Screening Of The Strains That Having Ability For Producing α-glucosidase Inhibitor

Diabetes Mellitus is a common,chronic metabolic diseases.With the improvement of living standards and changing lifestyles,the number of diabetic patients have increased extremely.α-glucosidase inhibitors as the first-line oral hypoglycemic drugs that treat of typeⅡdiabetes,attracted widespread attention.Drugs ofα-glucosidase inhibitors applied to clinical,such as acarbose,has some side effects.Therefore,the search for safe and effective new naturalα-glucosidase inhibitors has become one of the important tasks of microbial resources research.In this study,three kinds of culture media,PDA,NA,G₁,were used to isolate endophytes from 19 kinds of medicinal plants that can regulate blood sugar.At last,we got endophytes pure culture strains 530.For the screening of strains that producingα-glucosidase inhibitor provide sources.In our research,theα-glucosidase inhibitory activity screening model was built-up used PNPG as substrate:In 96-well microtiter plates,0.1 mol/L phosphate buffer 80μL,Crude extraction of rat intestinalα-glucosidase was 80μL（protein concentration of 3.61 mg/m L）,25μL of sample to be tested（dissolved in 50%DMSO）were added and mixed,37℃incubated for 15 min,while incubating the substrate PNPG,then added PNPG 15μL,concentration of20 mmol/L,after mixing,reaction at 37℃for 30 min,and finally added 1 mol/L of Na₂CO₃100μL to terminate the reaction,measured OD_405nmvalue.Acarbose was used as a positive control,130 endophytic actinomycetes were screened with the above established model.As a result,there were 22 strains withα-glucosidase inhibitory activity more than 50%,wherein,The strain 193403 showed inhibition rate of 92%,can be used as objects for further study.The further study of 22 strains about 16S r DNA sequence phylogenetic analysis,result indicated that among of them,17 strains belonging to the Streptomyces,3 strains belong to Micromonospora,1 strain attributed to Nocardiopsis,1 strains attributed to Nocardia.Strain 193403 fermentation products were isolated and purified,and we have obtained the componentsⅠ,Ⅱ,Ⅲseparated from the aqueous phase.Among of them,the componentⅠand the componentⅢwere with higherα-glucosidase inhibitor activity which inhibitory rate were 99.63%and 99.53%,respectively.And they should be further separated and purified to obtain a monomer substance that havingα-glucosidase inhibitory activity.The pure component A was separated from ethyl acetate phase,and theα-glucosidase inhibitory rate was 58.23%,After further study to identify its substance application of ¹H-NMR,¹³C-NMR,HMBC,HSQC and HR-ESI-MS analysis,then it was identified as4-thio-2’-deoxyuridine.