Studies On The Chemical Constituents And Antumor Activity Of Wedelia Chinensis

Wedelia chinensis is one of the genus Wedelia plant belong to Compositae, which mainly distributed in south China, including Fujian, Guangdong and Guangxi [1]. It is used for the treatment of diphtheria, chincough, diarrhea, hemorrhoids, injuries from falls, faucitis, measles as a folk herbal medicine [2]. Now it is preferably used for the treatment of hepatitis and stomatitis in clinic [3]. But a few phytochemical investigation of W. chinensis has been reported, and recent studies showed that organic acids, sesquiterpenes, triterpenoids, triterponoid saponins, flavonoids and steroids were isolated from the plants of this genus [4]. Extensive bioactivitie studies have shown that crude extract of Wedelia plants exhibited hepatoprotective effects, antipyretic-analgesic effects and antibacterial effects, and the mechanism of action of pure compounds is waiting to be investigated.Object: To develop the natural resources, search for its active components, extend the clinical effects and try to find out a new drug from Traditional Chinese Medicine. We studied the components in the air-dried aerial parts of Wedelia chinensis with Silica gel column chromatography to fractionate the crude extracts; with preparative pre-TLC and pre-HPLC to separate and purify integrates and to get compounds in pure form. Using spectroscopic methods including UV, IR, HR-FAB-MS, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC and NOESY to establish the structures of the isolated pure compounds. The pure compounds are tested for antitumor activity against several human tumor cell lines.Methods: The air-dried aerial parts of Wedelia chinensis (3.0 Kg) were pulverized and extracted with 95% alcohol for three or four times, filtered, and eliminated chlorophyll with activated carbon. The alcohol extract was concentrated to yield the total crud extract (600 g). The total crud extract was suspended in salt water (1:1) and re-extracted with petroleum ether, dichloromethane and ethyl acetate in order. Accordingly three sections were obtained: the petroleum ether section (24.5 g), the dichloromethane section (15.0 g), and the ethyl acetate section (13.0 g). Each section was applied to silica gel column chromatography for preliminary fractionation, and every fraction eluted from column chromatography was monitored with TLC, the developed TLC was detected by UV light at 254 nm and followed by color reaction with spraying 10% H2SO4-ethanol reagent and heating at 140℃. Similar fractions were pooled and subjected to silica gel column chromatography, polyamide column chromatography and sephadex LH-20 column chromatography. Combined some similar subfractions and applied to preparative TLC and/or reversed phase preparative HPLC for further purification at a flow rate of 3 mL/min. At last, pure compounds were obtained and the chemical and spectroscopic methods (UV, 1H-NMR, 13C-NMR, 1H-1H-COSY, HMQC, HMBC) were used for their identification of chemical structure.Preliminary antitumor activitys are test against several human tumor cell lines. Logarithmically proliferating cells were plated into 96-well plates (1×104 cells/well) with the medium containing the test compounds at the indicated doses, followed by being cultured for 2 days. After the culture, the activity of mitochondrial succinic dehydrogenase was measured by further incubation of the cells with 5 mg/ml MTT (Sigma) for 4 h, followed by estimation of absorbance at 570 nm with a reference wavelength of 655 nm. Cell viability was calculated by absorbance as a percentage of the survival cells.Result: Column chromatography, preparative TLC and preparative HPLC were employed to isolate and purify the components in the ethanol extract of Wedelia chinensis, and 13 compounds were isolated from above plant. Variety of spectroscopic methods was used to elucidate the structures of the compounds. Among them, the structures of four compounds were identified on the basis of chemical and spectral analysis as: compound D 24-methylene-9,19-cycloartanol; compound Fβ-sitosterol; compound H oleanolic acid; compound K Oleanolic acid-11,13(18)-dien-3-O-β-D-glucuronopyranosyl methylester. Remain 9 compounds are undergoing spectral elucidation. Antitumor activity of six pure compounds were tested, and the result showed that compounds A, I, and M were active against gynecological tumor cell lines, especially endometrial adenocarcinoma cell line (HEC-1); Compounds A and M also exhibited strong inhibitory effect against glioma cell line (T-98); Compound H showed moderate antibacterial activity against cervical cancer cell line (HeLa), Ovarian clear-cell carcinoma cell line (HAC-2) and melanoma cell line (HMV-1).Conclusion: Separation of extract of the air-dried aerial parts of Wedelia chinensis by all kinds of methods including chromatography, preparative TLC and/or reversed phase preparative HPLC yielded 13 compounds. Among them 24-methylene-9,19-cycloartanol (compound D) and oleanolic acid (compound H) are isolated from genus Wedelia for the first time. Activity screening showed that compounds A, I, and M were active against gynecological tumor cell lines, especially to HEC-1; Compounds A and M also exhibited strong inhibitory effect against T-98; Compound H showed moderate antibacterial activity against HeLa, HAC-2 and HMV-1.