Structural Characterization And Anti-inflammatory Activity Of UV/H₂O₂-treated Polysaccharide From Gracilaria Lemaneiformis

Gracilaria lemaneiformis(G.lemaneiformis),a traditional and edible economical seaweed in China,is rich in various functional components such as polysaccharides,proteins,fatty acids and amino acids.Previous reports showed that polysaccharide from G.lemaneiformis had abundant biological activities.However,the poor solubility,high molecular weight and high viscosity of the water-extraction polysaccharide prepared from G.lemaneiformis have limited their application.Therefore,in the present study,the effects on structural characteristics of G.lemaneiformis polysaccharides(GLP)after UV/H₂O₂ treatment with different irradiation time were studied.The degradation product with the best anti-inflammatory effect was selected using the IEC-6 cell model with inflammation induced by Lipopolysaccharide(LPS)for further separation and purification.The structural characteristics of purified fractions were analyzed using high-performance liquid phase(HPLC),infrared spectroscopy(FT-IR),scanning electron microscopy(SEM),atomic force microscopy(AFM),and nuclear magnetic(NMR)methods.The anti-inflammatory activity and intestinal barrier protection of G.lemaneiformis polysaccharide were studied using enzyme-linked immunosorbent assay(ELISA),real-time quantitative PCR(RT-q PCR),cell scratch assay and flow cytometry.This study results would supply a theoretical foundation for the further development and utilization of G.lemaneiformis polysaccharide and the main results were listed as follows:(1)GLP was prepared by water extraction and the degradation products were obtained from GLP after UV/H2O2 treatment.The effects on chemical compositions and physiochemical properties of GLP after UV/H₂O₂ treatment with different irradiation time were studied.After UV/H₂O₂ treatment for 30 min,the content of total sugars,protein and uronic acids decreased and the content of sulfate did not show significant change(p>0.05).The molecular weights of three degradation products(5-GLP,15-GLP and 30-GLP)after UV/H₂O₂ treatment for 5,15and 30 min were 351.76,108.37 and 48.62 k Da,respectively(The molecular weight of GLP>700 k Da).The monosaccharide types of G.lemaneiformis polysaccharide before and after UV/H₂O₂ treatment had few changes,while the ratio of galactose increased and the ratio of glucose and galacturonic acid decreased in the degraded products.In addition,the particle size and apparent viscosity of GLP decreased and the microscopic morphology was changed.The functional groups of GLP were not changed significantly based on the FI-TR spectra and I₂-KI assay analysis.The degradation of GLP after might be attributed to free radicals of UV/H₂O₂system attacking the glycosidic bonds in the main chain.(2)In the IEC-6 cell model with inflammation induced by LPS,the degradation product(5-GLP)with the best anti-inflammatory activity was selected based on the production of nitric oxide(NO),tumor necrosis factor-?(TNF-?)and interleukin-6(IL-6)in IEC-6 cells.5-GLP was separated and purified using a DEAE Sepharose Fast Flow agarose gel column chromatography.Then three purified fractions(GLP-1.0M,GLP-1.4M and GLP-1.6M)with higher recovery rate were obtained and their recovery rate were 8.95%,10.56%and 21.37%,respectively.(3)The chemical compositions and structural characteristics of the three purified fractions from G.lemaneiformis polysaccharide were studied.The contents of sulfate in GLP-1.0M,GLP-1.4M and GLP-1.6M ranged from low to high,which showed that the acidity of purified fraction increased with the increase of the concentration of Na Cl.There were few proteins in the purified fractions.The molecular weight of GLP-1.6M was 250.39 k Da with the best homogeneity.The microscopic morphology of three polysaccharide purified fraction was quite different from 5-GLP before purification.The AFM images of GLP-1.6M presented a curved chain shape and indicated the roughness of GLP-1.6M increased compared to that of 5-GLP.The FI-TR spectrum and NMR spectroscopy analysis showed that GLP-1.6M was a typical red alga sulfated polysaccharide,containing the linear backbone of?-(1?3)-and?-(1?4)-linked galactosyl residues.The content of?-glycosidic bond was more than that of?-glycosidic bond.(4)The anti-inflammatory activity of G.lemaneiformis polysaccharide was studied using ELISA assay and RT-q PCR.The results showed that 5-GLP,GLP-1.0M,GLP-1.4M and GLP-1.6M could significantly inhibit the production of NO and pro-inflammatory cytokines(TNF-?,IL-6 and IL-1?).GLP-1.6M exerted anti-inflammatory activity by down-regulating the expressions of inducible NO synthase(i NOS),receptor(TLR-4)and pro-inflammatory cytokines(TNF-?,IL-6 and IL-1?),and up-regulating the expression of anti-inflammatory cytokine(IL-10)in IEC-6 cells.The results indicated that GLP-1.6M might improve the inflammatory response by regulating the TLR-4/NF-?B pathway.(5)The mechanism of intestinal barrier protection of G.lemaneiformis polysaccharide was studied using RT-q PCR,cell scratch assay and flow cytometry.The results illustrated that GLP-1.6M up-regulated the expressions of mucin(MUC-2)and tight junction proteins(ZO-1,Occludin and Claudin-1)to enhance the intestinal barrier function.The degradation product 5-GLP and purified fraction(GLP-1.6M)promoted IEC-6 cell migration and cell proliferation to protect the intestinal barrier.