Virtual Screening,activity Verification And Modification Design Of ?-amylase Inhibitors

Carbohydrates are the main source of energy for human,and long-term high carbohydrate diet can lead to the disorder of sugar metabolism and major diseases such as diabetes.Prevention and improvement of diabetes is becoming a research focus by interfering with glucose absorption and reduction postprandial blood sugar.?-amylase is responsible for catalyzing the hydrolysis of starch and is one of the main digestive enzymes.Finding and developing?-amylase inhibitors is an effective way to prevent and treat diabetes.Related studies have shown that the active components from many medicine food homology plants(MFHP)have obvious inhibitory effect on?-amylase,and have the potential to develop into?-amylase inhibitors and reduce postprandial blood glucose.At present,there is no systematic screening and mechanism research on the anti-?-amylase molecules of MFHP.Therefore,this project aims at delaying starch digestion and reducing postprandial blood sugar.The virtual screening of?-amylase inhibitors from MFHP compound database based on molecular docking with?-amylase as the target was performed along with the study on the mechanism of action of?-amylase inhibitors in delaying starch digestion.Moreover,new substances with improved activity can be further obtained through enzymatic modification,and its application as dietary supplement and nutritional supplement in the auxiliary treatment of diabetes mellitus and reducing postprandial blood sugar can be expanded.Main research contents and results was as follows:The?-amylase inhibitors in MFHP compound database were rapidly identified by virtual screening method based on molecular docking and vitro?-amylase inhibitory activity experiment.Firstly,the MFHP compound database was established,and the molecular docking between?-amylase and the small molecular compounds in the MFHP database was carried out by Autodock Vina.According to the sorting of binding energy,the 547 potential hit active compounds were selected for secondary cross docking using CDOCKER and Surflex-Dock.After comprehensive consideration of three scoring functions,24 potentially active compounds were screened out for bioactive verification.These 24 compounds can be divided into flavonoids,flavanols and isoflavones according to their molecular structures,indicating that only certain polyphenols were effective inhibitors against?-amylase.Hierarchical cluster showed that His305,Asp300,Glu233,Val163,Tyr62,Gln63,Trp59 were the key contributing amino acids to the interaction between the ligands and?-amylase.The inhibitive rate of EGCG,ECG and procyanidin against?-amylase was 9.98%,25.82%and 21.33%at the same concentration(0.5?mol/L),which was higher than that of other tested samples,indicating that flavan-3-ol polyphenols had good anti?-amylase activity.It was proved that virtual screening can be applied to rapid screening and identification of amylase inhibitors.The mechanism of flavan-3-ol polyphenols(ECG,EGCG and procyanidin)delaying starch digestion was studied from the two perspectives of inhibiting the activity of?-amylase and the interaction with starch.The interaction characteristics of flavan-3-ol polyphenols with?-amylase were studied from five aspects:enzyme inhibition experiment,inhibition kinetic type,fluorescence spectra,circular dichroism and computer simulation.The results showed that ECG,EGCG and procyanidin were effective?-amylase inhibitors,and their IC50 values were 172.21±0.22?g/m L,732.15±0.13?g/m L and 504.45±0.19?g/m L,respectively.The Lineweaver Burk double reciprocal curve showed that the inhibition types of ECG,EGCG and procyanidin against?-amylase were mixed inhibition,noncompetitive inhibition and mixed inhibition,respectively.The fluorescence spectra showed that ECG,EGCG and procyanidin could produce static quenching of?-amylase.Thermodynamic parameters,molecular docking and molecular dynamics simulation showed that hydrogen bond and van der Waals force were the main driving forces of ECG and EGCG-?-amylase complex,while procyanidin-?-amylase complex was driven by hydrophobic interaction.In addition,the spectra of starch iodine complex showed that ECG and EGCG had strong interaction with amylopectin and amylose,while procyanidin had weak interaction with amylopectin and amylose,and the interaction of EGCG with starch was stronger than that of ECG.The results of Englyst digestion in vitro showed that the addition of 10%EGCG,ECG and procyanidin significantly reduced RDS content(9.84%,3.64%,29.51%)and increased RS content(11.90%,6.18%,25.92%).Enzymatic synthesis of ECG acylated derivatives and their effects on starch digestion were investigated.The optimal conditions of enzymatic synthesis of ECG derivatives were determined by single factor experiment as follows:the amount of lipase TL IM was 10%(w/w substrate),the molar ratio of substrate ethylene/butyrate:ECG was 50,and the reaction temperature was 50?for 5 days.Under these conditions,the acetylation conversion of ECG and butyrylation conversion of ECG were 70.16%and 72.73%,respectively.The acetylated and butyrylated derivatives of ECG were separated by silica gel column chromatography using chloroform,methanol and formic acid as elution phase at the ratio of 9:1:0.1.The isolated compounds were identified as 5"-1-O-acetyl ECG(1A-ECG),3",5"-2-O-acetyl ECG(2A-ECG),5"-1-O-butyryl ECG(1B-ECG),3",5"-2-O-butyryl ECG(2B-ECG)by ~1H-NMR.The IC50 values of 1A-ECG,2A-ECG,1B-ECG and 2B-ECG against?-amylase were 144.22±0.11?g/m L,426.96±0.24?g/m L,235.76±0.15?g/m L and 545.34±0.27?g/m L,respectively.The fluorescence spectra showed that 1A-ECG,2A-ECG,1B-EC-G and 2B-ECG could produce static quenching of?-amylase.Thermodynamic parameters and molecular dynamics simulation show that hydrogen bond and van der Waals force are the main driving forces of 1A-ECG-?-amylase complexes,while 2A-ECG,1B-ECG,2B-ECG and?-amylase complexes are driven by hydrophobic interaction.In Vitro Englyst digestion results showed that compared with the negative control ECG,when the addition amount was 10%,the co-gelatinization of acylated products and corn starch could form more resistant starch.The content of RS formed by acetylated products of ECG did not change significantly with the increase of addition amount,while the content of RS increased gradually with the increase of addition amount of butyrylated products of ECG.These results indicated that selective substitution of ECG by enzymatic acylation can improve the enzyme inhibitory activity and form more resistant starch.