Study On The Anti-diabetes Associated Activity And Mechanism Of Flavonoids In Lu’an GuaPian

Lu’an Gua Pian as famous traditional Chinese tea,which is one of the top ten teas consumed in China,produced in Lu’an,Anhui province.The health effects of Lu’an Gua Pian have become a research hotspot.The main active ingredients in Lu’an Gua Pian are tea polyphenols,flavonoids,alkaloids,tea saponins,tea polysaccharides,vitamins,amino acids and mineral elements.At present,there are many studies on the hypoglycemic activity of tea polyphenols and tea polysaccharides.Whether the flavonoids Lu’an Gua Pian have hypoglycemic effect deserves further study.Therefore,in this study,the anti-diabetic effects of flavonoids and flavonoid glycosides were preliminary studied based on the compounds isolated in the previous studies,and then a more active and higher content of flavonoids glycosides in Lu’an Gua Pian was screened.The effects and mechanism of the screened compound on anti-diabetes associated activity were investigated.The combination of plasma protein binding and degradation kinetics in the liver provides an experimental basis for the future use as a lead compound.The main research contents and results were as follows:(1)Inhibition of flavonoids on α-glucosidase and α-amylase.The compound 15(kaempferol-3-O-glucoside)showed inhibitory activity against α-glucosidase,and compound 13(kaempferol-3-O-rutinoside)showed higher α-amylase inhibition.Molecular docking suggested that 15 interacted with α-glucosidase mainly by hydrogen bonding,which was the same interaction force between 13 and α-amylase.Intrinsic fluorescence of α-glucosidase and α-amylase was quenched by 15 and 13,respectively,through a static quenching mechanism.The spontaneous formation of 15-α-glucosidase and 13-α-amylase complexes was driven by van der Waals forces and hydrogen bonding.The combination of enzyme inhibitory activity and high content of 13,13 was selected as the study object.And anti-diabetes associated activity and its mechanism of 13 were in-depth studied.(2)Protective effect of kaempferol-3-O-rutinoside on lipopolysaccharide induced inflammatory injury of H9c2 cardiomyocytes.H9c2 inflammatory injury model was induced by lipopolysaccharide.After pretreatment with kaempferol-3-O-rutinoside,this could significantly increase the survival rate of cells,and effectively decrease the protein expression levels of TLR4,My D88 and NF-κB.The results suggested that the mechanism of protection of lipopolysaccharide injury of H9c2 cardiomyocytes by kaempferol-3-O-rutinoside may be related to the inhibition of TLR4/My D88/NF-κB signaling pathway.(3)Protective effect of kaempferol-3-O-rutinoside on high glucose-induced apoptosis of PC12 cell.PC12 cell apoptosis injury model was induced by high glucose.After pretreatment with kaempferol-3-O-rutinoside,this could significantly increase the survival rate of cells,and effectively decrease the protein expression levels of Bax,Caspase-3,Caspase-9 and Caspase-12.The results have shown that kaempferol-3-O-rutinoside protect PC12 cell damage induced by high glucose by inhibiting the neuronal apoptosis pathway.(4)Determination of plasma protein binding of kaempferol-3-O-rutinoside.Determination of plasma protein binding rate of kaempferol-3-O-rutinoside by using ultrafiltration method,the results showed that the plasma protein binding of kaempferol-3-O-rutinoside was low,indicating that its blood content was high and its tissue distribution was more.(5)Study on the in vitro degradation kinetics of kaempferol-3-O-rutinoside.Liver homogenate can significantly degrade kaempferol-3-O-rutinoside,and this metabolic rate is accelerated with the increase of liver homogenate.The degradation kinetics of kaempferol-3-O-rutinoside in vitro is a first-order degradation reaction.In liver homogenates at concentrations of 5%,10%,and 25%,the half-lives were 7.92,7.12,and 6.14 h,respectively.The determination of the degradation half-life of kaempferol-3-O-rutinoside provides a certain experimental basis for the design of future pharmaceutical dosage forms.The highlights of this study were that we have systematically studied the anti-diabetes associated activity and its mechanism effect of kaempferol-3-O-rutinoside(13)in Lu’an Gua Pian,and also investigated tissue distribution and degradation kinetics in vitro,which will provide experimental evidence for its future clinical application and development as a lead compound.