Study On Antioxidant, Antimicrobial And Inducing Cancer Cell Apoptosis Of Phloridzin And Its Derivatives

Phloridzin?PZ?,a flavonoid compound,had a wide range of biological activity.However,low bioavailability limit its development.Phloridzin myristate?PME?was synthesized by lipase-catalyzed acylation of PZ and myristic acid.The inhibition of xanthine oxidase?XOD?and lipid oxidation,and the ability to clear hydrogen peroxide?H₂O₂?,secondary chlorine acid?HClO?and nitric oxide?NO?free radicals were studied.The differences of antioxidant properties between PZ and PME were compared.Four food-borne microorganisms were compared the difference between the antibacterial activity of PZ and PME and explored its antibacterial mechanism by study effects of paper diffusion,double gradient dilution,cell membrane permeability,intracellular protein and nucleic acid leakage,intracellular membrane integrity and cell morphology.The differences of PZ and PME on the apoptosis of hepatocellular carcinoma?Hep-2?cells were compared and explored their pro-apoptotic mechanism,through MTT,flow cytometry,lactate dehydrogenase?LDH?,superoxide anion?ROS?,mitochondrial membrane potential,and glutathione?GSH?.The results were as follows.?1?The lipase-catalyzed acylation of PZ with myristic acid was identified by ¹H NMR and ¹³C NMR,and the yield of PME was 82%at the position of 6"-OH of phlorizin glycoside chain.?2?PME and PZ were reversible competitive XOD inhibitors,and the inhibition of XOD was in a concentration-dependent manner.The semi-inhibitory concentration(IC₅₀)of PME and PZ was 120.11 and 170.39?g/mL,respectively.The suppression constant?Ki?was 58.50 and 104.80?g/mL,respectively.The IC₅₀0 of PME to inhibit lipid oxidation was 123.38?g/mL,which was significantly lower than value of PZ(IC₅₀=144.55?g/mL).The IC₅₀0 value of PME to inhibit H₂O₂ and HClO was 50.07and 166.23?g/mL,respectively,which was significantly lower than that of PZ to clear H₂O₂ and HClO 76.09 and 219.19?g/mL.PME and PZ had no significant difference in the scavenging ability of NO radicals,which IC₅₀0 of PME was 223.26?g/mL and the IC₅₀0 of PZ was 196.95?g/mL.The antioxidant capacity of PZ was improved after inoculation with myristate.?3?The inhibitory effect of PME and PZ on Staphylococcus aureus was stronger than that of Listeria monocytogenes,Escherichia coli and Salmonella typhimurium.So,Staphylococcus aureus was selected as the target strain for the further study.The minimum inhibitory concentrations?MIC?of PME and PZ against Staphylococcus aureus were 2.7 mg/mL and 5.4 mg/mL,respectively.With treatment time PME group,its conductivity,260 nm absorbance,protein and?-galactosidase leakage concentration were significantly higher than PZ treatment group and control group.Through field emission scanning electron microscopy,PME and PZ treated Staphylococcus aureus cell membrane surface with particulate matter and associated with abnormal cells.PME treatment group showed more severe pore-like damage structure,however,the blank control group of cell morphology showed normal.Therefore,PME and PZ showed antibacterial ability by changing the permeability of the cell membrane and cell morphology.?4?Through MTT cell viability screening test,the toxicity of PME to Hep-2 cells was significantly stronger than that of prostate cancer cells?PC-3?.PME and PZ did not show toxic effects on human normal throat cells?HBE?by MTT cell viability screening test.In a certain concentration and time range,toxicity of PME to Hep-2cells was concentration and time-dependent manner.The effect of PZ on Hep-2 cells was not significant in the whole process.The effect of 20 mmol/L N-acetylcysteine?NAC?+15?g/mL PME on Hep-2 cells was significantly lower than that of PME.Flow cytometry showed that the apoptotic rate of Hep-2 cells was 59.32%when PME concentration was 15?g/mL,which was significantly higher than that of 15?g/mL PME+20 mmol/L NAC treatment group and 30 mg/mL PZ treatment group were48.29%and 18.70%,respectively.With the increase of PME concentration,the LDH activity and the fluorescence multiplier of Hep-2 cells increased LDH activity and ROS fluorescence were 352.25 U/L and 1.96,respectively,which were significantly higher than those in the PZ group and 20 mmol/L NAC+15?g/mL PME treatment group.The mitochondrial membrane potential Fluorescence density,SOD activity and GSH content decreased with the increase of PME concentration.When the concentration of PME was 15?g/mL,the corresponding values were significantly lower than those of PZ treatment group and 15?g/mL PME+20 mmol/L NAC treatment group.In conclusion,the bioactivity of PME obtained by enzymatic modification was improved remarkably and the antioxidant activity of PZ was improved.Through changing the permeability and cell morphology to inhibit food-borne microorganisms.PME promoted apoptosis of Hep-2 cells,using intracellular free radicals and mitochondrial membrane potential changes.