Chemical Constituents Of Saussurea Lappa Roots

Saussurea lappa Clarke, is a perennial herb of the Asteraceae indigenous to Kashmir. Most plants in China grow wild in the alpine zone and adjacent regions of sichuan, yunnan, guangxi, guizhou at elevations of 3200-3800 m[1-2]. Dried root of S. lappa has been traditionally used for alleviating pain in abdominal distention and tenesmus, indigestion with anorexia, dysentery, nausea, and vomiting. Different extracts of this plant have been found to exhibit interesting properities such as anti-inflammation, anti-cancer, immunomodulation, free radical scavenging and anti-fatigue. S. lappa contains sesquiterpenes, sesquiterpene lactones, lignans, sterides, glycosides and is rich in the former two. It's reported that Costunolide and Dehydrocostus lactone showed strong suppressive effect on the expression of the hepatitis B surface antigen (HBsAg) in human hepatoma Hep3B cells, suggesting the potential to be developed as specific anti-HBV drugs in the future. Cynaropicrin, Reynosin, and Santamarine producd great inhibition of TNF-alpha production in murine macrophage-like cell (RAW 264.7 cells). So it is of great interest to get new compounds from S. lappa and develop the structure-activity relationship further. In this experiment, we systemically investigated the chemical composition of the root of S. lappa, and established the structures of the isolated pure compounds on the basis of spectroscopic techniques including 1D and 2D NMR methods.Objective: In order to develop the natural resources, enrich the diversities of Traditional Chinese Medicine and find out new compounds from the root of Saussurea lappa, we use the technologies of Silica gel column chromatography, polyamide 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 and gHMBC to characterize the structures of the isolated compounds. Besides, activity of several compounds were evaluated.Methods: Pulverized dried roots of Saussurea lappa (9.8 Kg) were extracted with 95% alcohol for three times. After filtered, the alcohol extract was concentrated in vacuum to yield the total crud extract of 3.0 Kg. The crud extract was suspended in water and re-extracted with petroleum ether, dichloromethane and ethyl acetate in order. Three fractions were obtained: petroleum ether fraction 312.2 g, dichloromethane fraction 156.4 g, and ethyl acetate fraction 58.0 g. The latter two fractions 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.Results: 19 compounds were isolated from the roots of Saussurea lappa, and 11 compounds were identified on the basis of chemical and spectral analysis including four sesquiterpenes, two triterpenes, two sterides, one anthraquinone, one coumarin and one phenol. They were elucidated as Betulinic acid (1), Santamarine (2), Dehydrocostus lactone (3), Betulin (4), Costunolide (5),β-Sitosterol (6), Chrysophanol (7), Dihydrodehydrocostuslactone (9), Pregnenolone (12), Bergapten (15), Vanillin (16). Compound 1, 7, 15, 16 were isolated for the first time from Saussurea lappa.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 roots of S. lappa. 19 compounds were obtained and 11 compounds were identified. Compound 1,2,4,5,6,12 showed no notable inhibition on Caspase-7, Thrombin, HLE, DHODH and the concentration of compounds in the four groups were 0.200 mg/mL, 0.050 mg/mL, 0.125 mg/mL, 0.125 mg/mL respectively.