Isolation And Identification Of Endophytes To Perform Biotransformation Of Polyphylin From Paris Polyphylla Smith Var. Yunnanensis

Polyphyllin is the main active ingredient of Paris polyphylla Smith var. yunnanensis,which has widely pharmacological actions. At present, the reports on using endophytes toconvert ingredient of the same plant were rare. In this study, endophytes were isolated fromParis polyphylla and then performed molecular biological identification. Depended on thebiotransform activity of polyphylla endophytes to polyphyllin (Polyphyllin I、Polyphyllin II、Polyphyllin VII) in Paris polyphylla, the strains were further screened. Afterwards, culturescale was enlarged, and then the transform products were purified and identified. The resultswere as the follows:(1) The endophyte of Paris polyphylla Smith var．yunnanensis was isolated andidentified.40and44endophytic bacteria were isolated from the root of cultivated or wildParis polyphylla Smith var．yunnanensis in Wenshan,Yunnan province. They were thenidentified and categorized as6and8genera by16S rRNA gene sequenceing. Bacillus andEnterobacter were dominant genera in cultivated of Paris polyphylla Smith var．yunnanensisbecause they accounted for35%and27.5%respectively in the identified bacteria; Bacillusand Klebsiella were dominant genera in wild Paris polyphylla Smith var．yunnanensis andaccounted for36.4%and20.4%respectively. The results reflected the difference andbiodiversity of endophytic bacteria between cultivated Paris polyphylla Smithvar．yunnanensis and wild ones. This research provides experimental and theoretical basis forsubsequent development of Paris polyphylla Smith var．yunnanensis.(2) The effect of the ratio of material to liquid of solvent, and the kind and concentrationof solvent, and to solvent on the yield of total saponin were studied. The optimum extractionprocess was that the plant materials were extracted by70%ethanol with material to solventratio1:8. Depending on thin layer chromatography method, three Polyphyllin monomers(PA22, PB2, and PD2) were obtained after silica gel column and gel column separation andpurification. Liquid chromatography tandem mass spectrometry (LC-MS) and nuclearmagnetic resonance (NMR) were used to identify these monomers. The results showed thatthe structures of PA22andPB2are same to Polyphyllin I; the structure of PD2is same toPolyphyllin VII.(3) The84endophytic bacteria in Paris polyphylla Smith var．yunnanensis were furtherscreened depending on the Polyphyllin I, Polyphyllin II and Polyphyllin VII conversionability of these strains. The results showed that strain YA09can convert Polyphyllin VII. Thetwo converted products-ZH-1and ZH-2were obtained by isolation and purification afteramplifying Polyphyllin VII conversion system by strain YA09. Liquid chromatography tandem mass spectrometry and nuclear magnetic resonance were used to identify theseproducts. The results showed that the structure of ZH-1is same to Polyphyllin VI; thestructure of ZH-2may be pennogenin-3-O--L-rhamnopyranosyl(1→4)-[-L-rhamnopyranosyl(1→2)]-β-D-glucopyranoside. This study indicated that it is great impossible todiscover new sources by utilizing plant endophy, and amplify the action of traditional Chinesemedicine endophy to host pesticide effect by bioconversion method.