Study On Biotransformation And Separation Of Ursolic Acid By Microbe

Ursolic acid has anti-carcinogenic, anti-tumor promotion and anti-angiogenesis, is an excellent new anticancer drugs. Currently, ursolic acid derivatives are synthesized mainly chemically. Compared with organic synthesis, biotransformation with high stereoselectivity, regioselectivity and chemoselectivity, mild reaction conditions, small environmental pollutional, and helps to improve conversion rates and reduce costs. In this paper, microbial conversion technology was used, and ursolic acid was catalyzed by Alternaria longipes. The way to assay for conversion rate was established, and the medium and culture condition was optimized, and three compounds were isolated from the biotransformation products.First, the analysis method of the main components was found. The method of Thin Layer Chromatography which employed silica gel was used to qualitatively analyze the substrates and products, cyclohexane:ethyl acetate=7:3and chloroform:methanol=9:1(V/V). A High Performance Liquid Chromatography was also established to analyze the conversion rate of Alternaria Longipes3.2875. When the liquid medium substrate concentration at0.3mg/mL, the conversion rates was42.04%. Because the UV absorption of ursolic acid is weak, this method selected acetonitrile-water (85:15) as the mobile phase, and204nm as detection wavelength. When injection volume of ursolic acid was in the range of4.0-16.Oμg, peak area and sample size showed a good linear relationship.Second, the bioconversion conditions were optimized. The transformation of the optimal conditions for shake flask dosage concentration at9mg/mL, reaction time3days, bioconversion temperature at28℃, shaking speed140r/min, medium initial pH=5, MgSO4dosage of0.25mg/mL, K2HPO4dosage of1.0g/mL, FeSO4dosage of0.083g/mL. The conversion rate could be μp to86.20%.Furthermore, the converted products were separated by macroporous resin column chromatography, silica gel column and RP-HPLC column chromatography. Three compounds were isolated from transformation products. These three compounds were3-carbonyl ursolic acid, ursolic acid-3-O-β-D-glucopyrano side, ursolic acid-28-O-β-D-glucopyranosyl ester. The in vitro cytotoxic activities of metabolites against one human cancer cell lines were determined by the MTT method. Transformation products on the inhibitory activity of Hela cells was not obvious.