Biotransformation Of Pulsatilla Saponin And Separation Of The Hydrolysate

The purpose of this paper is to prepare low sugar pulsatilla saponins.Total pulsatilla soponins was extracted from traditional medicine the root of Chinese pulsatilla and separated to obtain pure saponins.Then,biotransformation method was used to convert the substrate.The product was also purified.Firstly,total pulsatilla soponins was prepared.Ethanol was used to extract the total saponins from Chinese pulsatilla.Macroporous adsorption resin AB-8 and ion exchange resin D-296 was used to desugar and decolor the crude product,respectively.The yield of crude product was 7.71%;the yield of extraction method by n-butyl alcohol was 9.86%.To investigate the transformation of pulsatilla saponin by biotransformation method,the two main saponins were separated,i.e.,sample a and anemoside B4.The strain Absidia sp.POOr was screened from 6 stains.The enzyme activity was highest when the strain was incubated at 30℃ for 5 d using pulsatilla as inducer.The optimal reaction condition was:a substrate concentration was 2%,pH5,a temperature of 40℃,reaction for 24 h.To confirm the hydrolysis of the pulsatilla saponin-glycosidase,this enzyme was purified and its molecular weight was 114.7kDa.This enzyme can hydrolyze sample a and anemoside B4 to produce sample c and anemoside A3.Finally,the transformation was carried out using 50 g total pulsatilla saponin as substrate.After desugar and decoloration,34.2 g hydrolysate was obtained.Then further purification was performed by silica gel chromatography and recrystallization,3.61 g of sample e was obtained,the yield of sample e was 10.6%,and its purity was 98%;2.03g of sample f was obtained,the yield of sample f was 5.97%,and its purity was 82%;5.10g of mixture containing sample c anemoside A3 was separated,after further purification of crystallization and recrystallization,0.63g of sample c was obtained,and its purity was 99.41%;1.37 g of anemoside A3 was obtained,and its purity was 93.01%.