Inhibitory Effects Of Six Phytochemical Polyphenolic Compounds On Glucosidase

Diabetes mellitus is a kind of chronic metabolic syndrome with no radical cure,which is only second to tumors,cardiovascular and cerebrovascular diseases.Glucosidase is a kind of enzymes which mainly affects the degradation of starch and other carbohydrates into glucose,and eventually affects the absorption and utilization of glucose in human blood.Glucosidase inhibitors are some substances which can inhibit the activity of glucosidase,and delay the decomposition and absorption of carbohydrates in human body resulting in reducing the level of postprandial blood glucose.Polyphenolic compounds are a kind of natural products with high biological activities as an inhibitor of glucosidase,and can be potentially used to control hyperglycemia.For the purpose to select out the effective glucosidase inhibitors from natural plant polyphenolic compounds,invertase,α-amylase and amyloglucosidase were used as representative glucosidases and their optimal enzyme reaction systems were established,respectively,in a physiological simulation condition of human body,by using starch and sucrose as natural substrates of glucosidase and by single-factor optimization and Box-Behnken design experiment with substrate additions,enzyme dosage and reaction time as reaction factors.The established optimal enzyme reaction systems were employed to test the effects of the polyphenolic extracts obtained from six plants of cinnamon,pine bark,blueberry,lophatherum leaf,olive leaf and persimmon leaf on their inhibitory activity of glucosidase in vitro with international recognized acarbose as control.The results of the various inhibitory rates on glucosidase activity of these six plant polyphenolic compounds combined with their IC₅₀values were further used to evaluate the potential of these polyphenolic compounds to control the level of blood glucose.1.For the purpose to effectively detect the inhibitory effects of phytochemical polyphenolic compounds under the optimal reaction system,the enzyme reaction systems of 5%sucrose with 5 U/m L invertase,1%soluble starch with 1 U/m Lα-amylase and 1%soluble starch with 1 U/m L amyloglucosidase by single-factor optimization and Box-Behnken design experiment were established,respectivel.The enzyme reaction rate of invertase reached 15.59±2.10（mmol/L·min）under the optimal enzyme reaction system for invertase with 0.750 m L sucrose,0.500 m L invertase,and 17.5 min reaction time,and that ofα-amylase reached 19.53±1.74（mmol/L·min）under its optimal enzyme reaction system with 0.600 m L soluble starch,0.150 m Lα-amylase,and 10.0 min reaction time,and that of amyloglucosidase reached 15.17±1.84（mmol/L·min）under its optimal enzyme reaction system with 0.515 m L soluble starch,0.115 m L amyloglucosidase,and 7.0min reaction time.The predicted values of the enzyme reaction rate of the above three models were highly consistent with the predicted ones,indicating that the established model was able to predict the enzyme activity actually and to screen the inhibitors effectively.2.The inhibitory effects of different concentrations of six plant polyphenolic compounds on invertase,α-amylase and amyloglucosidase were investigated by using the established enzyme optimal reaction system with acarbose as control.The results show that all the six polyphenolic compounds exerted some inhibitory effects on invertase,α-amylase and amyloglucosidase.Cinnamon exhibited the strongest inhibition on invertase while the other extracts of blueberry,persimmon leaf,pine bark,lophatherum leaf,acarbose and olive leaf showed less effect in order（P<0.05）,and the inhibitory effect of persimmon leaf,pine bark and lophatherum leaf polyphenolic compounds on invertase was similar（P>0.05）,indicating that the inhibitory effect of these extracts on invertase was better than that of acarbose except for the extract of olive leaf.The extract inhibitory effect of cinnamon,pine bark,blueberry,persimmon leaf,lophatherum leaf and olive leaf onα-amylase decresed in order,but all of them lower than that of acarbose（P<0.05）.The extract inhibitory effect of cinnamon was,however,close to acarbose at5 mg/m L and reached to almost 100%inhibitory rate.Additionally,activity ofα-amylase was almost completely inhibited by blueberry extract at the concentration of 50 mg/m L.Though the extract inhibitory effect of cinnamon,blueberry,pine bark,lophatherum leaf,persimmon leaf,and olive leaf on amyloglucosidase decreased in order and all lower than that of acarbose（P<0.05）,the extracts of blueberry and lophatherum leaf polyphenolic could achieve the effective high inhibitory level.3.It could further study the potential effectiveness of these polyphenolic compounds through combination.By analyzing the inhibitory effects of plant polyphenolic compounds on invertase,α-amylase and amyloglucosidase at the concentration of 10 mg/m L,it was found that the extract of cinnamon showed the most potent glycosidase inhibitory activities.Therefore,cinnamon polyphenolic compounds were selected as the target compound with the other five polyphenolic extracts in order to perform the assay and screen out the remarkable effects of combinations.The results indicated that cinnamon polyphenolic compounds and other plant polyphenolic extracts,except for the extract of olive leaf,had synergistic effects in different degrees on invertase,α-amylase and amyloglucosidase.Among them,the inhibitory effect of blueberry and cinnamon polyphenolic combinations on invertase was the best;the inhibitory effect of pine bark,lophatherum leaf and cinnamon polyphenolic combinations onα-amylase was the best;the inhibitory effect of pine bark,blueberry,lophatherum leaf and persimmon leaf and cinnamon polyphenolic combinations on amyloglucosidase was similar and were superior to the olive leaf polyphenolic compounds group.According to the experimental results,it has been suggested to study pine bark,blueberry,lophatherum leaf and cinnamon as ideal inhibitor of glucosidase in exploring inhibitory effects of multiple ratios,optimum ratios and combinational enzymes,so as to find more effective measures to inhibit glucosidase,and further explore and verify the hypoglycemic potential of plant polyphenolic compounds in the field of diabetes.