| Background:With the improvement of living standard and the accelerating pace of life,the number of diabetes patients is increasing year by year,which is a serious threat to human life and health.Because of its low toxicity,high safety,mild and lasting effects,TCM has shown great advantages in the prevention and treatment of diabetes.Sanye tablet is derived from the name old doctor of traditional Chinese medicine experience,mulberry leaf,lotus leaf,hawthorn leaf,salvia miltiorrhiza,red paeony root of five kinds of traditional Chinese medicine?TCM?,extracted by modern technology made into tablets,is applicable to phlegm turbidity caused by intrinsic despair an illusion of spleen,namely pre-diabetes,clinical recommendations to dose for each time 3 pills,three times a day.The previous study of the research group concluded that Sanye tablet could improve the function of islet of islet of diabetes rats.It can inhibit the activity of small intestinal sucrase and maltidase in kk-ay mice.It can also reduce the postpranational blood glucose level of patients and healthy people,and has the function characteristics similar to acarbose.On the basis of the above studies,this experiment took sucrose,a common carbohydrate in human diet,as the research object to investigate the effect of Sanye tablet on blood glucose and the characteristics and mechanism of sucrase activity in healthy people after sucrose consumption.Objective:To observe the hypoglycemic effect of sanye tablet on healthy subjects after taking sucrose,and to investigate the characteristics and mechanism of sanye tablet on?-sucrase by using in vitro enzyme inhibition kinetics,so as to provide data support for the study on the hypoglycemic mechanism of sanye tablet.Methods:1.A single-center,five-cycle randomized cross-control experiment was conducted to select 12 healthy volunteers who met the standards.The volunteers were randomly divided into 5 experimental groups according to their weight,with each group being 3,3,2,2 and 2.Volunteers in each group were randomly treated with 5intervention methods,namely glucose,glucose,glucose,sucrose and sucrose plus 3sanye tablet,to observe the effect of sanye tablet on postpranpranastic blood glucose in healthy people after taking sucrose and the effect on glycemic index and glycemic load of sucrose.2.Using sucrose as the substrate,in vitro enzyme inhibition kinetics method was used to investigate the inhibitory activity,inhibition characteristics and mechanism of sanye tablet on?-sucrase.?1?different concentrations of inhibitors?sanye tablet extract?,quantitative sucrase?intestinal enzyme extract?and excess substrates?sucrose?were tested to calculate the inhibition rate of sucrase from sanye tablet extract with different concentrations.The inhibition curve was fitted to obtain the IC50value of half inhibitory concentration.?2?reaction of sucrase of different concentrations and quantitative inhibitors with excess substrates was carried out,and the enzyme-dose-velocity diagram?E-V diagram?was drawn to judge whether the inhibition effect was reversible;?3?substrates of different concentrations,inhibitors of a certain concentration and sucrase were tested.The substrate concentration-reaction rate double reciprocal inhibition curve?[S]-v double reciprocal curve?of extracts from sanye tablet extract to sucrase was drawn by Lineweaver-Burk mapping method,and the Km value was calculated to determine the type of sucrase inhibition by sanye tablet to?-sucrase.Results:1.Clinical research:?1?As a reference,there was no significant difference between the three baseline glucose tests at the blood glucose collection points?P>0.05?,indicating good repeatability.?2?The blood glucose peaks in the glucose,sucrose and sucrose+3 leaf groups all occurred at 30min after meal.?3?Sanye tablet can significantly reduce the blood glucose level at 60min and120min after sucrose consumption in healthy people?p<0.05?,and the blood glucose level at other time points also has a tendency to decrease,but the effect is not obvious?p>0.05?.?4?The increment area of glucose response curve?IAUC?of sucrose could be significantly reduced in sanye tablet.P=0.04?P<0.05?.?5?The sucrose GI value?87.97±33.36vs.66.85±34.59,P<0.05?and GL value?44.09±32.70vs.32.73±33.89,P<0.05?were reduced after the administration of sanye tablet.2.Mechanism research:?1?In the selection of optimal substrates,sucrose?substrates?showed a zero-order reaction at a concentration of 0.224mol/L.In order to ensure the excess of substrates in the reaction system,the experimental concentration was 0.448mol/L.?2?When the concentration of intestinal enzyme was 7mg/ml or below,the enzymatic reaction was linear within 14min.In order to maximize the initial reaction rate,the reaction time of 7mg/ml intestinal enzyme in the reaction system was 14min as the condition for the kinetic analysis of the enzyme reaction.?3?Inhibition of sucrase activity by sanye tablet extracts and acarbose was dose-dependent.When the concentration of sanye tablet extracts was 2.2×10-2mg/ml,the inhibition rate could reach 86.37%.The IC50 value of half inhibitory activity of the extracts from sanye tablet was?1.53±0.07?×10-3mg/ml.The IC50 of the positive control drug acarbose was?3.94±0.07?×10-3mg/ml.?4?The enzyme-dose-velocity plots of the sanye tablet extracts in the two groups(2.8×10-3mg/ml,2.2×10-2mg/ml)showed that the inhibition curves were both approximately through the origin,indicating reversible inhibition.?5?The double reciprocal curves of the two groups(2.8×10-3mg/ml,2.2×10-2mg/ml)[S]-v were almost parallel to the normal control group,showing anti-competitive inhibition.Compared with the normal control group,acarbose group showed competitive inhibition on the Y-axis.Conclusion:1.Clinical studies have shown that sanye tablet can reduce postprandial blood glucose level,glycemic index and glycemic load of healthy individuals after sucrose consumption.2.The results of enzyme inhibition kinetics in vitro showed that the activity of sucrase was inhibited by the sanye table in a reversible anti-competitive manner,and the IC50 was?1.53±0.07?×10-3mg/ml.Acarbose inhibited sucrase activity in a reversible competitive manner,and the IC50 was?3.94±0.07?×10-3mg/ml.3.Sanye table is a potential?-glucosidase inhibitor of Chinese medicine compound,and its hypoglycemic effect is similar to that of acarbose. |
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