Ginsenosides Of Rb <sub> 3 </ Sub>, Biotransformation, And Paecilomyces Sp.229 In Separation And Purification Of The Metabolite Identification

Paecilomyces Bainier sp.229 is a fungal strain isolated from the soil of ginseng plantation localities.It shows strong glycosidase activity that can transform ginsenoside Rb₁ to CK efficiently,and the Mol.conversion quotient reaches to 80%.Ginsenoside Rb₃ is a protopanaxadiol ginsenoside,similar to ginsenoside Rb₁ in chemical structure.Compared to others ginsenosides,it is abundant in natural products,especially in leaves and stems of Panax notoginseng.So far,none study on the microbial transformation of ginsenoside Rb₃ has been reported.A convenient High performance liquid chromatography standard method was established for determining Rb₃ content in Panax notoginseng saponins.And silica gel chromatography and reverse-phase silica gel chromatography were employed to separate the Panax notoginseng saponins,affording ginsenoside Rb₃ as the substrate for the advanced fermentation.After preparing enough ginsenoside Rb₃,Microbial-transformation was carried out,and the metabolic products were separated and identified by TLC, HPLC,as well as X-ray diffraction analysis.The metabolizing pathway of ginsenoside Rb₃ was also studied.The metabolites have been identified as ginsenoside Mx and ginsenosidesC-K.X-ray diffraction technology was firstly employed in studying ginsenoside Mx,and a High performance liquid chromatography standard method was established for determining ginsenoside Mx.To exploring the metabolizing pathway of ginsenoside Rb₃,Samples of microbial-transformation in different time,samples of products transformed by crude enzymes for different hours,samples of products transformed by immobilized crude enzymes for different hours,and samples of different quantities substrate transformed by immobilized crude enzymes,were analyzed by TLC and HPLC.And the metabolizing pathway of ginsenoside Rb₃ was speculated to be:Rb₃â†'Gypenosideâ…¨â†'Mxâ†'C-K.In this study,the influences of carbon source,nitrogen source,growth factor, and surface acting agent were studied,and then orthogonal experiment was carried out to optimize the conditions of fermentation.The optimal conditions were determined as:3%Sucrose,3%yeast powder,0.1%wheat bran powder, 0.1%MgSO₄,0.15%CaCl₂,and 0.2%(NH₄)₂SO₄.Because of its abundant metabolites,microorganism gets more and more attention.Apart from antibiotics,lots of compounds with kinds of activities were found in the metabolites,including flavones,alkaloids and saponins etc.microbic metabolites have been one of the important sources of drugs.In this study,the metabolites of Paecilomyces Bainier sp.229 were partly separated,affording two steroid compounds,the crystals of which were prepared,and were identified as Ergosterol Peroxide(EP) and Ergcoterol.This is the first time that these two compounds were separated from Paecilomyces Bainier sp.229,which is a new way to prepare these two compounds.