Ginsenoside Isolation & New Cloning Ginsenosidase Reaction

In this paper, The isolation of ginsenosides and the reactions by new cloning ginsenosidase were studied. The ginsenosides were extracted from ginseng stem by water extraction method, and then ginsenoside Rb1, Rc, Rd were separated from total ginsenoside by silica gel column. In this foundation, the optimum reaction conditions of new cloning ginsenosidase were stadied.The extracted yield of total ginsenosides was 6.02% in ginseng stem, and was 5.01% in gen-seng stem extracted by water, respectively. TLC and HPLC methods were used to detect the content of total ginsenoside. In ginseng stem, the main ginsenosides were Re, Rgl, Rd and Rc, the content of Re, Rg1 and Rd was the highest, it was 53.7% in total ginsenoside. In gen-seng stem, the main ginsenosides was Rd, Rb3, Re and Re, the content of Rb3, Rd was the highest, it was 38.3% in total ginsenoside. In notoginseng stem, the main ginsenosides were Rb3, Rc and Rd, the content of Rb3 was the highest, was 17.2%in total ginsenoside.40 g ginsenoside was purified by silica gel column,4.64 g Rb1 was obtained, and the yield was 11.6%, the purity was 96.0%.4.14 g Rc was obtained, the yield was 10.4%, the purity was 82.1%.5.38 g Rb1 was obtained, the yield was 13.8%, and the purity was 95.0%.With the substrate was preparation foundation, the optimum reaction conditions of new cloning ginsenosidase was studied. The optimal temperature and pH for the new cloning ginsenosidase were 37-45℃ and 7.0 (sodium phosphate buffer), respectively.0.5% Rb1 was hydrolyzed to Gyp 17 in 3 h completely, and then the Gyp 17 was further hydrolyzed to Gyp75. The substrate was most suit to solubilize in sodium phosphate buffer containing glycerol, and best concentration of reaction was 2% in 24h. The new cloning ginsenosidase could hydrolyze 3-O-multi-glycosides of PPD ginsenoside. It hydrolyzed Rbl to Gyp17, and then the Gyp17 was further hydrolyzed to Gyp75; it hydrolyzed Rb2 to C-O, and then the C-O was further hydrolyzed to C-Y; it hydrolyzed Rc to C-Mc1, and then the C-Mc1 was further hydrolyzed to C-Mc; it hydrolyzed Rd to F2, and then the F2 was further hydrolyzed to C-K.At the optimum reaction conditions of new cloning ginsenosidase,3.12g ginsenoside Gyp17 was obtained from the hydrolyzation of 4g Rb1, the percentage conversion was 78.0%. 1.30g ginsenoside C-Mc1 was obtained from the hydrolyzation of 2g Rc, the percentage conversion was 65.0%.The new cloning ginsenosidase only hydrolyzed 3-O-β-D-(1â†'2)-glucoside of PPD ginsenoside into Gyp 17 from Rb1 or C-Mc1 from Re in 24h. If prolonging the reaction hours, Gyp17 and C-Mcl would be subsequently transformed to Gyp75 and C-Mc by the enzyme, respectively. The interesting product could be obtained through controlling the reaction conditions.