Chemical Constituents Of Inula Cappa Flowers

Inula cappa is a subshrub of the genus Inula. The rhizome is very thick and multi-branched. The stems are erect and about 70～200 cm high, which have white or beige and silk-like or cotton-like dense villus on the surface. It is distributed in the semitropics, tropical low mountain and moist or dry hills, wasteland or shrub in the area of semialpline, and it also often be found in the acidity earth, sandy clay and clay. The provinces such as Sichuan, Yunnan, Guizhou, Guangxi, Guangdong, Jiangxi and Fujian in our country are the major place of production of Inula cappa. This species can also grow in the Vietnam, Myanmar, Thailand, Malaysia and India and so on. Its roots and whole plants were used as medicine with pharmacological effect such as antitussive, expelling phlegm, promoting blood circulation to restore menstrual flow, wound and so on [1]. It is usually used as a folk medicine with pharmacological effect such as detumescence, anti-inflammation by Zhuang minority in the districs of wenshan and xichou in the Yunnan province [2]. Inula cappa, named"Na Han"in Dai nationality, was one of the primary ingredients of the ethnic medicine formula"Ya Jiao Ha Dun San", which was efficiently used for the treatment of rheum, laryngotracheitis, irregular menstrual periods, abdominal pain and so on. So far the chemical constituents of sesquiterpenoid, triterpenoids, steroids, anthraquinones, flavonoids, balmy compounds, amides, organi acids have been obtained from the roots of Inula cappa. Among of them, the triterpenoids and steroids were the most predominant constituents. Only one sesquiterpenoid was found. These informations indicated that chemical constituents of Inula cappa are not investigated sufficicently. We systemically investigated the chemical composition of the flowers of I. cappa, and established the structures of the isolated pure compounds through spectroscopic methods. The antitumor activities of some compounds were also evoluated. Objective: In order to develop the natural resources, enrich the diversities of Traditional Chinese Medicine and find out new compounds from the flower of Inula cappa, we use the technologies of Silica gel column chromatography, sephadex gel column chromatography, preparative TLC, HPLC and recrystallization to systemically isolate and purify the compounds of this plant. And use the spectroscopic methods including UV, IR, MS, 1H-NMR, 13C-NMR, 1H-1H-COSY, gHSQC, gHMBC and NOESY to characterize the structures of the isolated compounds. Besides, activities of several compounds were also evoluated.Methods: The air-dried flowers of Inula cappa (10 Kg) were extracted through reflux with 95% alcohol for three times. After filtered, the alcohol extract was concentrated in vacuum to yield the total crud extract of 2.3 Kg. The crud extract was suspended in water and re-extracted with petroleum ether, dichloromethane and ethyl acetate in order. Three fractions were obtained: the petroleum ether fraction 225.4 g, the dichloromethane fraction 70 g, and the ethyl acetate fraction 59.6 g. The three fractions were applied to silica gel column chromatography for preliminary fractionation in turn; each fraction was monitored with TLC and combined the similar fractions together. Combined subfractions were subjected to Silica gel column chromatography, sephadex gel column chromatography, preparative TLC for further separation and purification to get pure compounds. The spectroscopic methods including 1D and 2D NMR methods were used for the structural identification of these compounds.Results: 20 compounds were isolated from the flowers of Inula cappa, and 9 compounds were identified on the basis of chemical and spectral analysis including one sesquiterpenoid, two triterpenoids, one steroid, one lignanoid, one aromatic compound and three flavonoids. They were elucidated as Friedelin (1), Lupeol acetate (2), Stigmasterol (4), Ineupatorolide B (8), Cleomiscosin C (or D) (12), Luteolin (13), 3, 4-dihydroxy-Benzoic acid (14), Apigenin and Fortuneletin (15), Luteolin 4'-methyl ether (16). Compounds 2, 8, 12, 14, 16 were isolated for the first time from Inula cappa. Conclusion: Results of our experiment indicated that the solvents and methods of extraction used in this experiment were practicable. Silica gel column chromatography, sephadex gel column chromatography and preparative TLC were employed to isolate and purify the components of the flowers of I. cappa. 20 compounds were obtained and 9 compounds were identified. Compounds 8, 15-19 showed strong activity in the test of primary bioactive screening on HeLa tumor cells. Compounds 8, 17-18 showed strong activity on HOC-21 tumor cells. Compounds 8, 16-17 showed strong activity on T-98 tumor cells. Compounds 8, 17-18 exhibited strong activity on U251SP tumor cells. Compounds 8, 15, 17-18 demonstrated strong activity on A549 tumor cells. Compounds 4, 5, 8, 15-18 showed strong activity on QG-56 tumor cells. Compounds 8, 16-19 showed significant activity on PC-6 tumor cells. Compounds 8, 15-17 demonstrated strong activities on QG-90 tumor cells and HLE tumor cells. Compound 8 exhibited significant activity on MCF-7 tumor cells. Compounds 6, 8, 15-18 showed strong activity on MM1-CB tumor cells. Compounds 8, 15-18 exhibited potential activity on HMV-1 tumor cells.