Study On Enzymatic Glycosylation Modification Of Total Bufadienolides From Venenum Bufonis

Venenum Bufonis?VB?,also called ChanSu or toad venom,is an important Traditional Chinese Medicine derived from the parotoid gland secretion of giant toads?Bufo bufo gargarizans or Bufo melanostictus?.It is applied in various preparations,such as Liu-Shen-Wan,She-Xiang-Bao-Xin pill and ChanSu injection,for the treatment of heart failure,pains,and various cancers in clinics.The total bufadienolides are known to be the major active constituents of VB in these preparations and more than 50bufadienolides have been isolated.However,the high toxicity,low bioavailability,and poor water solubility of total bufadienolides limit the clinic applications.Glycosylation is a universal approach to increase the water solubility,which would also improve their pharmacodynamic and pharmacokinetic properties.Therefore,it is of great significance to develop an efficient strategy for the glycosylation of total bufadienolides in VB,which would possibly alleviate its toxicity and facilitate the clinical applications.Chemical synthesis of steroid glycosides is hindered by many drawbacks,such as poor regio-and stereoselectivities,requirement of protection and deprotection steps,use of toxic catalysts,and production of chemical waste.Instead,biocatalysis has been recognized as an alternative way to solve these problems,which is environment-friendly and cost-effective.It was reported that the cell suspension cultures of several specific plant species are capable of glycosylation of cinobufagin,such as Catharanthus roseus,Platycodon grandiflorus,and Saussurea involucrate.However,low yields and generation of by-products makes it hard to purify the target steroid glycosides.Recently,significant progress has been made in enzymatic glycosylation of steroid compounds.For examples,the microbial glycosyltransferase?GT?OleD ASP,YjiC1 and plant GT UGT74AN1 showed robust catalytic efficiency toward cardenolide or bufadienolide aglycons,which makes it possible to establish an efficient strategy for glycosylation of total bufadienolides in VB.In this thesis,the glycosylated VB was produced by glycosylation of crude VB extracts through the enzymatic transformation of bufadienolide aglycones into the corresponding monoglycosylated or diglycosylated compounds by using permissive GTs.Three GTs were firstly compared and YjiC1 was found to be the most efficient one.The results showed that the main components were glycosylated with high conversion rates?90%?with YjiC1 catalysis.The toxicity on zebrafish indicated that compound arenobufagin-3-O-glucoside?3a?showed much weaker(>5-fold,LD₅₀=83.37±0.04?M)toxicity as compared to the corresponding bufadienolide aglycone;The inhibitory activity on NKA indicated that compound 3a demonstrated much stronger(about 3-fold,IC₅₀=0.52±0.04?M)activities than the corresponding aglycones.We prepared the glycosylated product by glycosylating the total bufadienolides in VB.Then the glycosylated bufadienolides mixture was used as the starting material and made into injection with reference to the preparation method of toad venom injection.Finally,the toxicity of the two preparations was evaluated by the zebrafish model.At the same concentration,the toxicity of the glycosylated chansu injection was weaker than the toad venom injection.