Inhibitory Effect And Mechanism Of Catechins On Glycosidase And Protein Non-enzymatic Glycation

Type Ⅱ diabetes characterized by hyperglycemia is a non-insulin-dependent metabolic disease,and blood glucose levels higher than 140 mg/dL or 7.8 mmol/L at2 hours after a meal is generally one of its initial evidence.α-Amylase andα-glucosidase are two key glycosidase responsible for carbohydrate digestion,and the inhibition of them can delay the digestion and absorption of post-prandial carbohydrates to prevent postprandial hyperglycemia.In addition,in hyperglycemia,the non-enzymatic glycation of proteins（such as hemoglobin,albumin,low-density lipoprotein and lens protein）occur,which damages the structural and functional groups of the protein to form advanced glycated end products（AGEs）,leading to the development of diabetic complication.Tea known as“national drink”in China is one of the world’s three famous beverages,possessing the reputation of“healthy liquid,soul drink”.Catechins are the main polyphenolic compounds and the main functional components in tea,having many physiological activities including antioxidant,hypoglycemic,anti-inflammatory,hypolipidemic and anti-atherosclerotic,however,their inhibitions onα-amylase,α-glucosidase and protein non-enzymatic glycation are still relatively rare.Therefore,exploring the inhibitory effect and mechanism of catechins onα-amylase,α-glucosidase and protein non-enzymatic glycation helps to interpret the unique nutritional value of catechins,providing a theoretical basis for dietary prevention of diabetic and its complications using catechin as the functional factor,further offering scientific guidance and experimental basis for dietary nutrition.These are of great significance for promoting the application of catechin as food-borne inhibitor in the food industry to assist the development of tea industry.In this paper,three major components in catechins including epigallocatechin gallate（ECG）,epigallocatechin gallate（EGCG）and gallocatechin gallate（GCG）were selected as inhibitor ofα-amylase andα-glucosidase to analyze their inhibitory effect,inhibition kinetics,the binding properties and their effects on enzymatic structure,and investigating the interactions between catechin component and acarbose/catechin component in inhibiting enzyme activity.In addition,bovine serum albumin（BSA）–fructose and BSA–methylglyoxal（MGO）were used as models to investigate the inhibitory effect and mechanism of catechins on protein non-enzymatic glycation.Moreover,the effect of vitamin C or malic acid on EGCG inhibiting protein non-enzymatic glycation was explored.The main content and results were as follows:（1）ECG,EGCG and GCG had strong inhibitory effects onα-amylase,and their half-inhibitory concentrations(IC₅₀)were（45.30±0.22）,（137.15±0.13）and（30.02±0.23）μg/mL,respectively.The inhibition ability was from large to small:GCG>ECG>EGCG,their IC₅₀ were higher than the positive control acarbose(IC₅₀=27.76±0.19μg/mL),and the inhibition types ofα-amylase by three catechins were mixed type.Whenα-glucosidase was inhibited,the inhibitory types of them were mixed,mixed and non-competitive with IC₅₀ values of（4.03±0.01）,（1.05±0.02）and（1.15±0.32）μg/mL,respectively.The inhibition abilities were EGCG≥GCG≥ECG,they were significantly stronger than acarbose(IC₅₀=79.41±0.19μg/mL).In addition,the main interaction of inhibitingα-amylase was antagonistic when catechin was combined with acarbose/catechin,in the case ofα-glucosidase,the main interaction was synergistic between catechins,while it was additive effect between catechins and acarbose.（2）ECG、EGCG bound toα-amylase/α-glucosidase via hydrogen bonds and hydrophobic interactions with only one binding site,the interaction force between GCG andα-glucosidase was the same with it,however,Van der Waals force and hydrogen bonds drove the binding of GCG toα-amylase,these forces promoted the binding of catechins withα-amylase/α-glucosidase to form ground state complexes and quench the endogenous fluorescence of the enzymes.At 25℃,the binding constants（K_a）were（1.39±0.21）×10⁵ L/mol,（5.49±0.21）×10⁵ L/mol and（4.47±0.03）×10⁴ L/mol between the three catechins andα-amylase,and the values were（3.46±0.20）×10⁴ L/mol,（2.49±0.16）×10⁵ L/mol and（7.31±0.05）×10⁴ L/mol when they bound toα-glucosidase.The affinity of EGCG for both enzymes was stronger than ECG and GCG.Synchronous fluorescence and circular dichroism spectroscopy showed that ECG,EGCG and GCG caused changes in the tryptophan,tyrosine microenvironment and secondary structure ofα-amylase/α-glucosidase.Molecular simulations indicated that ECG and EGCG bound to the active center ofα-amylase/α-glucosidase to occupy the active site of enzyme,preventing the substrate from interacting with active amino acid residues to inhibit the enzyme activity,while GCG bound to a site close to the active center ofα-glucosidase causing the change of enzymatic conformation,occupying the channel of substrate access to the active site resulting in a decrease in enzyme catalytic activity.（3）Inhibitory effects of ECG,EGCG and GCG on non-enzymatic glycation of BSA induced by fructose were studied,including the phase products（fructosamine,dicarbonyl compounds and fluorescent AGEs）,functional groups of BSA（carbonylation and free sulfhydryl groups）,protein oxidation products（AOPP）,protein cross-linked structures as well as protein conformations and hydrophobicity effects.The results showed that ECG,EGCG and GCG could inhibit the formation of staged products,the inhibition activities for fructosamine and fluorescent AGEs were higher than that of dicarbonyl compounds;They can reduce the content of protein carbonylation,but the effect of preventing the reduction of sulfhydryl content was not obvious;They inhibited the formation of AOPP and amyloid cross-βstructures,alleviated secondary structure changes of BSA induced by fructose to some extent,and inhibited the decrease of the hydrophobicity of proteins caused by glycation.（4）The abilities of ECG,EGCG and GCG to scavenge free radicals,trap MGO,chelate Fe²⁺,reduce Fe³⁺and bind BSA were studied,the inhibitory effects and mechanisms of them on AGEs in BSA–MGO system were analyzed.The results showed that ECG,EGCG and GCG had strong scavenging activities（92.9%,50.2%and 85.3%）on O₂.^-,and the abilities to chelate Fe²⁺and reduce Fe³⁺were strong,which may prevent the oxidation process in the system.The catechins were able to efficiently trap MGO as an important precursor of AGEs,and when ECG trapped MGO（molar ratios were 1:3 and 1:10）to form ECG–mono-MGO adduct and ECG–di-MGO adduct,while two GCG–mono-MGO adducts were observed when GCG trapped MGO at the molar ratios of 3:1 and 6:1.ECG,EGCG and GCG were able to bind with BSA,the binding constant（K_a）was 10⁴ orders of magnitude,and the K_a value between ECG and BSA was larger.Inhibitory activities of the three catechins on AGEs in the BSA–MGO system were 98.1%,97.3%and 96.7%.The mechanism of catechin inhibit protein non-enzymatic glycation to reduce AGEs formation may be related to their inhibition activities of the oxidation process in the system,MGO trapping and BSA binding.（5）Impact of vitamin C or malic acid on EGCG inhibiting non-enzymatic glycation of BSA was investigated,including trapping MGO,inhibition of BSA carbonylation and AGEs formation,scavenging O₂.^-and binding affinity with BSA.The results showed that vitamin C promoted the trapping efficacy of EGCG while malic acid only exhibited a promoted effect for 8.9μg/mL EGCG when vitamin C/malic acid in the concentration range of 1.7–35.2μg/mL was present in EGCG–MGO system;At the same time,vitamin C promoted the inhibitory activity of EGCG on protein carbonylation and AGEs,while malic acid was not obvious;The effect of 17.6μg/mL vitamin C/malic acid promoted 8.9 and 35.4μg/mL EGCG scavenging O₂.^-was best;Both vitamin C and malic acid increased the binding affinity between EGCG and BSA,the binding constant（K_a）value increased from（6.86±0.19）×10⁴ L/mol to（7.42±0.13）×10⁴ L/mol and（7.76±0.18）×10⁴ L/mol),respectively;Compared with malic acid,vitamin C promoted the inhibition of protein non-enzymatic glycation by EGCG may be due to its enhanced efficiency in the respect of trapping MGO,scavenging O₂.^-and binding affinity with BSA.