Isolation And Purification Of Gynostemma Pentaphyllum Tea Polysaccharides And Their Immunomodulatory Activities On RAW264.7 Cells

Gynostemma Pentaphyllum is the medicine and dietary dual-purpose plant released by Ministry of health.The Gynostemma Pentaphyllum tea is a natural health tea,which is made of the buds,tender stems and leaves after withering,fixing,rolling and drying process.The G.pentaphyllum tea has obvious function of reducing blood fat,anti aging and improving immunity,because of its rich in saponins,polysaccharides,flavonoids andother active ingredients.Long-term drinking of the tea has no side effect to the body,so that the G.pentaphyllum tea is also called "second ginseng".At present,the research of G.pentaphyllum tea is mainly focused on the efficacy and mechanism of saponin.There are less studies about G.pentaphyllum tea polysaccharide.Therefore,this paper takes it as the research object.Given this,in this paper,Gynostemma pentaphyllum tea polysaccharides was taken as the object of study and its three polysaccharides fractions were extracted,isolated and purified.Their preliminary structural characterization,antioxidations and immunomodulatory activities in RAW246.7 cells were compared and then the mechanism of immune regulation activity of GTPP-3 was also studied.The results were as follows:1.Extraction and purification of polysaccharides from Gynostemma pentaphyllum tea.Ultrasound-assisted hot water extraction and ethanol precipitation were employed to get Gynostemma pentaphyllum tea polysaccharides and the extraction yield was 4.25%.Its three purified fractions named GTPP-1,GTPP-2 and GTPP-3 were further isolated and purified by Cellulose DEAE-52 and Sephadex G-150 chromatography.2.The preliminary structure characterizations were discussed by combine with UV,FT-IR,HPLC-UV,HPGPC,Congo-red experiment,I2-KI experiment and SEM methods.In addition,the antioxidant activities were further studied.The results indicated that the total carbohydrate contents measured by the phenol-H₂SO₄ assay for GTPP-1,GTPP-2 and GTPP-3 were 99.68%,99.29%and 99.47%.All the three GTPP fractions had no absorption peaks at 260 nm and 280 nm in UV spectrum,indicted that there were no nucleic acid and protein The monosaccharide compositions of GTPP-1,GTPP-2 and GTPP-3 were identified and quantified by the HPLC-UV analysis.The results showed that three of the components were all acidic heteropolysaccharides being mainly composed of mannose,glucose and galactose,and the proportions that sum of the three kinds of monosaccharide in each fraction were were 77.92%,77.82%,75.23%.According to HPGPC results,three fractions was homogeneous polysaccharides and the molecular weights of GTPP-1,GTPP-2 and GTPP-3 were 1695kDa,1723kDa and 2485kDa,respectively.FT-IR spectra of them all contained the characteristic absorption peaks of pyranoid rings.The results of SEM reflected that GTPP-1,GTPP-2 and GTPP-3 all covered more than two kinds of structures.GTPP-3 turned out to have triple-helix structure while other two components had no triple-helix structure according to Congo-red results.Finally,the results of I2-KI showed that three components of GTPP might have longer side chains or more branched chains.The antioxidant activities of GTPP-1,GTPP-2 and GTPP-3 were evaluated by antioxidant evaluation system in vitro.The results indicated that antioxidant activity of GTPP-3 was the strongest among the three fractions with certain antioxidant activities.3.RAW264.7 cells were treated with a given dose of GTPP-1,GTPP-2 and GTPP-3,respectively.Immunoregulatory activity was evaluated by observing the morphological changes and detecting cell proliferation activity,phagocytosis ability as well as secretion level of nitric oxide（NO）and cytokines（TNF-α,IL-1β,IL-6）.The results showed that macrophages were amplified slightly,and the cell edges grow pseudopodia,showing that the morphological characteristics of activated macrophages after stimulation with three components of GTPP.GTPP-1,GTPP-2 and GTPP-3 had no toxic effect on macrophages,and not only enhanced the phagocytosis of macrophages,but also remarkably promoted the secretion production of NO and cytokines.Especially,the immunoregulation of GTPP-3 was relatively better.4.Western blot and immunofluorescence staining were used to study the mechanism of GTPP-3 activation in RAW264.7 cells.TLR4 receptor blockers inhibited the release of NO and TNF-α induced by GTPP-3,suggesting that TLR4 is involved in the activation of macrophages by GTPP-3.Western Blot analysis showed that GTPP-3 could induce the phosphorylation of pERK1/2,JNK,p38 and Akt and the nuclear translocation of NF-κB p65 which were all located in TLR4 downstream.In addition,the specific inhibitors of the five proteins significantly inhibited the induction effect of GTPP-3 on NO and TNF-induced by macrophages.These results indicated that GTPP-3 activated macrophages probably through the MAPKs,PI3K/Akt and NF-kB signaling pathways.