Analysis Of Portulaca Oleracea L. Polysaccharide And Evaluation Of Its Anti-ultraviolet And Whitening Efficacy

Objective:A variety of polysaccharides were extracted and separated from Portulaca oleracea L.(PO)by different enzyme assisted extraction and gradient ethanol precipitation,the fraction with the strongest whitening and anti-photoaging activities were screened and their compositions were analyzed.The anti-photoaging and whitening activity mechanism of polysaccharide of PO(POP)was investigated in vitro and in vivo.The aim is to laid the foundation for further development and application of PO in cosmetics industry.Methods:Firstly,16 fractions were extracted by using four kinds of enzymes(cellulase,α-amylase,β-glucanase,Viscozyme)assisted extraction combined with step-gradient ethanol precipitation.By measuring the scavenging activities of the 16 fractions on DPPH free radical and hydroxyl free radical,the fraction with the strongest antioxidant ability in vitro were selected for further experimental study.The contents of polysaccharide,uronic acid,proteins alkaloids,flavonoids and polyphenols in the active component VPOP3were determined by phenol-sulfuric acid method,M-hydroxybiphenyl method,Bradford method,acid dye colorimetry method and colorimetric method.HPLC,GPC and FT-IR were used to analyze the monosaccharide composition,molecular weight(MW)and main functional groups of VPOP3.In vitro experiments,600m J/cm~2UVB induced Ha Ca T cells were used to establish photoaging damage model in vitro.MTT method and DCFH-DA probe were used to detect cell survival rate and intracellular ROS production.Hoechst33342staining and flow cytometry were used to analyze cell apoptosis.The expression of Bcl-2,Bax,caspase-3 and Cyto-C proteins in the apoptotic pathway was detected by western blot to verify the effect of VPOP3 at the protein level,so as to evaluate the anti-photoaging activity of VPOP3 in vitro.B16-F10 cells were induced by 1.5μMα-MSH to secrete excessive melanin,and then treated with different concentrations of VPOP3.The intracellular melanin content and tyrosinase activity were detected,and the expression levels of MITF,TYR,TRP-1 and TRP-2 proteins in the melanin synthesis pathway were detected to evaluate the whitening activity of VPOP3.Using zebrafish as an animal model,ROS,apoptosis and lipid peroxidation in zebrafish after UVB induction were detected with different concentrations of active fraction,and the content of melanin and tyrosinase activity in zebrafish afterα-MSH induction were detected.The anti-photoaging and whitening activities of VPOP3 were evaluated in vivo.Results:In the 16 fractions,using Viscozyme to assist extraction,the polysaccharide VPOP3 obtained by 80%vol ethanol precipitation showed the strongest antioxidant ability.Analysis of chemical composition of VPOP3 showed that the content of polysaccharides in VPOP3 was 56.8%,the content of uronic acid was 20.7%,and there were a small amount of proteins and polyphenols in VPOP3.The monosaccharide composition and molar ratio of VPOP3 was composed of Man,Rha,Glu A,Gal A,Glu,galactose Gal,and Ara,with a molar ratio of 0.49:1.37:0.11:1.00:0.79:1.89:2.18,arabinose has the highest molar ratio.The MW of VPOP3 is mainly distributed in 0.71 KDa,the chain of polysaccharide molecule is composed of pyran sugar ring andβ-glycosidic bond.In vitro studies have shown that VPOP3 can scavenge excessive ROS in Ha Ca T cells induced by UVB,improve cell survival rate,up-regulate the expression of Bcl-2 in cells,reduce the expressions of Bax,caspase-3 and Cyto-C proteins expression,and inhibit cell apoptosis.VPOP3 can reduce melanin production and tyrosinase activity in B16-F10 cells,inhibit the expression of MITF,TYR,TRP-1 and TRP-2 in the melanin synthesis pathway,and play a whitening role.In vivo experiments have shown that VPOP3 can inhibit ROS generation and cell apoptosis in zebrafish,inhibit lipid peroxidation,melanin synthesis and tyrosinase activity in zebrafish,and exert its anti-photoaging and whitening effects.Conclusion:VPOP3 is a low MW acid heteropolysaccharide,the MW is mainly distributed in 0.71KDa,the main monosaccharide of VPOP3 was mannose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose and arabinose,arabinose has the highest molar ratio.VPOP3 effectively inhabited ROS production in cells and zebrafish and reduced lipid peroxidation levels in zebrafish.In addition,VPOP3 inhibited UVB-induced apoptotic body formation and apoptosis by down-regulating caspase-3,Cyto-C and Bax and up-regulating Bcl-2 in mitochondria-mediated apoptotic signaling pathway.On the other hand,high concentration of VPOP3 can significantly down-regulate the expressions of MITF,TYR,TRP-1 and TRP-2 in the melanin synthesis signaling pathway,inhibit the synthesis of melanin and the activity of tyrosinase to achieve whitening effect.