Chemical Constituents Of Inula Heleium Roots And Structural Modification Of Sesquiterpene Lactones

Inula helenium, knowed as"qimuxiang"or"zangmuxiang"in traditional Chinese medicine, is a perennial herb of the Inula genus compositae family, grow wild in Europe, North America and the eastern part of Asia. It is distributed in northeast, northwest, and central region of China and cultivated in the province of Hebei, Shanxi, Henan, Hubei, Sichuan and so on. Dried roots of I. helenium have been traditionally used for dyspepsia, abdominal distention, vomiting, diarrhea, dysentery in traditional Chinese medicine. Extracts or the roots have been found to exhibit activity of anti-bacteria, antiinflammatory, anti-cancer, helminthicide and decompression. The common components of I. helenium were sesquiterpenes, sesquiterpene lactones, triterpenes, glycosides, amino acids. Among sesquiterpene lactones, alantolactone and isoalantolactone are the major and important active constitutent of I. helenium. In 2005, a thymol derivative was isolated from the I. helenium roots cultures and it showed moderate antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Escherischia coli, Pseudomonas aeruginosa and Candida albicans, similar to the activities of isoalantolactone. Isocostunolide is a sesquiterpene lactone isolated from the roots of I. helenium, it was proved that could effectively induce cytotoxicity in three cancer cell lines of A2058, HT-29, and HepG2 with an IC50 of 3.2, 5.0 and 2.0μg/mL, respectively. In the anti-tumor activities of sesquiterpene lactone on the human tumor cell cultivated in vitro, isoalantolactone exhibited significant anti-growth activities to U251SP,HLE and MM1-CB and the portability tumor in mice and showed upstanding dose-dependent relationship. Lawrence et al. preceded a study onα-aminomethyl substituted lactone of alantolactone and isoalantolactone and all of the prepared compounds induced apoptosis and acted as an alkylating agents. It is interesting to find new active compounds and establish structure-activity relationship further for the research and development of I. helenium. In this experiment, we systemically investigated the chemical composition of the root of I. helenium, and established the structures of the isolated pure compounds on the basis of spectroscopic techniques including 1D and 2D NMR methods and bioactivity screening for part of monomer.Objective: In order to develop the natural resources, enrich the diversities of traditional Chinese medicine and find out new compounds from the root of I. helenium, we use the technologies of Silica gel column chromatography, polyamide column chromatography, sephadex gel column chromatography, preparative TLC, HPLC and re-crystallization to systemically isolate and purify the compounds of this plant and prepare the constituents of the sesquiterpene lactone of the plant. Use the spectroscopic methods including MS, 1H-NMR, 13C-NMR, 1H-1H-COSY, HSQC and HMBC to characterize the structures of the isolated compounds. Besides, activities of several compounds were evaluated.Methods: Lamellate dried roots of I. helenium (9.8 Kg) were extracted with 95% alcohol for three times and dipping fifteen days for one time. After filtered, the alcohol extract was concentrated in vacuum to yield the total crud extract of 3.0 Kg. The crud extract 1.5 Kg was re-extracted with petroleum ether in ultrasonic sound with perseverative agitation, concentrated and obtained the petroleum ether fraction 333.9 g. The remaining residue was suspended in saturated salt solution and re-extracted with dichloromethane, ethyl acetate and n-butanol in order. Three fractions were obtained: dichloromethane fraction 3.5 g, ethyl acetate fraction 7.8 g and n-butanol fraction 34.7g. The last fraction were applied to 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, polyamide column chromatography, Sephadex gel column chromatography, preparative TLC for further separation and purification to get pure compounds. Spectroscopic methods including 1D and 2D NMR methods were used for the structural identification of these compounds.Dissolve the sesquiterpene lactone in suitable solvent and add in the reactant fractional with the accelerant existed, heating and perseverative agitation and Check the progress with thin-layer chromatography. Retrieve solvent after the reaction finished and use the technologies of silica gel column chromatography to isolate and purify the admixture, characterize the structures of the synthetical compounds with the same methods mentioned above.Results: Eight compounds were isolated from the roots of I. helenium, and five compounds were identified on the basis of chemical and spectral analysis: two sesquiterpenes, two sterides and one anhydride. They were 4, 4-dimethylsterols (2), isoalantolactone (4), alantolactone (5),β-sitosterol (6), caffeic acid anhydride (7).Conclusion: Studies on the chemical components of the roots of I. helenium were practicable supported by our results. Application of silica gel column chromatography, polyamide column chromatography, sephadex gel column chromatography, preparative TLC in the studies and variety of spectroscopic methods were used to establish the structures of the compounds. We obtained 8 compounds in all, and the structures of 5 compounds were identified.