatrogrossidentadiona, rhamnofolano, latirano, β-sitosterol, α-amirina, β-amirina, taraxasterol.

 

Introduction

Various medicinal properties have been attributed to plant species of the large genus Jatropha (Euphorbiaceae). For instance, the fresh latex of many plants belonging to this genus is used in folk medicine for the treatment of mouth blisters [1], pimples [2], and scabies [3]; similarly, leaf infusions are used to treat ulcers [4], infected wounds [5], and diarrhea [6]; finally, both the leaves and seeds of some Jatropha spp. are used as laxatives [7, 8]. Phytochemically, the genus Jatropha is recognized as an important source of numerous structural classes of secondary metabolites, including alkaloids [9], diterpenes [10-12], lignans [13], triterpenes [14], and cyclic peptides [15]. A number of biological activities have been detected in natural products from Jatropha spp., these include antimicrobial [16] and both antitumor and cytotoxic [17], and tumor-promoting [18] activities.

One of the most frequently used plants in Yucatecan traditional medicine is Jatropha gaumeri Greenm., a tree commonly known in Mayan language as "pomolché". The plant grows at sea level in the jungles of Guatemala and Belize, and in the states of Quintana Roo and Yucatan, in Mexico [19]. When cut, the tree exudates a milky resin which is used to alleviate skin rashes and mouth blisters, as well as to treat fever and bone fractures [1, 20].

As part of an ongoing screening project directed towards the search for biologically active metabolites from the Yucatecan flora [21], we wish to describe herein the bioassay-directed purification of the root and leaf extracts of J. gaumeri, which resulted in the isolation and identification of the antimicrobial diterpene 2-epi-jatrogrossidione (1) and 15-epi-4E-jatrogrossidentadione (2), together with β-sitosterol, α-amyrin, β-amyrin and taraxasterol.

 

Results and discussion

A bioassay-guided purification of the antimicrobial root organic crude extract of J. gaumeri, yielded 2-epi-jatrogrossidione (1), a diterpene with a rare rhamnofolane carbon skeleton previously isolated from the root bark of J. grossidentata [22]. A complete assignment of the ¹³C NMR spectrum signals for the structure of 1, not previously reported in the literature, is detailed in Experimental. 2-epi-Jatrogrossidione showed significant antimicrobial activity when tested at 25 µg against B. subtilis using the agar-overlay method [21]. The antimicrobial activity of 1 was comparable to that shown by the commercial antibiotic amikacin sulfate when tested at 250 µg.

Inactive 15-epi-4E-jatrogrossidentadione (2) was isolated during the purification of 1. This lathyrane-type diterpene was previously isolated from the roots of J. grossidentata [23].

The methanolic extract obtained from the leaves of J. gaumeri showed significant antioxidant activity when tested in both the reduction of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the inhibition of the bleaching of β-carotene assays [21]. A bioassay-guided purification of the leaf organic crude extract yielded two main fractions showing secondary metabolites with antioxidant activity; one of the fractions contained a single component which was identified as β-sitosterol by comparing its spectroscopic data with those reported in the literature [24-26] and by direct comparison on TLC with an authentic sample.

The second, less-polar, fraction showed the presence of two main components having very similar R_f values on TLC; all attempts to separate the components in their natural form proved unsuccessful. Esterification and oxidation of the two-component mixture produced the corresponding acetylated and keto derivatives, which could then be separated by means of PTLC on AgNO₃-impregnated silica gel plates [27, 28]. Hydrolysis of the acetylated derivatives yielded each of the original components as a single spot on TLC; one of the components was identified by GC/MS as an inseparable mixture of α- and β-amyirin and their identity was confirmed by both comparing their spectroscopic data with those reported in the literature [29, 30] and by direct comparison on TLC with a mixture previously obtained in our laboratory. The second component, obtained in pure form, was identified as the triterpene alcohol taraxasterol by comparing its spectroscopic data with those reported in the literature [29,30].

There are a limited number of reports in the literature on the biological activities of both sterols and triterpenes; β-sitosterol, when tested at 0.02 and 0.04%, apparently exhibits a higher antioxidant activity than that of the well known commercial antioxidants α-tocopherol or butylated-hydroxytoluene (BHT) [31]. It has also been reported that triterpenes such as α-amyrin, β-amyrin, and taraxasterol have significant antitumor and anti-inflammatory activities in mice [32] and that their capacity to inhibit lipid peroxidation might be due to their high lipophylicity [33].

 

Experimental

General

IR spectra were obtained on a Nicolet Magna Protégé 460 FT-IR instrument, using CHCl₃ (Merck, Uvasol) as solvent. ¹H (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on a Bruker Avance 400 apparatus using CDCl₃ as solvent and TMS as internal reference. GC-MS analyses were carried out in a Hewlett Packard 5890 gas chromatograph coupled to a HP-5971A mass selective detector [GC conditions: split injection of 1 µL of sample; HP-Ultra 2 column (crosslinked 5% Ph Me silicone; 25 m × 0.32 mm × 0.52 µm film thickness); flow rate: 1 mL/min; oven temperature: 280 to 300 °C; gradient: 5 °C/min; injector: 290 °C; detector: 300 °C]. Analytical TLC was performed on aluminum sheets impregnated with silica gel 60 F₂₅₄ (Merck, 0.20 mm thickness). Preparative TLC (PTLC) was performed on glass plates impregnated with silica gel 60 F₂₅₄ (Merck, 0.50 mm thickness, 20 × 20 cm). Chromatographic purifications were run using silica gel 60 (Merck, 70-230 mesh) for open column, silica gel 60 GF₂₅₄ for VLC, and silica gel 60 (Merck, 200-400 mesh) for flash column. Chromatograms were examined under UV light (365 and 254 nm) using a UV-viewing cabinet (Spectroline, model CX-20) and by spraying with 4% phosphomolybdic acid containing a trace of ceric sulfate in 5% H₂SO₄.

Plant material

Roots and leaves of J. gaumeri were collected in April 2001 in an area located 2 Km from Dzemul, Yucatan, Mexico, and identified by one of us (FMP). A voucher specimen (FMay-1883) representing this collection has been deposited in the herbarium of CICY.

Extraction and isolation

The roots (2.2 Kg) were cut and dried, first at room temperature (five days) and then in an oven at 50-60 °C (72 h); the dry material was ground and extracted three times with MeOH (12 L) at room temperature. The extracts were combined and the solvent eliminated under reduced pressure to yield 206.5 g of crude methanolic extract 1A. The extract was suspended in a mixture of H₂O/MeOH (3:2, 1 L) and partitioned successively with hexane (2:1, 3×) and ethyl acetate (2:1, 3×) to produce the corresponding low (2A, 36.6 g) and medium (2B, 26.2 g) polarity crude fractions. Testing of both the methanolic crude extract 1A and the resulting crude fractions 2A and 2B for antimicrobial activity using the agar-overlay method [21], indicated that fraction 2A had the strongest activity against Bacillus subtilis (ATCC-6633). A bioassay guided purification of fraction 2A (17.8 g) using VLC (7.0 cm height × 5.0 cm width; hexane/AcOEt, gradient elution) yielded nine fractions (3A-I). Fraction 3H (633 mg) was further purified by flash CC (4.0 cm width × 50.0 cm height; CHCl₃/acetone, 98:2) to afford fractions 4A-G. A final PTLC purification of fraction 4C (290 mg) [hexane/acetone/MeOH, 80:15:5, multiple elution (3×)] resulted in the isolation of 1 (13 mg) and 2 (6.8 mg) in pure form.

The leaves were also air and oven dried, and then manually ground to produce 479 g of plant material which was extracted with methanol (3 × 2 L) at room temperature to yield 38.9 of organic crude extract 5A. The organic crude extract was suspended in a 3:2 mixture of H₂O/MeOH (500 mL) and successively partitioned with hexane and ethyl acetate to produce the corresponding low (6A, 10.3 g) and medium (6B, 6.0 g) polarity fractions. Both the crude extract and the crude fractions showed antioxidant activity when tested in the inhibition of the bleaching of β-carotene and the reduction of the DPPH assays. The low polarity fraction was subjected to a VLC purification (10.0 cm width × 5.0 cm height; hexane/AcOEt, gradient elution) to produce 10 major fractions (7A-J). One of the fractions (7F) showed a single component on TLC and GC which was identified as β-sitosterol. Further purification of fraction 7D by flash column chromatography [5 cm diameter × 20 cm height, hexane/EtOAc/MeOH (95:4:1)] afforded 11 major fractions (8A-K).

Esterification of fraction 8F. A mixture of 8F (33 mg), acetic anhydride (1 mL) and pyridine (0.5 mL) was allowed to stir overnight at room temperature. The reaction mixture was poured over water (15 mL) and the resulting suspension was extracted with EtOAc (3×, 1:1). The organic layer was successively washed (1:1) with 5% HCl, water, 3% NaOH, and brine and then dried over anhydrous sodium sulfate. Evaporation of the solvent yielded 30 mg of crude acetylated product, which was purified by PTLC using silica gel plates impregnated with 5% AgNO₃ to give two main fractions (9A and 9B), each identified by GC/MS as a mixture of the acetylated derivatives of α- and β-amyrin (9A) and as the acetylated derivative of taraxasterol (9B).

Hydrolisis of the acetylated derivatives 9A and 9B. Each fraction (9A, 8.2 mg and 9B, 10.0 mg) was separately hydrolyzed by stirring overnight with 5 mL of 30% KOH in MeOH at room temperature. The reaction mixture was poured over water (15 mL) and the resulting suspension was extracted with EtOAc (3×, 1:1). The organic layer was washed with water and brine and dried over anhydrous sodium sulfate. Evaporation of the solvent yielded the corresponding hydrolyzed product in good yield, i.e. 8.0 mg of the α- and β-amyrin mixture and 10 mg of taraxasterol.

Oxidation of fraction 8F. 70 mg of pyridinium chlorochromate (PCC, Corey's reagent) was added to a solution of fraction 8F (20 mg) in CH₂Cl₂ (3 mL) and the mixture was allowed to stir overnight at room temperature. The reaction mixture was passed through a silica gel bed (70-230 mesh, 3 cm high) and the adsorbent washed with CH₂Cl₂ to produce 18 mg of crude oxidized product. PTLC purification of the crude product using silica gel plates impregnated with 5% AgNO₃ yielded two main fractions (10A and 10B), each identified by GC/MS as a mixture of α- and β-amyrenone (10A) and as taraxasterone (10B).

Antimicrobial assay by the agar overlay method. Bacillus subtilis (ATCC-6633) was used for testing antimicrobial activity. Tripticasein soy agar (Bioxon) was used as solid medium. Inoculum was prepared by suspending the microorganism in sterile water in an approximate concentration of 1 × 10⁸ cell/mL. Just before applying the overlay, a portion of the inoculum, with a final concentration of approximately 1 × 10⁶ cell/mL, was added to the culture medium kept in a water bath (45 °C). Samples (25 µg) were applied to TLC plates prepared in the laboratory. The TLC plates were placed in a sterile Petri dish and covered with 4.5 mL of inoculum. Once the medium had solidified, the plates were incubated for 15 h at 36 °C. After this time, TLC plates were sprayed with an aqueous solution of methylthiazolyltetrazolium chloride (MTT, 2.5 mg/mL, Fluka) and incubated for another 3 h. Biological activity was observed as clear inhibition zones against the purple background. Amikacin was used as positive control.

Inhibition of bleaching of β-carotene. A TLC plate spotted with 100 µg of sample was eluted using a mixture of CHCl₃/MeOH (95:5). After drying, the plate was sprayed with a 0.05% solution of β-carotene (Sigma) in CHCl₃ and left at room temperature for 12 h until decoloration of the background occurred. Active products remained as orange spots on a white background. Vitamin C (10 µg) was applied to the plates as positive control. All experiments were carried out in duplicate.

Reduction of DPPH. A TLC plate spotted with 100 µg of sample was eluted using a mixture of CHCl₃/MeOH (95:5). After drying, the plate was sprayed with a 0.2% solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH, Fluka) in MeOH and left at room temperature for 8 h. Active products appeared as yellow spots against a purple background. Vitamin C (10 µg) was applied to the plates as positive control. All experiments were carried out in duplicate.

2-epi-Jatrogrossidione (1): Colorless gum; R_f 0.42 [CH₂Cl₂/acetone (95:5)]; IR ν_máx (film): 3498, 2925, 2852, 1715, 1649, 1137 cm^-1; ¹H- and ¹³C-RMN: Table 1; LREIM m/z: 312 [M]⁺, 284 [M - CO]⁺, 269 [284 - CH₃]⁺, 241 [269 - CO]⁺, 69, 55.

15-epi-4E-Jatrogrossidentadione (2): IR ν_máx (film): 3399, 2918, 2851, 1703, 1650, 1455, 1369, 1135, 1107, 979 cm^-1; ¹H- and ¹³C-RMN: Table 1; LREIM m/z: 332 [M]⁺, 314 [M - H₂O]⁺, 271, 257, 215, 175, 163, 161, 124, 109, 95, 67, 55.

 

Acknowledgements

The authors wish to thank Fabiola Escalante-Erosa and Karlina García-Sosa for technical assistance.

 

References

1. Flores, J.S.; Ricalde, R.V. J. Herbs Spices Med. Plants 1996, 4, 53-59.         [ Links ]

2. Zamora-Martinez, M.C.; Pola, C.N.P. J. Ethnopharmacol. 1992, 35, 229-257.         [ Links ]

3. Manandhar, N.P. Econ. Bot. 1995, 49, 371-379.         [ Links ]

4. Gupta, M.P.; Monge, A.; Karikas, G.A.; Lopez de Cerain, A.; Solis, P.N.; De Leon, E.; Trujillo, M.; Suarez, O.; Wilson, F.; Montenegro, G.; Noriega, Y.; Santana, A.I.; Correa, M.; Sanchez, C. Int. J. Pharmacog. 1996, 34, 19-27.         [ Links ]

5. Giron, L.M.; Freire, V.; Alonzo, A.; Caceres, A. J. Ethnopharmacol. 1991, 34, 173-187.         [ Links ]

6. Coee, F.G.; Anderson, G.J. Econ. Bot. 1996, 50, 71-107.         [ Links ]

7. Barrett, B. Econ. Bot. 1994, 48, 8-20.         [ Links ]

8. Klinar, S.; Castillo, P.; Chang, A.; Schmeda-Hirschmann, G.; Reyes, S.; Theoduloz, C.; Razmilic, I. Fitoterapia 1995, 66, 341-345.         [ Links ]

9. Staubmann, R.; Schubert-Zsilavecz, M.; Hiermann, A.; Kartnig, T. Phytochemistry 1999, 50, 337-338.         [ Links ]

10. Denton, R.W.; Harding, W.W.; Anderson, C.I.; Jacobs, H.; McLean, S.; Reynolds, W.F. J. Nat. Prod. 2001, 64, 829-831.         [ Links ]

11. Brum, R.L.; Cavalheiro, A.J.; Monache, F.D.; Vencato, I. J. Braz. Chem. Soc. 2001, 12, 259-262.         [ Links ]

12. Adolf, W.; Opferkuch, H.J.; Hecker, E. Phytochemistry 1984, 23, 129-132.         [ Links ]

13. Das, B.; Anjani, G. Phytochemistry 1999, 51, 115-117.         [ Links ]

14. Tinto, W.F.; John, L.M.D.; Reynolds, W.F.; McLean, S. J. Nat. Prod. 1992, 55, 807-809.         [ Links ]

15. Baraguey, C.; Blond, A.; Correia, I.; Pousset, J.L.; Bodo, B.; Auvin-Guette, C. Tetrahedron Lett. 2000, 41, 325-329.         [ Links ]

16. Naengchomnong, W.; Thebtaranonth, Y.; Wiriyachitra, P.; Okamoto, K.T.; Clardy, J. Tetrahedron Lett. 1986, 27, 2439-2442.         [ Links ]

17. Evans, F.J.; Taylor, S.E., in: Progress in the Chemistry of Organic Natural Products, Vol. 44, Herz, W.; Grisebach, H.; Kirby, G.W., Eds., Springer-Verlag, Wein, 1983, 1-99.         [ Links ]

18. Adolf, W.; Opferkuch, H.J.; Hecker, E. Phytochemistry 1984, 23, 129-132.         [ Links ]

19. Villamar, A.A. Atlas de las Plantas de la Medicina Tradicional Mexicana, Vol. 2. Instituto Nacional Indigenista, México, 1994, 881, 1162, 1186.         [ Links ]

20. Comerford, S.C. Econ. Bot. 1996, 50, 327-336.         [ Links ]

21. Sánchez-Medina, A.; García-Sosa, K.; May-Pat, F.; Peña-Rodríguez, L.M. Phytomedicine 2001, 8, 144-151.         [ Links ]

22. Jakupovic, J.; Grenz, M.; Schmeda-Hirschmann, G. Phytochemistry 1988, 27, 2997-2998.         [ Links ]

23. Schmeda-Hirschmann, G.; Tsichritzis, F.; Jakupovic, J. Phytochemistry 1992, 31, 1731-1735.         [ Links ]

24. Adler, J.H. Phytochemistry 1983, 22, 607-608.         [ Links ]

25. Garg, V.K.; Nes, W.R. Phytochemistry 1984, 23, 2919-2923.         [ Links ]

26. Goad, L.J., in: Methods in Plant Biochemistry, Vol. 7, Dey, P.M. and Harborne, J.B., Eds., Academic Press, London, 1991, 369-434.         [ Links ]

27. Stahl, E.; Jork, H. Thin-layer Chromatography. A Laboratory Handbook. Springer-Verlag, 2^nd edition, New York, 1969, 227-229.         [ Links ]

28. Fried, B.; Sherma, J. Thin-layer Chromatography: Techniques and Applications, Vol. 17, Chapter 14. Marcel Dekker, USA, 1982, 217-220.         [ Links ]

29. Ogunkoya, L. Phytochemistry 1981, 20, 121-126.         [ Links ]

30. Conolly, J.D.; Hill, R.A., in: Methods in Plant Biochemistry, Vol. 7, Dey, P.M. and Harborne, J.B., Eds., Academic Press, London, 1991, 331-359.         [ Links ]

31. Weng, X.C.; Wang, W. Food Chem. 2000, 71, 489-493.         [ Links ]

32. Akihisa, T.; Yasukawa, K.; Oinuma, H.; Kasahara, Y.; Yamanouchi, S.; Takido, M.; Kumaki, K.; Tamura, T. Phytochemistry 1996, 43, 1255-1260.         [ Links ]

33. Anton, R.; Beretz, A.; Haag-Berrurier, M., in: Biologically Active Natural Products, Chapter 8, Hostettmann, K. and Lea, P.J., Eds., Clarendon Press, Oxford, 1987, 123-124.         [ Links ]