The Synergistic Mechanism Of Flavonoids And 1-DNJ In Inhibiting α-Glucosidase

Diabetes is a serious public health issues and is extremely harmful to human.Inhibiting α-glucosidase can effectively control hyperglycemia.Several oral hypoglycemic agents which have been used clinically to inhibit the activity of α-glucosidase usually have many side effects,such as abdominal distention,flatulence.It is important to find safe and effective natural α-glucosidase inhibitors.Flavonoids and 1-DNJ have been reported to inhibit α-glucosidase and lower blood sugar.However,few studies have been conducted on its synergetic hypoglycemic effect.And the synergistic mechanism of flavonoids and 1-DNJ is still unclear.No studies have been conducted on the α-glucosidase subunit.In order to solve the above problems,this paper has carried out research from the following aspects:Firstly,the recombinant MGAM-C and MGAM-N were constructed and purified.And the inhibitory activity of flavonoid alone and 1-DNJ in combination was evaluated.The purity of MGAM-C/N proteins was up to 99% and the total yield was about 2 mg/L.For the tested flavonoids,scutellarein was noticeably the most effective with IC50 values of 80.1±3.8 μM,the activity decreased by the order of baicalein,quercetagetin,quercetin,and gallacetophenone.The 3’,4’-hydroxyl groups and the 5,6,7-hydroxyl groups was important for the inhibitory activity.The five compounds were combined with 1-DNJ,the MGAM-N was the major target of the flavonoid aglycones and 1-DNJ.The calculated CI values of baicalein and 1-DNJ were between 0.18 and 0.47,which showed the strongest synergistic inhibition.Only baicalein showed synergistic inhibitory effect with 1-DNJ against both MGAM-C and MGAM-N.The combination of baicalein and 1-DNJ decreased postprandial blood glucose and AUC levels in normal mice than the two individuals compounds in vivo,and its hyperglycemic effects is comparable with clinical dosage of acarbose.This result suggested that the PBG lowering effect of baicalein and 1-DNJ resulted from the inhibition of carbohydrate digestion.Secondly,the synergetic mechanism of baicalein and 1-DNJ investigated by a series of techniques including enzymatic assay,enzyme kinetics,molecular docking,fluorescence spectroscopy,and CD spectrum at the subunit level.The date indicated that the baicalein can bind to the non-competitive site of MGAM to influence the configuration of the active site of MGAM and enhance the affinity of the enzyme for 1-DNJ,consequently improving the inhibition of 1-DNJ against α-glucosidase.Finally,the traditional Chinese medicine food which can inhibit α-glucosidase was screened.Different extraction methods were evaluated,and the synergistic inhibitory activity of selected traditional Chinese medicine food and 1-DNJ was further evaluated.The results showed that 70% ethanol thermal reflux extraction of flavonoids was best.Among the seven kinds of traditional Chinese medicine food,the flavone content of sophora japonica is the highest,and the content of polysaccharides in longan was the highest.The extract of mulberry branch has a strong maltose inhibitory activity of mammalian α-glucosidase.HPLC-ESI-MS analysis showed that the main components of mulberry branch were flavonoids,and the combination of flavonoids and 1-DNJ could synergistic inhibit the maltose activity of α-glucosidase.This research provides new insights into the inhibitory mechanism of flavonoids and 1-DNJ on α-glucosidase and valuable information for the pharmacotherapy by combinations of different natural α-glucosidase inhibitors.