Isolation,Purification Of Polygonum Multiflorum Polysaccharide And Its Antioxidant,Anti-Inflammatoryactivity

Polygonum multiflorum is the dried tuberous root of polygonaceae,which contains anthraquinone,stilbene glycosides,polysaccharides and other active components,and has various pharmacological activities such as antioxidant,hypolipidemic,antitumor and so on.Polysaccharides are one of the main active substances in polygonum multiflorum,which have high research value.OBJECTIVE:to investigate the antioxidant activity of Polygonum multiflorum homogeneous polysaccharides in vitro and their protective effects against lipopolysaccharide（LPS）-induced inflammation and anti-imflammatory mechanism in RAW264.7 cells and zebrafish.METHODS:the crude polygonum multiflorum polysaccharides（PMP）were obtained by water extraction and alcohol precipitation.Then the crude polysaccharides were deproteinized and decolorized by macroporous resin,and they were separated by DEAE-52 cellulose column chromatography to obtain different polysaccharides fractions.After that,those fractions were sieved by Sephadex G-100dextran gel column chromatography to obtain homogeneous fractions of polygonum multiflorum polysaccharides.The antioxidant effect of Polygonum multiflorum polysaccharides in vitro were evaluated by using the scavenging ability of ABTS and DPPH free radicals.The inflammation model of LPS-induced RAW264.7 cells in vitro were used to evaluate the pro-inflammatory factor inhibitory activity of polygonum multiflorum polysaccharides by measuring interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）and nitric oxide（NO）levels,meanwhile,the inflammation model of LPS-induced zebrafish in vivo was constructed to further evaluate the anti-inflammatory activity of polygonum multiflorum polysaccharides by measuring hatching rate,heartbeat rate,yolk area,ROS production rate and NO production rate.RESULTS:The neutral polysaccharide PMP-1 and the acidic polysaccharide PMP-2 were obtained from the extraction and purification of polygonum multiflorum,in which the glyoxylate contents of PMP-1 and PMP-2 respectively were 1.27%and12.53%.The experiment of free radical scavenging efficiency showed that polygonum multiflorum polysaccharides have strong antioxidant ability.when the concentration of polygonum multiflorum polysaccharides was 12 mg·m L^-1,the scavenging rates of ABTS by PMP-1 and PMP-2 respectively were 67.70%and 75.02%,when the concentration of polygonum multiflorum polysaccharides was 4 mg·m L^-1,the scavenging rates of DPPH by PMP-1 and PMP-2 respectively were 35.03%and46.96%.The experiments on inflammation in RAW 264.7 cells in vitro showed that when the concentrations of polygonum multiflorum polysaccharides reached above 50μg·m L^-1,the polysaccharides-treated groups under the concentrations were able to significantly inhibit the secretion of IL-6,TNF-αand NO compared with the LPS-induced group（P<0.01）,showing good anti-inflammatory activity.Then,the experiments on inflammation in zebrafish showed that the polysaccharides-treated groups had significant anti-inflammatory effects in vivo.The10μg·m L^-1polysaccharides-treated groups could slow down the heart rate of zebrafish larvae,which showed significant differences with the heart rate of LPS-induced group（P<0.05）,in addition,50μg·m L^-1polysaccharides-treated groups could cause the yolk sac area to gradually become smaller in zebrafish larvae,and also could make the levels of ROS and NO decrease,showing significant differences compared with the LPS-induced group（P<0.05）.Finally,it could be learned that the acidic polysaccharides PMP-2 showed higher activity than the neutral polysaccharides PMP-1 in scavenging ABTS and DPPH free radicals,inhibiting the secretion of pro-inflammatory factors TNF-α,IL-6 in RAW264.7 cells and reducing ROS and NO levels in zebrafish,which showed significant differences between them.CONCLUSION:This study demonstrated polygonum multiflorum polysaccharides have good antioxidant and anti-inflammatory activities,providing a scientific basis for the use of polygonum multiflorum polysaccharides as a natural and effective anti-inflammatory component.