Studies On Biotransformation Of Active Constituents In Dioscoreae Nipponicae Rhizoma By Endophytic Fungus And Component Identification

Dioscorea nipponica is the drying rhizome of perennial vine Dioscorea nipponica Makino which belongs to Dioscoreaceae and dioscorea contains abundant diosgenin.Diosgenin is one of the most important precursors to synthetize many kinds of steroid hormones and contraceptives.But with the development of the pharmaceutical industry and the increasing demand of domestic and international market for diosgenin,the resources of Dioscorea nipponica have become less.It was found that Dioscorea diosgenin content was in a substantial increase by microbial conversion method,and to some extent alleviated the shortage of yam and other medicinal resources.Now,the endophytic fungi C39 was isolated from the rhizome and leaf of D.nipponica Makino and then fermented it with herbs,which can significantly improve the content of diosgenin.To study more about the mechanism of biotransfo-rmation by C39 and find the precursors of biotransformation,through the experiments,we found that the avtive part which can biotransformation by C39 is in the n-butyl alcohol parts of diosgenin.This experiment is based on biotransformation,combined with TLC,HPLC and UPLC/Q-TOF-MS as techniques,which does a preliminary research on biotransformation of steroidal saponins in herbal ingredients by the fungus C39,and we have a discussion on how to improve the mechanism of diosgenin in Dioscoreae Nipponicae Rhizoma.First of all,extract the active part of Dioscoreae Nipponicae Rhizoma by 70%ethanol.Then we use the technologies of macroporous adsorption resin to purification,and we got aqueous part,30%ethanol part,50%ethanol part,70%ethanol part,95%ethanol part and water-insoluble part.Having biotransformation by C39,we found that in aqueous part,30%ethanol part,70%ethanol part 95%ethanol part and water-insoluble part,there is no active part which can improve the content of diosgenin,but 50%ethanol part have.In order to find the saponins which are transformed through the biotransformation,we use silica gel column with 50%ethanol part.We got 12 parts including methylene chloride and methanol 11:1、9:1、7:1(1-12)、7:1(13-24)、7:1(25-34)、5:1(1-14)、5:1(15-35)、5:1(36-47)、5:1(48-94)、3:1、1:1and aqueous part.By HPLC analysis,methylene chloride and methanol 11:1、9:1、7:1(1-12)have little ingredient,so we choose methylene chloride and methanol 7:1(13-24)、7:1(25-34)、5:1(1-14)、5:1(15-35)、5:1(36-47)、5:1(48-94)、3:1、1:1 and aqueous part as substrate,these possible substrate were transformed by C39.Combined with the TLC result,we found that furostanol saponins was gradually reducing,but spirostanol saponins are gradually increasing.So a conclusion was drew that furostanol saponins transformed into spirostanol saponins in the process of biotransformation.Combined with the HPLC result,the precursors peaks which disappear in all of methylene chloride and methanol are 6,besides,in7:1(13-24)、7:1(25-34)、3:land aqueous part is one,in 5:1(1-14)、5:1(48-94)are two,in 5:1(15-35)are three,in 5:1(36-47)are seven.The generated peaks after biotransformation of furostanol saponins in all of methylene chloride and methanol are one,besides,in 7:1(25-34)、5:1(1-14)、5:1(48-94)、3:1、1:1and aqueous part is one,in 5:1(36-47)are two,in 5:1(15-35)are three.The generated peaks after biotransformation of spirostanol saponins in all of methylene chloride and methanol are five,bisedes,in7:1(13-24)、7:1(25-34)、6:1(48-94)、3:1、1:1 and aqueous part is one.The peak which appear then disappear in the biotransformation,in the methylene chloride and methanol 5:1(15-35)is none,in other part is one.Besides,in 5:1(1-14)、5:1(36-47)and aqueous part is one,in 7:1(13-24)、7:1(25-34)、5:1(48-94)are two,in 3:1are three.Last,combined with UPLC-TOF-MS,in the biotransformation,the saponins which disappear are Prosapogenin A of dioscin,deltonin,colletinside,dioscin DC,diosgenin diglucoside,pregnadienolone(3-O-β-gracil-limatriose)、Lilioglycoside K、Protodeltonin.In the biotransformation,the content of saponins which decreased are trill in,pseudoprotodioscin,Zingiberenin is new saponin and protodioscin.In the biotransformation,the production of Yamogenin increased by 51.80 times,and the production of dioscin increased by 245.64 times,diosgenin triglucoside is new.