Study On Purification,structural Identification And Biological Activities Of Polyphenols From Platycladus Orientalis(L.) Franco

Polyphenols possess special chemical and physiological activities.In recent years,polyphenols are more and more applied to cure or prevent diseases as natural healthy foods and herbal drugs.Franco leaves contains abundant polyphenols and have already been approved by CFDA to be used in health food.In this study,the purification,structure identification and biological activities of polyphenols from Platycladus Orientalis(L.)Francoleaves were investigated.Purification of polyphenolsfromFrancoleaveswas conducted with HPD-722 macroporous resin for theadsorption and desorption rates were the highest among the 10 different types of resin(HPD-300?D101?ADS-5?ADS-F8?AB-8?HPD-722?HPD-450?ADS-17?HPD-500 and NKA-9).The desorption kineties,static and dynamic adsorption discipline on HPD-722 resin were studied and the optimal conditions for the purification of polyphenols from Platycladus Orientalis(L.)Franco leaves with HPD-722 resin can be stated as follows: the optimum concentration of total polyphenols in loading solution was 1.4 mg/mL;the injection amount was 250 m L;the flow rate was seted as 1.0 m L/min;the ethanol concentration of eluant was selected as 40% and 80%(v/v).At the concentration of 0.2-1.4 mg/m L,the adsorption isotherm results illustrated that the polyphenols adsorption mechanism of HPD-722 was consistent with Langmiur and Freundlish model and there was no need to raise the temperature in the process of purification,that is to say,room temperature(25 oC)is the most suitable temperature.At last,the total polyphenols was separated into two fractions with different concentration of alcohol eluent which were named PF-A and PF-B.In the process of aging or disease,abundant radical ions are produced and the matter of the reaction is redox reaction.In order to explore the radical anion scavenging activity of PF-A and PF-B,the DPPH radical scavenging assay,hydroxyl radical scavenging assay,superoxide anion free radical scavenging assay and ABTS scavenging assay were conducted.Vc and gallic acid were used as positive control.Results showed that the IC50 value of PF-A for DPPH radical scavenging assay,hydroxyl radical scavenging assay,superoxide anion free radical scavenging assay and ABTS scavenging assay were 0.42 mg/mL,0.50 mg/mL,0.56 mg/m L and 1.27 mg/m L;the IC50 value of PF-B for DPPH radical scavenging assay,hydroxyl radical scavenging assay,superoxide anion free radical scavenging assay and ABTS+ scavenging assay were 0.33 mg/m L,0.61 mg/m L,0.64 mg/m L and 1.15 mg/m L.Trauma infection,the common disease of most organs and common and frequent disease all belongs to chronic inflammation.In order to investigated the anti-inflammatory effects produced by PF-A and PF-B,the experiment was conducted in a model of LPS-stimulated THP-1 cell line with the PF-A and PF-B concentration of 1.0,1.3,2.0 and 4.0 ?g/m L.The results demonstrated that both PF-A and PF-B inhibited gene expression and the secretion of LPS-induced pro-inflammatory cytokines(IL-6,Pro-IL-1? and TNF-?).Anti-inflammatory effect was maximum when the concentration of PF-A was 2 ?g/m L,while PF-B was 1.3 ?g/m L.Structural identification of PF-A and PF-B was conducted using HPLC-MS/MS and the main components in PF-A and PF-B were further determined by comparing the mass sepctrometry or the tandem mass sepctrometry of polyphenols published earlier.Analysis results showed that the main compounds of PF-A were Phellodendrine,Retusaphenol,Chlorogenic acid,Brevifolincarboxylic acid,rutin,Cynaropicrin,Afzelin,Vicenin,Aromadendrin,Myricetin;The main compounds of PF-B were Luteolin,Esculin,Apigenin,Kaempferal,Isorhoifolin,Neocryptomerin,Quercitrin,Phloretin,Amentoflavone,Quercetin.To further explore the inhibition mechanism of PF to xanthine oxidase,the suppress experiment was conducted to evaluate the inhibitory activity of PF-A and PF-B to xanthine oxidase,results indicated that the inhibitory activity of PF-B was higher than PF-A.Molecular docking was used to preliminarily evaluate the inhibition mechanism of the main polyphenols in Platycladus Orientalis(L.)Franco leaves on xanthine oxidase.Results indicate that luteolin,apigenin,myricetin,quercetin,rhizoma kaempferal all have strong inhibitory activity to xanthine oxidase and quercetin,myricetin,kaempferal inhibit xanthine oxidase by binding to the active site of the enzyme.While luteolin and apigenin inhibite the activity of xanthine oxidase by forming hydrogen bonds to ARG and THR amino acids of xanthine oxidase.Through the comprehensive analysis of the inhibitory effect on xanthine oxidase,antioxidant activity and anti-inflammatory activity of PF,we found that the inhibitory activity of PF on xanthine oxidase appears positive correlation with the DPPH free radical-scavenging activity,ABTS free radical-scavenging activity and anti-inflammatory activity.