Degradation Behavior Of Dihydromyricetin In Ampelopsis Grossedentata And Bioactivity Of Its Products

This study evaluated the detection method of total flavone content,the separation,purification,degradation and degradation product structure of dihydromyricetin from the Ampelopsis grossedentata.And the activities of dihydromyricetin and its degradation products were studied by antioxidant,pro-oxidant,enzyme inhibition and antibacterial tests in vitro.This provides a reference for the rational development and utilization of dihydromyricetin.The specific research results are as follows:1.Six components of total flavonoids in Ampelopsis grossedentata?TF?were identified by LC-MS,which were dihydromyricetin,quercetin,myricetin,myricetin,dihydroquercetin and kaempferol.The direct method,differential spectrophotometry,AlCl₃ colorimetric method and Al?NO₃?₃-NaNO₂-Na OH colorimetric method?Al?NO₃?₃ colorimetric method?were compared in the determination of TF.It was found that the TF could be accurately determin by the differential spectrophotometry.The TF with single interference could be accurately determin by the direct method and the Al?NO₃?₃ colorimetric method.The TF with complex interferences could be accurately determin by AlCl₃ colorimetric method.Dihydromyricetin?DMY?was purified by preparative HPLC from TF.The purity of DMY was 99.50±0.27%and the yield was57.99±0.21%when the mobile phase was methanol-water?35:65,V/V?,the elution time was 30 min,the flow rate was 8 mL/min,and the sample volume was 600?L.2.The degradation of DMY in cell culture conditions?DMEM medium,37°C,pH 7.4?and simulated physiological conditions?PBS,37°C,p H 7.4?was detected by HPLC,and the structure of degradation products was analyzed by HPLC-QTOF-MS/MS.The results showed that the degradation rate of DMY in DMEM and PBS was first-order kinetics with half-life of 4.01 d and 5.50 d,respectively.The degradation products of DMY in DMEM and PBS were the same.According to the MS²information,it is inferred that the three degradation products may be produced by dehydrogenation,oxidative polymerization and reduction of DMY.3.The antioxidant and pro-oxidant activities of DMY and dihydromyricetin degradation products mixture?DDPsM?in vitro were investigated and evaluated by chemical antioxidant experiments and cell-based antioxidant and pro-oxidant experiments.The results of chemical antioxidant experiments in vitro showed that DMY,TF and DDPsM had a certain scavenging ability to ABTS and DPPH,and DMY had a better activity.FRAP results showed that DMY had better reduction ability.Antioxidant experiments based on erythrocytes showed that DMY(erythrocyte hemolysis rate IC₅₀=125.52±1.79?g/mL)had a better inhibitory effect on AAPH-induced hemolysis and lipid peroxidation than DDPsM(erythrocyte hemolysis rate IC₅₀=131.72±2.16?g/mL).The results of cell-based pro-oxidation experiments showed that the promotion effect of DDPs M was stronger than DMY.4.The hypoglycemic and bacteriostatic activities of DMY,TF and ddpmsm were investigated by in vitro enzyme inhibition and bacteriostatic experiments.The results of in vitro enzyme inhibition experiments showed that DDPsM(IC₅₀=0.132±0.0031 mg/mL)and TF(IC₅₀=0.391±0.010 mg/mL)inhibited the activity of?-glucosidase more strongly than DMY(IC₅₀=2.986±0.073 mg/mL)and acarbose(IC₅₀=1.511±0.027 mg/mL).DDPsM(IC₅₀=1.114±0.020 mg/mL)and DMY(IC₅₀=1.442±0.029 mg/mL)inhibited the activity of?-amylase more than TF(IC₅₀=1.589±0.033 mg/mL),but both were weaker than acarbose(IC₅₀=0.202±0.0053 mg/mL).The results of in vitro bacteriostatic experiments showed that Staphylococcus aureus was the most sensitive to DMY?minimum bactericidal concentration,MBC=0.125 mg/mL?,TF?MBC=0.125 mg/mL?and DDPsM ?MBC=0.0625 mg/mL?,followed by Pseudomonas aeruginosa.And the antibacterial activity of DDPs M and TF was stronger.