The Inhibition Effect And Mechanism Of Triterpenoids Extracted From Suspended Cultured Cyclocarya Paliurus Cells On The Activity Of Carbohydrate Digestion Enzymes

Blood glucose is one of the most important human health indices.The digestion and absorption of carbohydrates is a key factor affecting postprandial blood glucose.The postprandial blood glucose rising speed could be effectively reduced by the inhibitors of carbohydrate digestion enzyme.At present,the commonly used carbohydrate digestion enzyme inhibitors were produved by chemical synthesis.It could effectively lower down the blood glucose,but its side effects are obvious.Therefore,it is of great significance to study the mechanism of natural and highly effective carbohydrate digestion enzyme inhibitors.Four kinds of carbohydrate digestion enzymes inhibitors micro screening model were optimised in this paper.Based on the models,I studied the inhibitory effect of triterpenoids from Cyclocarya paliurus suspension cell.In addition,I studied the mechanism of the inhibitory effect through fluorescence spectroscopy and molecular docking.The main results of this research were as follows:1.Four kinds of carbohydrate digestive enzyme inhibitor screening model were optimised:（1）The main parameters of the model which was used to screen Alpha-starch glycoside enzyme inhibitor are as follows:the enzyme concentration was 1 U/ml,the substrate concentration range of 0.5³ mM,the reaction temperature is 37℃,the reaction time is 8 minute;（2）The optimized parameters of invertase inhibitor screening are as follows:the enzyme concentration was 8 U/ml,the substrate concentration range of 0.1¹.5mM,the reaction temperature is 45℃,the reaction time is 8 minute;（3）The optimized parameters of porcine pancreatic amylase inhibitor screening are as follows:the enzyme concentration was 1.25 U/ml,the substrate concentration range of 0.5³ mM,the reaction temperature is 37.5℃,the reaction time is 40 minute;（4）a model of alpha glucosidase inhibitor screening in vitro was established.The optimized parameters of this model were as follows:the enzyme concentration,substrate concentration,reaction temperature,and reaction time were 0.05 U/ml,0.05¹ mM,37℃,and 6 minute respectively.2.Based on theα-glucosidase inhibitor screening model,The kinetic data processing and analysis method of enzyme inhibition reaction was studied.According to the data processing and the comparison of results,they showed that the five methods had distinct characteristics and the K_i、K_m and V_max calculated by these five methods differed slightly.Non-linear-Regression Analysis was more convenient,reasonable,and reliable,and therefore was the first choice for the data processing of enzyme kinetics.3.The four models were used to test the inhibitory effect of triterpenoids from Cyclocarya paliurus suspension cell.The results showed that triterpenes had no inhibitory effect on invertase and amylase.And it had a little inhibitory effect on alpha starch glycoside enzyme.The IC₅₀ value was 0.698μg/μl.It had good inhibitory effect on alpha glucosidase.And the IC₅₀ value was 0.123μg/μl.4.Determining the inhibitory effect of 12 batches of Cyclocarya paliurus suspension cells of with alpha glucosidase inhibitor screening model and analysising the inhibitory material basis and the inhibitory type.Results showed that CPSC batch had the best inhibitory effect on alpha glucosidase.Total triterpene of this batch of cells containing ursolic acid,oleanolic acid,corosolic acid,hawthorn acid,betulinic acid.Five triterpenoid content were 13.9277,8.2629,14.2097,8.9226,2.0653μg/mg.The inhibiton of Corosolic acid on alpha glucosidase was the strongest in five kinds of triterpenoid,followed by ursolic acid.While betulinic acid had no inhibitory effect on alpha glucosidase.Corosolic acid was competitive and non competitive inhibitors,ursolic acid,oleanolic acid,and hawthorn acid were noncompetitive inhibitors.5.To explore the inhibitory mechanism of Cyclocarya paliurus suspension cells through fluorescence spectroscopy and molecular docking.The quenching mechanism of corosolic acid,ursolic acid,oleanolic acid and malonic acid belonged to static quenching.Corosolic acid had strongest fluorescence quenching effect on alpha glucosidase,and hawthorn acid could highly combine with alpha glucosidase.Molecular docking results showed the inhibitory effect of corosolic acid on alpha glucosidase was higher than that of oleanolic acid and it mainly dued to the hydrogen bonding effect of corosolic acid was stronger than that of oleanolic acid and the free energy of corosolic acid with alpha glucosidase was lower than that of oleanolic acid.In summary,triterpenoid isolated from Cyclocarya paliurus suspension cells had good inhibitory effect on alpha glucosidase.Corosolic acid and ursolic acid is the major active components.they were non competitive inhibitors and fluorescent static quenching agent.The inhibitory effect of corosolic acid on alpha glucosidase was higher than that of oleanolic acid and it mainly dued to the hydrogen bonding effect of corosolic acid was stronger than that of oleanolic acid and the free energy of corosolic acid with alpha glucosidase was lower than that of oleanolic acid.