Isolation,Characterization And Anti-inflammatory Activity Of Gelidium Pacificum Okamura Polysaccharide

In recent years,polysaccharides isolated from seaweed have attracted more and more attention.It has been reported that sulfated polysaccharides derived from seaweed not only have antioxidant,anti-inflammatory and anti-tumor activities but also have few side effects.Therefore,it has broad market prospects in industries such as cosmetics and functional foods.Gelidium pacificum Okamura has a large amount of carbohydrates and is currently used for consumption or extraction of agar.At present,its comprehensive utilization is low,and research reports are relatively few.This study made a supplement to the research on Gelidium pacificum Okamura and provided theoretical basis for the isolation of polysaccharides and researched on its biological activities.In this paper,the research status of Gelidium pacificum Okamura was summarized,the isolation method of its polysaccharides was studied,and the structure of purified polysaccharides was analyzed.Its antioxidant activity and anti-inflammatory activity have also been studied.Compared with the traditional aqueous extraction,ultrasound-assisted extraction method had higher extraction rate and greatly saved extraction time.Therefore,ultrasonic-assisted extraction was used to extract the polysaccharide of Gelidium pacificum Okamura.After single factor and response surface experiments,the optimal extraction conditions for crude polysaccharide from Gelidium pacificum Okamura were determined as ultrasonic temperature 85?,ultrasonic time 50 min,liquid to material ratio 36 mL/g,and polysaccharide yield was approximately 31%.After removal of the protein using Sevag method,The crude polysaccharide was dialyzed,lyophilized and then purified.The polysaccharide was purified by DEAE-52 cellulose column and Sephadex G-100 gel column,eluted with 0.3 mol/L NaCl.The eluate was dialyzed and lyophilized to get a purified polysaccharide GPOP-1.The total sugar content,protein content,uronic acid content,and sulfate content were determined by phenol–sulphuric acid method,BCA method,sulfuric acid-carbazole method,and barium sulfate turbidimetry,respectively.Studies have shown GPOP-1 contains total sugar(88.93%)and uronic acid(19.76%),sulfate ester(8.80%),and almost no protein.The monosaccharide composition of GPOP-1 was mainly xylose,galactose and galacturonic acid in a molar ratio of 1 : 8.41 : 2.79,and its weight average molecular weight(Mw)is 28807 Da.Fourier Transform Infrared Spectroscopy(FT-IR)also showed that GPOP-1 contained uronic acid and sulfate ester.According to the above analysis and nuclear magnetic resonance spectroscopy(NMR)analysis,GPOP-1 comprised of 1,4-linked-?-D-Galp3 S,1,2-linked-?-D-Xylp and 1,3-linked-?-D-GalpA residues,respectively.The inflammatory cell model could established by LPS acting THP-1 cell line and was used to determine the anti-inflammatory activity of GPOP-1 by MTT assay,quantitative reverse transcriptase polymerase chain reaction(RT-PCR)analysis,and Western immunoblot analysis in vitro.GPOP-1 at a concentration of 5 ?g/mL fully protected the THP-1 cells against LPS-stimulated cytotoxicity.Measurement of NO production initially indicated that 5 ?g/mL of GPOP-1 significantly inhibited the LPS-induced inflammatory response.Therefore,the polysaccharide concentration was determined to be 5 ?g/mL,which was used for the following investigation.The effect of GPOP-1 on the expression of inflammatory factors TLR4,MyD88 and TRAF6 at mRNA and protein levels was studied by RT-PCR and Western blotting.The mRNA expression levels of TLR4,MyD88 and TRAF6 in LPS-stimulated cells were significantly inhibited by GPOP-1,and the protein expression levels of TLR4,MyD88 and TRAF6 were also markedly inhibited,demonstrating the reliability of the results.Therefore,GPOP-1 has good anti-inflammatory activity.