Study On The Gypenosides Enzymolysis Into Compound K

Gynostemma pentaphyllum(Thunb.)Makino(Cucurbitaceae)is widely distributed in southern China and Southeast Asian countries.Gypenosides belong a group of dammarane triterpene saponins which are main active ingredients in Gynostemma pentaphyllum.The gypenosides can be used to produce rare ginsenosides through enzymolysis because of the same skeleton structure as ginsenosides.The rare ginsenosides Compound K(CK)is produced from degradating panaxadiols by intestinal bacteria,and it is the main active compound that exerts pharmacodynamic effect in vivo.How to obtain large amounts of high-purity CK efficiently and at low cost has been the focus of scientific research since trace of CK exist in ginseng and difficult to meet application requirements through separation and extraction.Based on the similarity of the structure of gypenosides and ginsenosides,this study speculates that the structure modification can be achieved by enzymolysis to prepare the CK in vitro.First of all,panaxadiols-riched Gynostemma pentaphyllum was screened from 10 main growing areas.The Gynostemma pentaphyllum from Jinggangshan in Jiangxi Province was chosen for further study for the highest content of panaxadiols.The panaxadiols were enriched by HP-20 and RP-18 column chromatography and named as gyp A.Single factor and orthogonal test were used to optimize the extraction conditions of gyp A.The optimized extraction conditions were as follows:ratio of material to liquid was 1:20,extraction temperature at 60℃,ethanol concentration was 70%,extraction time was 70 min,and extraction rate reached 7.57%.And then,the enzymolysis effect of different glycosidases on gyp A were studied.The naringinase was the screened as the best invertase.The hydrolyzate was separated by HP-20 and RP-18 column chromatography,and the obtained compound was identified as CK by NMR.The single factor test and BBD response surface method were used to optimize the conditions for the enzymolysis of CK from gyp A.The optimized conditions were as follows:pH was 4.1,hydrolysis time was 71 h,enzymatic hydrolysis temperature at 50 ℃,theoretical yield of CK yield reached 63.93%,and verification experiment result was(65.44±4.52)%which was consistent with the prediction.Finally,the anti-hepatoma activity of substances before or after transformation were studied.The effect of the drugs on the cells were studied by MTT assay and cell morphology observation.Treatment of hepatoma cells Bel7402,SMMC7721 and normal liver cells Chang liver with different concentrations of gyps,gyp A,gyp A enzymatic hydrolysates and CK.The results showed that as the concentration of drugs increased,the number of death cells also increased.Anti-heptoma activities were in turns:gyps<gyp A<gyp A enzymatic hydrolysate<CK.In summary,gyp A can be effectively converted into CK by naringinase,and the anti-hepatoma activity was enhanced.This study might promote the development of gypenoside substitutes,and advance the development of the Gynostemma pentaphyllum industry.