Extraction,Purification And Bioactivity Of Polysaccharide From Gracitaria Rubra

As a primary source of natural products,seaweeds are rich in various nutrient compounds,including polysaccharides,proteins,polyunsaturated fatty acids(PUFAs),carotenoids,polyphenols,terpenoids,etc.Gracilaria is a kind of benthic macroalgae widely distributed in tropical and subtropical seas,belonging to Rhodophyta,Florideae,Gigartinales,Gracilariaceae.Polysaccharide is a primary component of Gracilaria species,which has significant effects on immune,antitumor,antioxidant,anti-inflammatory,antidiabetic,antibacterial and antiviral activities.Gracilaria rubra is an endemic species in China.But there is little information about characterization and biological activities of polysaccharides from G.rubra.In this experiment,crude polysaccharide was extracted from G.rubra and purification was conducted to obtain pure fractions,of which preliminary characterization,changes after simulated digestion and fermentation in vitro,antioxidant and immunomodulatory activities was investigated.1.Extraction,purification and physicochemical property of GRPSGRPS was extracted from G.rubra by hot water,and the resulting GRPS was purified with DEAE-52 cellulose column and Sephadex G-50 column to afford three fractions:GRPS-1-1,GRPS-2-1 and GRPS-3-2.The contents of carbohydrate in GRPS,GRPS-1-1,GRPS-2-1 and GRPS-3-2 were 47.16 ± 1.08,45.61 ± 1.44,54.78± 0.59 and 52.72 ± 0.91%,respectively.Notably,the contents of sulfuric radical in GRPS,GRPS-1-1,GRPS-2-1 and GRPS-3-2 were 12.42±0.40,5.96±0.91,8.46 ±0.75 and 12.03 ± 0.80%.It was also found that GRPS had low content of uronic acid(<2%).In addition,the protein contents GRPS-1-1 were 0.16 ± 0.04%,and no protein was detected in GRPS-2-1 and GRPS-3-2.2.Preliminary structure characterization of GRPSThe molecular weights of GRPS-1-1,GRPS-2-1 and GRPS-3-2 were 1310,691 and 923 kD,which were evaluated by HPLC.The monosaccharide compositions of GRPS were measured using HPLC based on the PMP derivatization method.The results showed that GRPS-1-1,GRPS-2-1 and GRPS-3-2 were composed of Gal and Fuc with different molar ratios of 1.79:1,2.16:1 and 2.76:1,respectively.Besides,the FT-IR spectra of GRPS-1-1,GRPS-2-1 and GRPS-3-2 exhibited typical characteristic peaks of sulfated polysaccharide.3.Simulated digestion and fermentation of GRPS in vitroThe structural and chemical changes of GRPS were investigated by simulating oral,gastric,small intestine digestion conditions.The results of molecular weights,monosaccharide and reducing sugar determination indicated that GRPS couldn't be degraded.Gut microbiota were separated from fresh fecal.In vitro fermentation of GRPS was carried out in the simulated gut microbiota fermentation environment.The changes of molecular weights,monosaccharide,pH,total carbohydrate content,short-chain fatty acids content and the microbia structure during In vitro fermentation were determined.The results showed that some of GRPS was degraded to lower-molecular-weight carbohydrate,which can be utilized directly by fecal microbiota.During GRPS fermentation,the pH kept a downward trend and the total SCFAs production increased especially acetic,propionic and isobutyric acids.GRPS administration could modulate the microbial structure,lower the ratio of Firmicutes to Bacteroidetes and increase the relative abundance of some certain bacteria which can depolymerize and metabolize polysaccharide to gain energy from GRPS to satisfy the growth of microbiota.4.Antioxidant and immunomodulatory activities of GRPSThe superoxide anion radical scavenging activities and lipid peroxidation inhibitory activities of GRPS,GRPS-1-1,GRPS-2-1,and GRPS-3-2 were noticeable.GRPS and three fractions exhibited some extent of DPPH radical and ABTS radical scavenging activities.Nevertheless,all samples except GRPS-1-1 exerted limited scavenging abilities for hydroxyl radical.GRPS,GRPS-1-1,GRPS-2-1 and GRPS-3-2 also showed poor ferrous chelating activity(<15%).Furthermore,GRPS-1-1,GRPS-2-1 and GRPS-3-2 had the ability to protect PC12 cells from H2O2 induced oxidative injury and increase PC 12 cell viability,exhibiting satisfactory antioxidant effects.GRPS-1-1,GRPS-2-1 and GRPS-3-2 could increase RAW264.7 cell viabilities.Particularly,GRPS-3-2 had a stronger promoting effect and was selected for further studies.Experimental results showed that the treatment of GRPS-3-2 significantly enhanced phagocytic activity,acid phosphatase activity and NO production of RAW264.7 cell.GRPS-3-2 exhibited strong immunomodulatory effects on murine macrophage RAW264.7 cells,which might have a correlation with higher sulfuric radical content.