Inhibitory Effect Of Three Flavonoids On ?-Glucosidase Activity And Advanced Glycation End Products Formation

Currently,diabetes and its complications are a growing threat to human health.Alpha-glucosidase is an important hydrolase in the body.It can hydrolyze oligosaccharides to release glucose,which ultimately leads to an increase of postprandial blood glucose levels.In the case of long-term hyperglycemia,the production of advanced glycation end products?AGEs?in the body will be accelerated.Excessive accumulation of AGEs can lead to complications of diabetes,such as diabetic nephropathy,diabetic retinopathy,and diabetic vascular complications,etc.Since the current inhibitors of?-glucosidase and AGEs formation have certain side effects in long-term use.Therefore,it is particularly important to find a"multi-target"and high-efficiency drug that can inhibit?-glucosidase and prevent the production of AGEs at the same time.Flavonoids are ubiquitous in various natural plants.They have small toxic and side effects and possess various biological activities.Screening of effective inhibitors of?-glucosidase and AGEs production from natural plant active ingredients such as flavonoids has become the current research hotspot.In this study,the inhibition of?-glucosidase by three kinds of flavonoids?astilbin,baicalein and naringenin?were studied by the enzyme inhibition and inhibition kinetics experiments in vitro.Ultraviolet absorption spectroscopy,fluorescence spectroscopy,synchronous fluorescence spectroscopy,three-dimensional fluorescence spectroscopy,circular dichroism spectroscopy and molecular docking method were used to study the interaction mechanisms between astilbin,baicalein,naringenin and?-glucosidase.By constructing the non-enzymatic glycosylation model of bovine serum albumin?BSA?-glucose in vitro,the inhibition and inhibition mechanisms of astilbin,baicalein and naringenin on AGEs production were studied by fluorescence spectroscopy,circular dichroism spectroscopy and molecular docking method.The results showed that astilbin,baicalein and naringenin had strong inhibition ability to?-glucosidase,and their IC₅₀ values were?0.55±0.02?,?0.43±0.04?and?0.12±0.06?mM,respectively.The inhibitory ability of astilbin,baicalein and naringenin were superior to the positive control group acarbose.The inhibitory effects of three flavonoids on?-glucosidase were reversible reaction,and astilbin and baicalein were noncompetitive inhibitors,and naringenin was a competitive inhibitor.Between three flavonoids and?-glucosidase could form a ground state complex driven by hydrogen bonding and van der Waals force to cause endogenous fluorescence quenching of the enzyme.There was only one binding site between astilbin,baicalein and naringenin and?-glucosidase.The binding constants were?3.85±0.08?×10⁴,?7.93±0.06?×10⁵ and?4.80±0.08?×10⁴ L·mol^-1,respectively.Synchronous fluorescence spectroscopy and three-dimensional fluorescence spectroscopy results showed that these flavonoids caused the microenvironmental changes around the tryptophan residues of?-glucosidase,thereby leading to conformational changes of the enzyme.The results of circular dichroism spectroscopy indicated that astilbin,baicalein and naringenin induced the changes in the secondary structure of?-glucosidase.Molecular docking results suggested that astilbin interacted with Glu-361,Ile-362,Lys-363,Leu-364,Arg-428,Glu-429,Ile-431,Glu-443,Phe-444,Val-446,Gln-447,Asn-448,Ile-451,His-561,Leu-563 and Asp-568 residues of?-glucosidase.Baicalein interacted with Arg-26,Pro-34,Asn-129,Asp-202,Lys-203,Tyr-341,Phe-342,Gly-433,Asn-434and Glu-435 residues of?-glucosidase.Naringenin interacted with Glu-361,Ile-362,Arg-428,Glu-429,Ile-431,Phe-444,Gln-445,Val-446,Gln-447,Ile-451,Gly-556,Arg-557,Thr-558,Leu-563 and Pro-564 residues of?-glucosidase.Fluorescence spectroscopy results indicated that three flavonoids showed strong inhibition to fluorescent AGEs production.When the concentrations of astilbin,baicalein and naringenin were 0.25 mM,the inhibition rate of fluorescent AGEs formation in the reaction system were?52.34±0.07?,?32.45±0.08?and?34.98±0.12?%,respectively.Among them,astilbin had the best inhibitory effect.The results of circular dichroism spectroscopy showed that three flavonoids had protective effects on the changes of secondary structure of BSA induced by AGEs,and increased the content of?-helix.Molecular docking results showed that astilbin interacted with Arg-217,Lys-294,Glu-339,Tyr-340,Ala-341,Val-342,Glu-443,Pro-446 and Asp-450 residues of BSA.Baicalein interacted with Leu-197,Ser-201,Trp-213,Ser-343,Leu-346,Ser-453,Leu-456,Leu-480,Val-481,Arg-483 and Arg-484 residues of BSA.Naringenin interacted with Lys-273,Lys-275,Glu-276,Cys-277,Cys-288,Glu-291,Val-292 and Glu-293residues of BSA.In addition,astilbin,baicalein and naringenin had hydrogen bonding interactions with Arg-217,Arg-484 and Lys-273 residues on BSA,respectively.These results indicated that three flavonoids could inhibit the production of AGEs.The above results suggested that three flavonoids had good ability to inhibit?-glucosidase activity and production of advanced glycation end products.This study would provide theoretical and experimental basis of astilbin,baicalein and naringenin on the prevention and treatment of type 2 diabetes mellitus and its complications in the future.