Inhibitory Activity And Mechanism Of Calycosin And Calycosin-7-O-β-D-Glucoside On α-glucosidase

Objective:Flavonoids are the main active components of Astragalus membranaceus（AM）.It has been reported that Astragalus flavone can reduce the blood glucose level of diabetic mice.But the biological activity and mechanism of the single compound of Astragalus flavones are not entirely clear.α-Glucosidase（α-Glu）is a key enzyme in the hydrolysis process of polysaccharides.The decrease of enzyme activity plays a key role in reducing the blood glucose level of diabetes with type 2.Therefore,in this study,the interaction between the two active compounds of Astragalus flavone,calycosin（CA）and calycosin-7-O-β-D-glucoside（CAG）withα-Glu was studied.Methods:1.The effects of CA and CAG onα-Glu activity were studied by enzyme activity inhibition experiment in vitro.2.The effects of CA and CAG on the intrinsic fluorescence and secondary structure ofα-Glu were studied by fluorescence spectroscopy,fourier transform infrared（FT-IR）spectroscopy and circular dichroism（CD）.3.The possible binding modes of CA and CAG with theα-Glu were analyzed by Autodock molecular docking method.Results:1.The results of enzyme activity inhibition experiment showed that acarbose,CA and CAG could decreaseα-Glu activity in a concentration dependent manner.In addition,the inhibitory ability of CA(IC₅₀=39.45μM)and CAG(IC₅₀=174.04μM)onα-Glu were better than acarbose(IC₅₀=471.73μM),which indicated that CA and CAG had inhibitory effect onα-Glu activity and the order of activity was CA>CAG>acarbose.2.CA and CAG could form spontaneously CA-α-Glu and CAG-α-Glu complexes withα-Glu.CA and CAG reduced the intrinsic fluorescence intensity ofα-Glu by static quenching.Both CA and CAG had high affinity forα-Glu.And hydrophobic interaction played an important role in the formation of the two complexes.FT-IR results showed that CA could induce the rearrangement of the polypeptide carbonyl hydrogen bonding pattern ofα-Glu,while CAG had little effect on it.The results of circular dichroism showed that CA and CAG could induce the change of the secondary structure ofα-Glu,which would influence the activity ofα-Glu.3.The results of molecular docking simulation showed that the binding energy of CA and CAG toα-Glu were-6.76 and-5.62 kcal·mol^-1,respectively.Hydrogen bond is another force to maintain the stability of CA-α-Glu and CAG-α-Glu complexes.Besides,CA might bind to the allosteric site of the enzyme and the binding site of CAG might be near the entrance of the enzyme active site ofα-Glu.Conclusion:In this study,the inhibitory effect of CA and CAG,two active components of Astragalus flavonoids,onα-Glu activity was determined.And the spectral results showed that CA and CAG could quench the intrinsic fluorescence of the enzyme by spontaneously form complexes withα-Glu,respectively.CA and CAG could induce the change of enzyme conformation.Besides,molecular docking results showed that CA might bind to the allosteric site of theα-Glu to form CA-α-Glu complex,and CAG might form CAG-α-Glu complex by binding near the active site of theα-Glu.These results suggested that CA and CAG could induce the change of theα-Glu conformation by forming complex with enzyme,which might eventually hinder substrate bind with the active site ofα-Glu and inhibit the enzyme activity.