Isolation And Purification Of Glycosides From Asp.niger 848 And Enzymatic Transformation Of The Acanthopanax Senticosides

Acanthopanax Senticosus is one of the traditional Chinese medicine, with a more extensive pharmacological effects and clinical value, and has a long medicinal history. In recent years, Acanthopanax Senticosus leaves contain much active component such as saponins, polysaccharides, flavonoids etc., as a result, they are widely used as a substitute for Acanthopanax roots. It was interested to research and develop Acanthopanax Senticosus leaves due to the saponins with various physiological activities in them. In this thesis, we focused on one monomeric saponin and did some exploring.The purification and enzymatic properties of glycosidase from the liquid fermentation of Asp.niger FFCC848 were investigated. After the successive steps of 30% to 70% ammonium sulfate precipitation, dialysis and DEAE-Sepharose FF anion exchange chromatography, highly purified glycosidase from the fermentation broth of Aspergillus niger was obtained. It was shown that there was only one clear band in SDS-PAGE, and the pure glycosidase had a molecular weight of 65 k Da. Glycosidase was purified by 13.82 folds with an enzyme activity recovery of 13.30%, and the specific activity of it was 6932.54 U/mg. Furthermore, the activity of glycosidase was stable at a temperature range from 20 oC to 50 oC and a pH range from 3.0 to 6.0, with the optimum activity of 50 oC and pH 4.5, respectively. Meanwhile, the enzyme activity was enhanced by K+, Ca²⁺ and Na+, while it was strongly inhibited by Fe³⁺, SDS and EDTA-Na2.The structure of acanthopanax senticosides B and its hydrolysate were analyzed. Chomatography results showed that the effect of glycosidase on acanthopanax senticosides B was obvious significantly, and Rf value of 0.32 of product was formed. By the means of infrared spectroscopy and mass spectrometry to analyse, it was indicated that acanthopanax senticosides B produced only one secondary saponin, which was probably resulted by removing the C-28 site of α-1,4 rhamnose linked to the end of the sugar chain.The evaluation method of antioxidant activity were used to analyze the activity of acanthopanax senticosides B and its hydrolysate. The results of DPPH and Fenton methods showed the antioxidant activity of secondary saponin was improved to some extent, which may be related to changes in its structure.