Isolation,Purification And Anti-Inflammatory And Antioxidant Activities Of Phenolic Compounds From Camellia Ptilophylla Chang

The paper aims to explore the functional characteristics of the active ingredients of the Camellia ptilophylla Chang.In this study,Camellia ptilophylla Chang was used as a material,extracted by 30%ethanol/ethyl acetate.Then using a hydroxypropyl dextran gel(Sephadex LH-20)column.Separation and purification by chromatography and preparative high-performance liquid chromatography,three kinds of polyphenolic monomer substances were obtained.The monomer structure was identified by high-performance liquid chromatography-tandem mass spectrometry,nuclear magnetic resonance,and infrared spectroscopy.To comprehensive evaluation of the antioxidant activity of three polyphenolic monomer substances,four kinds of Chemical methods involved DPPH,ABTS,FRAP ORAC,CAA method,and L-O₂hepatocyte oxidative stress model method.Using mouse macrophage RAW264.7 inflammation model,The anti-inflammatory activity and mechanism of polyphenolic monomer substances isolated from the leaves of Camellia ptilophylla Chang were studied by technical methods,including ELISA,RT-PCR and Western blot.The main results of this study are as follows:1.Isolation,Purification and Structural Identification of Polyphenols from Camellia ptilophylla ChangUsing Camellia ptilophylla Chang as experimental material,after 30%ethanol extraction and ethyl acetate extraction,30%ethanol/ethyl acetate extract was obtained,and the substance 1,substance 2 and substance 3 were separated and purified,and the purity of substance 1,substance 2 and substance 3 was 98%,96%and 97%,respectively.the yields were 2.45%,0.55%and 0.31%,respectively.The structure of the components was identified by high-performance liquid chromatography-tandem mass spectrometry,nuclear magnetic resonance and infrared spectroscopy.The substance 1 was identified as gallocatechin gallate(GCG),and the substance 2 was 1,2,4,6-tetragalylglucose(1,2,4,6-GA-glc),substance 3 is catechin-3,5-digallate(GC-3,5-di GA),the latter two are special polyphenols in Camellia ptilophylla Chang.2.Antioxidant activity of polyphenol monomer(1)Basically,the antioxidant results of DPPH,ABTS and FRAP are the same:GCG>GC-3,5-di GA>1,2,4,6-GA-glc,but the result of the ORAC method is opposite,1,2,4,6-GA-glc is stronger than GCG and GC-3,5-di GA.Considering GCG,1,2,4,6-GA-glc and GC-3,5-di GA has chemical resistance Oxidation ability,in which GCG has strong ability to scavenge free radicals.(2)CAA results showed that GCG,GC-3,5-di GA and 1,2,4,6-GA-glc inhibited DCF production in a dose-dependent manner;in H₂O₂-induced L-O₂cell oxidative damage model,GCG,GC-3,5-di GA and 1,2,4,6-GA-glc can significantly increase the cell survival rate,reduce the intracellular ROS content,and inhibit the release of LDH in the cell supernatant.Two kinds of anti-oxidation studies based on cell level showed that these three polyphenolic monomers have good antioxidant activity,among which 1,2,4,6-GA-glc have the strongest cellular antioxidant capacity,GC-3,5-di GA is second,and GCG is the weakest.3.Anti-inflammatory activity of polyphenolic monomer(1)GCG and GC-3,5-di GA can significantly inhibit NO and PGE₂production after mixed with 1?g/m L LPS for 24 h,GCG and GC-3,5-di GA inhibited NO production by IC₅₀values of 92.37±10.04 and 58.17±3.90?g/m L.At 100?g/m L,the inhibition rates of PGE₂production by GCG and GC-3,5-di GA were 38.75±5.42%and 24.64±2.42%,respectively.(2)GCG and GC-3,5-di GA can significantly inhibit TNF-?,IL-6 and IL-10 generated in cell supernatants with increasing concentration in the concentration range of 25-100?g/m L.(3)GCG and GC-3,5-di GA can down-regulate the transcription levels of i NOS,COX-2,TNF-?,IL-6 and IL-10 m RNA in RAW264.7 cells.(4)GCG and GC-3,5-di GA can down-regulate the expression of i NOS and COX-2proteins.