Extraction And Purification,structure Identification And Anti-inflammatory Activity Of Polysaccharide From Lignified Okra(Abelmoschus Esculentus(L).Moench)

Okra(Abelmoschus esculentus(L.)Moench)is a kind of food with high nutritional and medicinal value,which is favored by lots of consumers in China.The lignified okra is a byproduct of the processing of ripe okra for harvesting the seeds,and the polysaccharide is the main bioactive component of the lignified okra.In this paper,lignified okra polysaccharides were purified and characterized,and lipopolysaccharide(LPS)was used to stimulate mouse macrophages RAW 264.7 to construct an inflammation model to investigate the anti-inflammatory activity and mechanisms of polysaccharides.The main research contents and results are as follows.(1)Acquisition of polysaccharides from the lignified okra.AP1,AP2 and AP3 were extracted from the lignified okra by alcohol precipitation.Eight fractions,AP1-a,AP1-b,AP1-c,AP1-d,AP2-a,AP2-b,AP2-c and AP2-d,were separated from AP1 and AP2 by DEAE-Cellulose ion exchange chromatography.AP1-b,AP1-c,AP2-b and AP2-c were homogeneous polysaccharide components.(2)The physicochemical and rheological properties of lignified okra polysaccharides.The contents of total sugar of AP1,AP2 and AP3 were 49.97%,54.76% and 68.34%,respectively.Protein contents of AP1,AP2 and AP3 were 3.70%,4.36% and 7.73%,respectively.Uronic acid contents of AP1,AP2 and AP3 were29.99%,39.90% and 46.63%,respectively.The results of monosaccharide composition analysis showed that all the three polysaccharides were heteropolysaccharides containing various monosaccharides.Thermogravimetric analysis showed that the heat resistance of AP3 was worse than that of AP1 and AP2.Rheological studies showed that three kinds of polysaccharides showed pseudoplastic fluid behavior.AP2 and AP3 exhibited an elastic gel structure,while the gel structure of AP1 was greatly affected by concentration.(3)Physicochemical properties and structure characterization of purified polysaccharides from lignified okra.The molecular weight of AP1-b,AP1-c,AP2-b and AP2-c were 659 k Da,658 k Da,160 k Da and 669 k Da,respectively.The analysis of monosaccharide composition showed that AP1-b,AP1-c and AP2-b were composed of rhamnose,galactose and a small amount of arabinose and glucose.The molar ratios of AP1-b,AP1-c and AP2-b AP1-b,AP1-c and AP2-b were gal:rha:glu:ara=1.98:1.00:0.15:0.32,1.85:1.00:0.24:0.67 and 11.70:4.08:1.00:1.30,respectively.AP2-c was composed of galactose and rhamnose in the ratio of 1.44:1.00.The structure characterization of the homogeneous polysaccharide fractions revealed that,the backbone of AP1-b consisted of →4)-α-D-Rhap-(1→,→4)-β-D-Galp-(1→and →6)-β-D-Galp-(1→,branched chains were α-L-Araf-(1 and β-D-Glcp-(1→.The main chain of AP1-c consisted of →3,4)-α-D-Rhap-(1→ and →4)-β-D-Galp-(1→ with branched chains α-L-Araf-(1→ and β-D-Glcp-(1→.The main chain of AP2-b consisted of→3,4)-α-D-Rhap-(1→,→4)-β-D-Galp-(1→and →3,6)-β-D-Galp-(1→ with branched chains α-L-Araf-(1→ and β-D-Glcp-(1→.The backbone of AP1-b consisted of→3,4)-α-D-Rhap-(1→ and →4)-β-D-Galp-(1→,branched chain was β-D-Glcp-(1→.(4)Analysis of biological activities of polysaccharide fractions from the lignified okra.AP1-a,AP1-b,AP1-c,AP1-d and AP2-a,AP2-b,AP2-c,AP2-d were no cytotoxic to macrophages in the concentration range studied.In the research of LPS induced NO secretion by macrophage RAW 264.7,it was found that AP1-b and AP2-c could significantly inhibit LPS induced NO secretion by macrophage.RT-PCR analysis showed that AP1-b and AP2-c could significantly inhibit m RNA expression of related inflammatory mediators,which proved that AP1-b and AP2-c had remarkable anti-inflammatory activity.In the evaluation for the mechanism of anti-inflammatory activity,western blot analysis of NF-κB inflammatory pathway suggested that AP1-b and AP2-c could inhibit the activation of NF-κB pathway by effectively inhibiting the phosphorylation of IκB and p65 induced by LPS.AP1-b and AP2-c exerted anti-inflammatory activity by inhibiting the expression of related inflammatory mediators and the activation of NF-κB pathway.