Screening Of Endophytic Producing α-Glucosidase Inhibitor Siraitia Grosvenorii And Study On Its Active Components

Currently,there are 463 million adults suffering from diabetes worldwide.α-glucosidase inhibition（α-GI）can effectively inhibitα-glucosidase（α-Glu）on the small intestinal mucosa,so as to maintain normal postprandial blood glucose.It is now widely used in the treatment of type 2diabetes.At present,such drugs as acarbose have some problems in clinical practice,such as fewer kinds of drugs,high price and large gastrointestinal reactions.Therefore,it is of great significance to develop safe and efficient newα-GI.Siraitia grosvenorii has hypoglycemic effect.Based on the symbiosis theory,the endophytes may also produce metabolites with hypoglycemic activity.In this study,the endophytic strains producingα-GI and their metabolites were studied by activity tracing method,in order to lay a foundation for the development and utilization ofα-GI from endophytes of Siraitia grosvenorii.Main contents and results of the study:1.Selection and identification of endophytes that produceα-GI in Siraitia grosvenoriiIn this study,8 strains withα-GI inhibition rate of over 20%were screened from 36endophytic strains of Siraitia grosvenorii by p-nitrophenyl-α-D-glucopyranoside method.The inhibition rate of 50μL of strain PD-14 fermentation broth onα-Glu was 60.70%.The results of colony morphology,gram staining and spore staining showed that the strain was gram-positive bacteria,and was identified as Bacillus preliminarily.The 16S r DNA of strain PD-14 was further sequenced.sequence alignment and phylogenetic tree construction showed that strain PD-14 was closest to the known strain Bacillus velezensis LZLJ01,with 100%homology.2.Optimization of fermentation conditions for the production ofα-GI by strain PD-14In order to explore the best fermentation conditions for strain PD-14 to produceα-GI,PDB medium was selected from seven different media as the best medium.Under these conditions,50μL of fermentation broth had the highest inhibition rate forα-Glu.It was 60.79%.The fermentation conditions forα-GI production of strain PD-14 were optimized by orthogonal experiments,and the optimal fermentation conditions forα-GI production were obtained as follows:liquid content of 100 m L/250 m L triangular flask,fermentation time of 4 d,fermentation temperature of 30℃,inoculation volume of 7%.Under these conditions,the inhibition rate of 50μL fermentation broth onα-Glu was 91.05%,which was 1.5 times of that before optimization.3.The active site ofα-GI produced by strain PD-14 was tracedBased on the inhibition rate of the fermentation product of strain PD-14 onα-Glu,the active site was traced.It was determined thatα-GI is mainly concentrated in the extracellular,that is,in the fermentation broth,and the effective phase is the n-butanol phase and the aqueous phase.By preliminary experiments on chemical components,the main chemical components are proteins,organic acids,flavonoids,steroids or triterpenoids.4.Study on reaction kinetics ofα-GI enzyme inhibitorIn order to judge whether the inhibitory effect ofα-GI is reversible,the concentration of enzyme solution E was taken as the abscissa and the reaction rate V was the ordinate.The inhibition kinetic curves ofα-Glu by three different concentrations(0,75 and 150 mg·m L^-1)of the inhibitor,namely the aqueous phase sample solution,were ploted.As a result,the three straight lines obtained all pass through the origin,and the slope of the straight line becomes smaller as the concentration of the added inhibitor increases.Therefore,the inhibition ofα-GI in aqueous sample is reversible.The Lineweaver-Burk double reciprocal curves ofα-Glu at different inhibitor concentrations(0,1.2 and 2.4 mg·m L^-1)were plotted by fixing the concentration ofα-Glu at 0.125U·m L^-1,the reciprocal of substrate concentration（1/S）as the abaxial axis and the reciprocal of reaction rate（1/V）as the vertical axis.The results show that the three lines intersect in the second quadrant,and with the increasing of the inhibitor concentration,V_max decreases and K_m increases,indicating that the inhibition type is mixed inhibition.5.Research on the influencing factors ofα-GIIn order to explore the stability ofα-GI,the effects of temperature,p H and metal ions on the activity ofα-glucosidase inhibitors were studied.The results showed that theα-GI was stable at40℃and 60℃,and there was no significant difference between the two groups（P>0.05）.The inhibition rate ofα-GI under acidic condition（65.81%-73.06%）was higher than that under alkaline condition（65.69%-65.79%）,and the difference was significant（P<0.01）.Both K⁺and low concentration of Mg²⁺could increase the inhibition rate ofα-GI,Na⁺could decrease the activity ofα-GI,and Ca²⁺had no effect on the activity ofα-GI.6.Study on purification of active ingredients ofα-GIBefore and after protein removal by Sevage method,the inhibition rates ofα-Glu in effective phase aqueous phase of PD-14 fermentation broth were 75.34%and 78.27%,respectively,and the change of inhibition rates was extremely significant（P<0.01）,indicating that the protein removal effect was good.It was preliminary separated and purified by AB-8 type macroporous adsorption resin column chromatography.The results show that the 70%ethanol eluate has the highest inhibitory activity onα-Glu,the inhibition rate is 70.56%when the concentration is 1 mg·m L^-1,and the inhibition rate of acarbose solution with the concentration of 1μg·m L^-1 was 95.12%.Further purification was carried out by silica gel column chromatography.When the eluent is ethanol:methanol ratio of 2:3,and the sample concentration was 0.5 mg·m L^-1,the inhibition rate was 80.46%,which was 24.47%higher than that before silica gel column chromatography.The inhibition rate of the positive control acarbose solution with a concentration of 1μg·m L^-1 was95.03%.The results showed that silica gel column chromatography was effective in separation and purification.HPLC method was used to detect the sample after purification by silica gel column chromatography（eluent ethanol:methanol is 2:3）,a very small amount of material was washed out after the retention time was 1 min,and a single peak appeared at 4 min.Preliminary experiments on chemical components show that the components are flavonoids.In conclusion,the endophytic strain PD-14 producingα-GI of Siraitia grosvenorii was screened in this study and identified as Bacillus.The active sites ofα-GI were mainly extracellular,and the effective phases were n-butanol and aqueous phase.The inhibition ofα-GI in aqueous phase was reversible,and the inhibition type was mixed inhibition.Theα-GI is resistant to high temperature and is more suitable for processing under acidic conditions.The inhibition rate ofα-GI can be improved by both K⁺and low concentration Mg²⁺.After separation and purification,the active ingredient was preliminarily identified as flavonoids.