Cytotoxic Activity and Structural Analogues of Guaianolide Derivatives from the Flower of Chrysanthemum coronarium L.
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(6) ZKHUHDVDQGVKRZHGZHDN DFWLYLWLHVDJDLQVWKXPDQFDQFHUFHOOOLQHVVXFKDV$ 3&DQG+&7 Key words: Chrysanthemum coronarium L., sesquiterpene lactone, guaianolide, cytotoxicity.. Throughout the world, more than 250,000 species of higher plants have been used as medicines, and approximately 75% of the world’s population continues to rely on these and other traditional methods of healing.1) Plant-derived pharmaceuticals currently make up over 25% of the contemporary prescription medicines2) and, in Korea, the medicinal plants used are increasing with growing concerns on health-improving foods and medicines. Chrysanthemum coronarium L., called Crown Daisy or Garland Chrysanthemum, has been widely cultivated in Korea as a vegetable for a long time. It originated in the Mediterranean region, spreading to Europe, Africa, and Asia.3) C. coronarium L. not only has iron, potassium, calcium, and dietary fiber,4) but also is rich in carotenoids and vitamins compared to other vegetables.5,6) Gins et al.7) reported that C. coronarium L. prevented cardiac vascular diseases. The methanol extract of C. coronarium L. has been reported to have anticarcinogenic enzyme inducing activity.8) Polyacetylenic compounds have been isolated from the aerial parts of the plant,9) some of which were proved to have insect antijuvenile hormone activity.10) Furthermore, C. coronarium L. contains strong antioxitants, such as chlorogenic acid derivatives, 3,5-dicaffecylquinic acid and 4-succinyl-3,5dicaffeoylquinic acid,11) More than 15 essential oils including camphor (29.2%), α-pinene (14.8%), and β-pinene (9.5%) were also isolated from the flower of C. coronarium L.12) *HQHUDOO\SODQWPHGLFLQHVRI&RPSRVLWDHIDPLO\DUHZHOO NQRZQWRKDYHDQWLWXPRUDQWLLQIODPPDWRU\DQGDQWL *Corresponding author Phone: 055-751-5467; Fax: 055-757-0178 E-mail: [email protected]. PLFURELDO DJHQWV 6HYHQ VHVTXLWHUSHQH ODFWRQHV VXFK DV FXPDPEULQ $ DQG GLK\GURFXPDPEULQ $ GRXJODQLQH DQG UH\QRVLQ GLK\GURFKU\VDQROLGHHSLGLK\GURFKU\VDQROLGH DQG αK\GUR[\GHVR[RWDPLULQ. ZHUH LVRODWHG IURP & FRURQDULXP / DPRQJ ZKLFK FXPDPEULQ $ KDV D VWURQJ HIIHFW RQ EORRGSUHVVXUH UHGXFWLRQ ,Q VSLWH RI WKH YDULRXV ELRORJLFDODFWLYLWLHVRI&FRURQDULXP/VWXGLHVRQLWVJHQHWLF FRPSRQHQWVSDUWLFXODUO\VHTXLWHUSHQHODFWRQHVKDYHQRWEHHQ SHUIRUPHGVXIILFLHQWO\ In this study, sesquiterpene lactones were isolated from the flower of C. coronarium L., and their chemical characteristics and biological activities were determined.. Materials and Methods Materials. Flowers of C. coronarium L. were collected from a plantation at Chinju, Korea in June 1999. A voucher specimen (Park. K. H. 109) of this raw material was deposited at the herbarium of Gyeongsang National University (GNUC). Instruments. UV-VIS spectra were obtained using a Beckmann DU650 spectrophotometer. Optical rotation values were recorded using a Perkin-Elmer polarimeter. IR spectra were recorded using a Bruker IFS66 infrared Fourier transform spectrophotometer (KBr). Low-resolution EIMS data was collected by a Jeol JMS-110A spectrometer. 1H-, 13 C-, and 2D-NMR data were obtained with a Bruker AM 500 spectrometer in CDCl3 (1H-NMR at 500 MHz, 13CNMR at 125 MHz). ([WUDFWLRQDQGLVRODWLRQ 7KHGULHGIORZHUV NJ
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(20) Identification. Dihydrocumambrin A (1): Colorless crystal; mp 177.5oC [174oC]20); [α]25D = +60.3o (c 1.0, CHCl3)lit.] [α]24D + 50 (c 0.08, CHCl3); IR (KBr) νmax: 3478, 2929, 1765, 1715 and 1233 cm−1; EI-MS m/z (%): 306 (M+, 7), 290 (25), 248 (29), 230 (71), 167 (100) and 107 (48); UV (CHCl3) λmax = 230 nm; 13C-NMR (see Table 1). Cumambrin A (2): Colorless crystal; mp 178-180oC [177179oC] 20); [α]25D = +101 (c 0.68, CHCl3) [+97o (c 0.6, CHCl3)] 24); IR (KBr) νmax: 3500, 2940, 1750, 1715 and 1233 cm−1; EI-MS m/z (%): 306 (M+, 4), 246 (17), 228 (46), 165 (22), 81 (48) and 43 (100); UV (CHCl3) λmax = 235 nm; 13C-NMR (see Table 1). α$QJHOR\OR[\αK\GUR[\VORYHQROLGH
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(48) Tigeloylcumambrin B (4)27): Colorless oil; mp 130oC; [α]20D=+76.5o (c 0.36, CHCl3) +84.1 (c 0.22, MeOH); IR (KBr) νmax: 3477, 2927, 1757, 1641 and 1266 cm−1; EI-MS m/ z (%): 347 (M+, 25), 229 (tignoyl, 60), 154 (61) and 83 (100); UV (CHCl3) λmax = 229 nm; 13C-NMR (see Table 1); 1H-NMR (CDCl3, 500 MHz) δ1.24 (3H, s, H-14), 1.85 (3H, dd, J = 7.1, 1.2 Hz, H-4'), 1.89 (1H, dd, J = 16.8, 1.4 Hz, H-9a), 1.88 (3H, t, J = 1.2 Hz, H-5'), 1.92 (3H, d, J = 1.0 Hz, H-15), 2.10 (1H, m, H-2a), 2.22 (1H, m, H-2b), 2.34 (1H, dd, J = 16.8, 5.7 Hz, H-9b), 2.60 (1H, m, H-1), 2.78 (1H, dd, J = 9.6, 8.3 Hz, H-5), 3.96 (1H, m, H-7), 4.02 (1H, dd, J = 9.6, 9.0 Hz, H-6), 5.20 (1H, m, H-8), 5.47 (1H, d, J = 3.1 Hz, H-13b), 5.51 (1H, m, H3), 6.15 (1H, d, J = 3.1 Hz, H-13a), 6.94 (1H, m, H-2'). Sulforhodamin B assay. Human cancer cell lines were cultivated in humidified incubators (37oC and 5% CO2). The cells were grown in an RPMI 1640 medium with glutamine 1. (300 mg l−1), 1% penicillin/streptomycin, and 10% fetal calf serum. The cells, free from mycoplasm contamination as revealed through routine tests, were seeded in 24-well plates and allowed to grow for 24 h before the treatment. Activities of compounds were monitored at various concentration levels against three kinds of human tumor cell lines: A549 (Lung cancer cell), PC-3 (Prostate adenocarcinoma), and HCT15 (Colorectal adenocarcinoma). Cytotoxicity was determined as described previously28) and calculated as the survival of treated cells over control cells × 100 [% T/C].. 1. 1. 1. Results and Discussion Four compounds (1, 2, 3, and 4) were isolated from the chloroform extracts of flowers of C. coronarium L. by the repeated silica gel column chromatography and recrystallization (Fig. 1). In all four compounds, an absorption maximum at 220-235 nm in ultraviolet absorption spectra and strong bands at 1750 and 1690 cm−1 in the infrared absorption spectra revealed the presence of two carboxyl groups. Infrared (3500 cm−1) and mass (M+-18) spectra indicated the presence of a hydroxyl group in all four compounds. Additionally, mass (M+-60 for 1, M+-60 for 2, M+-83 for 3, and M+-83 for 4) spectra indicated the presence of acetyl, acetyl, angeloyl, and tigloyl groups, respectively (Table 1). All four compounds have similar correlations in 1H-1H COSY spectra due to the identical carbon skeleton. In particular, twofold stronger peaks at δH 5.5 and 6.2 were shown in α,β-unsaturated (C-13 methylene group for 2 and 4) and saturated (methyl group for Table 1. 13C-NMR data of compounds 1, 2, 3, and 4. Position C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-1' C-2' C-3' C-4' C-5' a. Compoundsa 1. 2. 3. 4. 53.5d 33.6t 125.6d 143.4s 54.5d 80.5d 51.2t 74.3d 41.7t 73.3s 41.9d 170.1s 15.3q 31.5q 17.5q 178.1s 21.3q -. 54.3d 33.5t 125.5d 138.5s 54.4d 80.4d 46.5d 73.6d 38.9t 73.6s 143.7s 169.5s 121.4t 33.5q 17.9q 170.2s 21.4q -. 53.4d 33.6t 125.6d 143.4s 54.6d 80.5d 51.4d 74.2d 41.9t 73.3s 42.0d 166.9s 15.3q 31.7q 12.1q 178.1s 126.4s 138.4d 14.5q 17.5q. 54.0d 33.6t 125.4d 138.5s 54.5d 80.3d 46.7d 73.4d 38.7t 73.8s 143.7s 166.9s 121.4t 33.5q 17.9q 169.6s 138.7d 128.4s 12.1q 14.6q. Recorded at 125 MHz in CDCl3; multiplicity by DEPT..
(49) Isolation of Guaianolide Derivatives. 31. PHWK\OHQHγODFWRQHPRLHW\ $OWKRXJKFRPSRXQGVDQG KDYHEHHQSUHYLRXVO\ LVRODWHGIURPVHYHUDOSODQWV DQGDUHILUVWLVRODWLRQV IURP WKLV SODQW VSHFLHV &RPSRXQGV DQG VKRZHG VWURQJ F\WRWR[LFDFWLYLWLHV *, µg · ml−1
(50) . References Fig. 1. Structures of compounds 1, 2, 3, and 4. Table 2. In vitro cytotoxicity of compounds 1, 2, 3, and 4 and adriamycin toward human cancer cell lines. Compounds 1 2 3 4 Adriamycin. Cell line GI50 (µgÁml−1)d A549a >30 ± 0.6 5.3 ± 0.5 >30 ± 0.8 5.2 ± 0.5 0.3 ± 0.1. PC-3b >30 ± 0.4 3.8 ± 0.7 >30 ± 0.5 3.8 ± 0.4 0.4 ± 0.1. HCT-15c >30 ± 0.5 3.2 ± 0.3 >30 ± 0.7 1.6 ± 0.1 0.7 ± 0.1. a. A549, Lung cancer cell; bPC-3, Prostate adenocarcinoma; HCT15, Colorectal adenocarcinoma; dGI50, 50% inhibition.. c. 1 and 3) lactone groups. Furthermore, compounds 3 and 4 have angeloyl and tigloyl groups, respectively, instead of EtOAc (1) at H-8 position. In the case of 4, H-2' (δH 6.94) observed H-4' (δH 1.88) and H-5' (δH 1.85) in allylic position and cross peak, when compare coupling constant each other with 1H-1H COSY spectrum, 4 could know that 3 are nearly the same of chemical shift value and the same skeleton. All of compounds were identified as guaianolide derivatives in sesquiterpene lactones. These sesquiterpene lactones are formed to the condensation of three isoprene molecules from mevalonate, and have the α-methylene-γ-lactone group with the characteristic functionality of the majority.30-33) These functional groups represent reactive receptor sites for biological nucleophiles, in particular, thiol and amino groups. Therefore, among the naturally occurring plant products the sesquiterpene lactones represent one of the largest groups with demonstrated cytotoxic13-15) and anti-microbial activity17-19). 7KHVH IRXU VHVTXLWHUSHQH ODFWRQHV DQG
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(58) KDYLQJ VDWXUDWHG ODFWRQH JURXS ZHUH QRW VHQVLWLYH WR KXPDQ FDQFHU FHOO OLQHV /HH HW DO UHSRUWHG WKDW RQH RI WKH VWUXFWXUDO UHTXLUHPHQWVIRUF\WRWR[LFDVZHOODVDQWLPLFURELDODFWLYLWLHV RI WKHVH VHVTXLWHUSHQH ODFWRQHV LV WKH SUHVHQFH RI WKH α. 1. Duke, J. (1990) Promising phytomedicinals. In Advances in New Crops, Janick, J. and Simon, J. (eds.), pp. 491-498, Timber Press, Portland, OR. 2. Kutchan, T. (1995) Alkaloid biosynthesis-The basis for metabolic engineering of medicinal plants. The Plant Cell 7, 1059-1070. 3. Larkcom, J. (1991) Chrysanthemum greens. In Oriental Vegetables, pp. 76-78, John Murray Ltd., London. 4. Wills, R. B. H., Wong, A. W. K., Scriven, F. M. and Greenfield, H. (1984) Nutrient composition of Chinese vegetables. J. Agric. Food Chem. 32, 413-416. 5. Jung, S. H. (1984) Sterol compositions in Chrysanthemum coronarium. MS thesis, Korea University, Seoul, Korea. 6. Yang, S. B. (1988) The effect of fertilization level, sowing distance and sowing date on the growth and quality of Chrysanthemum coronarium L. MS thesis, Korea University, Seoul, Korea. 7. Gins, V. K., Kolesnikov, M. P., Kononkov, P. F., Trishin, M. E. and Gins, M. S. (2000) Oxyanthraquinones and flavonoids from garland chrysanthemum. Prikl Biokhim Mikrobiol 36, 344-353. 8. Kim, S., Ryu, S., Choi, H., Kim, S., Kim, J. and Kim, J. (1998) Screening for Korean vegetables with anticarcinogenic enzyme inducing activity using cell culture system. J. Food Sci. Nutr. 3, 277-281. 9. Sanz, J. F., Falco, E. and Marco, J. A. (1990) New acetylenes from Chrysanthemum coronarium L. Liebigs Ann. Chem. 303-305. 10. Bowers, W. S. and Aregullin, M. (1987) Discovery and identification of an antijuvenile hormone from Chrysanthemum coronarium. In Mem. Inst. Oswaldo Cruz 82 (Suppl. III) (Intern. Symp. on Insects), pp. 51-54, Rio de Janeiro. 11. Chuda, Y., Ono, H., O-Kameyama, M., Nagata, T. and Tsushida, T. (1996) Structural identification of two antioxidant quinic acid derivatives from garland (Chrysanthemum coronarium L.). J. Agric. Food Chem. 44, 2037-2039. 12. Alvarez-Castellanos, P. P., Bishop, C. D. and Pascual-Villalobos, M. J. (2001) Antifungal activity of the essential oil of flowerheads of garland chrysanthemum (Chrysanthemum coronarium) against agricultural pathogens. Phytochemistry 57, 99-102. 13. Picman, A. K. (1986) Biological activities of sesquiterpene lactones. Biochemical Systematics and Ecology 14, 255-285. 14. Cassady, J. M. and Suffness, M. (1980) Anticancer agents based on natural products models, pp. 201-269, Academic press, London. 15. Misra, R. and Pandey, R. C. (1981) Antitumor compounds of natural origin: In Chemistry and biochemistry, Aszalos,.
(59) 32. Kyung Dong Lee et al.. A., vol. 2, pp. 145-192, CRC Press, Boca Raton. 16. Wong, H. R. and Menendez, I. Y. (1999) Sesquiterpene lactones inhibit inducible nitric oxide synthase gene expression in cultured rat aortic smooth muscle cells Biochem. & Biophys. Research Communi. 262, 375-380. 17. Lee, K. H., Ibuka, T., Wu, R. Y. and Geissman, T. A. (1977) Structure-antimicrobial activity relationships among the sesquiterpene lactones and related compounds, Phytochemistry 16, 1177-1181. 18. Picman, A. K. and Towers, G. H. N. (1983) Antibacterial activity of sesquiterpene lactones. Biochem. System. Ecol. 11, 321-327. 19. Goren, N., Woerdenbag, H. J. and Bozok-Johansson, C. (1996) Cytotoxic and antibacterial activities of sesquiterpene lactoenes isolated from Tanacetum praeteritum. Planta Med. 62, 419-422. 20. EI-Masry, S., Abou-Dania, A. H. A., Darwish, F. A., AbouKarum, M. A., Grenz, M. and Bohlmann, F. (1984) Sesquiterpene lactones from Chrysanthemum coronarium. Phytochemistry 23, 2953-2954. 21. Lee, K. D., Ha, T. J., Park, K. H. and Yang, M. S. (2001) Isolation of eudesmanolides derivatives from the flower of Chrysanthemun coronarium L. Kor. J. Medicinal Crop Sci. 9, 269-274. 22. Lee, K. D., Yang, M. S., Ha, T. J., Park, K. M. and Park, K. H. (2002) Isolation and identification of dihydrochrysanolide and its 1-epimer from Chrysanthemum coronarium L. Biosci. Biotech. Biochem. 66, 862-865. 23. Hong, Y. G., Yang, M. S. and Park, Y. B. (1999) Effect of cumambrin A treatment on blood pressure in spontaneously hypertensive rats. Kor. J. Pharmacogn 30, 226-230. 24. Michael, T., Robin B. E. and Dougulas, E. A. (1985) Bitter. guaianolides from Eriocephalus punctulatus. Pytochemistry 31, 2165-2167. 25. Barrero, A. F., Herrador, M. M. and Arteaga, P. (1992) Sesquiterpene and phenylpropanoids from Seseli vayredanum. Pytochemistry 31, 203-207. 26. Barrero, A. F., Herrador, M. M. and Arteaga, P. (1994) Sesquiterpene lactones and other constituents of Seseli vayredanum. Pytochemistry 37, 1351-1358. 27. Bohlmann, G., Zdero C., King, R. M. and Robinson, H. (1980) Sesquiterpene lactones from Eremanthus species. Phytochemistry 19, 2663-2668. 28. Skehan, P., Storeng, R., Scudiero, D., Monks, A., Mcmahon, J., Vistica, D., Warren, J. T., Bokesh, H., Kenney, S. and Boyd, M. R. (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 82, 1107-1112. 29. Lee, K. H., Ibuka, T. and Wu, R.Y. (1974) Beta unsubstituted cyclopentenone, a structural requirement for antimicrobial and cytotoxic activities Chem. Pharm. Bull. 22, 22062208. 30. Chappell, J. (1995) The biochemistry and molecular biology of isoprenoid metabolim. Plant Physiol. 107, 1-6. 31. Mohr, H. and Schopfer, A. (1995) Primary and secondary metabolism. In Plant physiology, pp. 275-281, Springer Verlag, Berlin. 32. Hoffmann, H. M. R. and Rabe, J. (1985) Synthesis and biological activity of -methylene--butyrolactones. Angew. Chem. Int. Ed. Engl. 24, 94-110. 33. Charlwood, B. V. and Banthorpe, D. V. (1991) Terpenoids. In Methods in Plant Biochemistry Dey, P. M. and Harborne, J. B. (eds.), vol. 7, pp. 187-211, Academic press, London..
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