Purification, Bioactivity And Structure Of Polysaccharides From The Brown Seaweed Sargassum Pallidum

Sargassum pallidum belongs to Phaeophyta Sargassaceae and are extensively distributed in China Yellow Sea and Bohai Sea.Due to not being edible,S.pallidum is used in relatively narrow scope at present,and only a small part is used as feed,algin and raw materials of pharmaceutical industry,and most of them has not yet been fully utilized.Alga glycine is a kind of miscellaneous polysaccharide.It has a variety of physiological functions,and is an important bioactive substance in brown alga.However,little information about S.pallidum polysaccharides(SPP) and biological activity is available compared with those of the other Sargassum genus at present.In this paper S.pallidum collected from Rongcheng in Shandong Province was studied,including the antitumor activity and antioxidant activity in vitro of SPP,and identification,of polysaccharides structure.This paper is composed of four parts:extraction of polysaccharides from S.pallidum, purification and identification of physical-chemical characters,antitumor activity and antioxidant activity of SPP in vitro,and identification of polysaccharides structure.Main results are listed as follows:1.Extraction of S.pallidum polysaccharidesSupercritical CO₂(SC-CO₂) extraction was used to degrease the lipophylic substances in S.Pallidum.The effect of extraction pressure,extraction temperature and extraction time were investigated.As results,the optimum operating conditions for treatment were as follows:temperature at 55℃,pressure at 45 MPa,a flow rate of CO₂ at 20 L/h,and extraction time for 6 h,and the yield was 8.53‰.Extraction of S.pallidum polysaccharide was optimized by the method of reSPPonse surface analysis(RSA).The effects of the main variables namely:ultrasonic time,extraction temperature and extraction time as well as the mutual interaction between variables were studied.The maximum yield of polysaccharides was up to 2.48%under the conditions as follows:ultrasonic time 469.54 s;extraction temperature 90.94℃;extraction time 4.96 h. The conditions of deproteinization by trichloroaeetie acid(TCA) were optimized.TCA final concentration was about 2%,and precipitation time was 2 h or longer.Membrane separation was performed at 0.5 MPa and at 20℃.The deproteinized polysaccharides solution was pumped to membrane surface(tangential flow) with different nominal molecular weight cut-offs(0.1μm,100 KDa,50 KDa,10 KDa,3 KDa),in turn. The results showed that S.pallidum polysaccharides were mainly included in the fractions with molecular weight more than 50 KDa.Due to the fraction with pore size more than 0.1μm contained much polysaccharides,having complex composition,it was collected and analyzed singly.Fractions with molecular weight less than 50 KDa containing less polysaccharide were combined.In later experiments S.pallidum polysaccharides(SPP) were divided into 3 main crude polysaccharides,SPP-1(＞0.1μm),SPP-2(＜0.1μm and＞50 KDa) and SPP-3(＜50 KDa).Effect of the ratio(material to water) on membrane separation was studied.At the ratio(material to water) of 1:20(m/V),1:30,1:100 and 1:150,pressure at 0.5 MPa, temperature at 20℃,the deproteinized polysaccharides solution were treated by membrane separation.The results showed that a higher ratio(material to water) led to a less loss of polysaccharides in membrane separation.It was considered to be suitable to dilute the solution at the ratio of 1:150 before membrane separation.At 20℃,membrane separation was performed at the pressure of 0.1,0.3 and 0.5 MPa,respectively.The results showed that the operating pressure had no significant effect on polysaccharides loss.So the pressure between 0.1 and 0.5 MPa are suitable in the scope allowed by membrane separation equipment.2.Purification of S.pallidum polysaccharidesThe yield of SPP-1,SPP-2 and SPP-3 accounted for 72.15%,16.69%and 11.16%, respectively of total crude polysaccharides.The polysaccharides contents were 58.13%, 18.98%and 16.38%,respectively and the protein contents were 1.09%,1.75%and 1.73%, respectively.The sulfate contents were 15.68%,15.26%and 22.62%,respectively and the uronic acid contents were 14.22%,14.30%and 5.20%,respectively.A lyophilized fraction of polysaccharides SPP-1 was chromatographed on a DEAE Cellulose-52 anion-exchange column to yield two peaks,SPP-1-1 and SPP-1-2.In a similar manner,SPP-2-1,SPP-2-2 and SPP-2-3,SPP-3-1 and SPP-3-2 were obtained from SPP-2 and SPP-3,respectively.The recovery of SPP-1-1 and SPP-1-2 were 32.53%and 8.67%, respectively.The recovery of SPP-2-1,SPP-2-2 and SPP-2-3 were 10.53%,9.20%and 36.33%,respectively.The recovery of SPP-3-1 and SPP-3-2 were 19.52%and 57.27%, respectively.During the purification of crude polysaccharides a little part of polysaccharides were losed,while the protein in crude polysaccharide was almost removed. Fractions of polysaccharides SPP-1-1,SPP-1-2,SPP-2-1,SPP-2-2,SPP-2-3,SPP-3-1 and SPP-3-2,respectively were chromatographed on a Sephadex G-100 chromatography column.So they could be used for further structural analysis.3.Antitumor activity and antioxidant activity in vitro of S.pallidum polysaccharidesThe inhibition effects of 7 fractions purified by DEAE Cellulose-52 on the growth of HepG2 cells,A549 cells and MGC-803 cells were evaluated in vitro by MTT assay. Fractions of polysaccharides SPP-3-1 and SPP-3-2 presented significantly higher antitumor activity against the HepG2 cells,A549 cells and MGC-803 cells in vitro than blank control groups,and the inhibition ability was dose-dependent.SPP-2-3 only presented significantly higher antitumor activity against the HepG2 cells in vitro than a blank control, dose-dependently.It seems that SPP-3-1 and SPP-3-2 from SPP-3 with relatively lower molecular weight(＜50KDa) and higher sulfate contents(22.62%) showed significantly higher antitumor activity against the HepG2 cells,A549 cells and MGC-803 cells in vitro.At the concentration of 2mg/mL,scavenging of DPPH radical of crude polysaccharide SPP-1 and SPP-2 and SPP-3 were 23.838%,13.232%and 12.828%,respectively.The total antioxidant capacity of SPP-1 and SPP-2 and SPP-3 were 0.087,0.083 and 0.088, respectively(μmolFeSO4/mg).The reducing power(expressed as absorbanee at 700 nm) and OH radical scavenging of SPP-1,SPP-2 and SPP-3 increased with the increasing concentration in experimental scope.Their reducing power were 0.785,0.446 and 0.541, respectively at 2 mg/mL.Their OH radical scavenging were 68.391%,13.218%and 27.586%,respectively at 2 mg/mL.Effect of ethanol extraction temperature on total polyphenol content extracted from S. pallidum were studied.At 30℃total polyphenol could not been fully extracted from S. pallidum,while at 70℃the extraction time was too short to be controlled,so 50℃was selected as alcohol extraction temperature.S.pallidum was extracted by 70%ethanol at 50℃,and then extracted by chloroform,ethyl acetate and n-butanol in turn.The yeied of total extract was 8.07%.Ethyl acetate extract,n-butanol extract and extract residue accounted for 15.76%,5.28%and 78.96%,respectively of total extract,and their total polyphenol contents were 3.20 mgCHA /gDW,12.12 mgCHA/gDW and 52.08 mgCHA/gDW, respectively determined by Folin-Ciocalteu method.DPPH radical scavenging of ethyl acetate and n-butanol extract were 30.59%and 29.36%,respectively at 2 mg/mL.Their total antioxidant capacity were 0.520 and 0.345 (μmolFeSO4/mg),respectively.Reducing power of ethyl acetate extracts,n-butanol extract and extract residue were high.The descending sequence was extract residue,n-butanol extract and ethyl acetate extracts.Metal ions scavenging of ethyl acetate extracts,n-butanol extracts and extract residue were 48.25%,51.34%and 17.21%at 2 mg/mL.4.Structural analysis of S.pallidum polysaccharidesThe average molecular weight and purity of polysaccharides fractions SPP-2-3, SPP-3-1 and SPP-3-2 were defined by high performance of gel filtration chromatography (HPGFC).They were 132.999KDa,5.874KDa and 7.249KDa,respectively. Monosaccharide composition and molar ratio of SPP-2-3,SPP-3-1 and SPP-3-2 were analyzed by Gas chromatography(GC).They were rhamnose:xylose:fucose:mannose: glucose:galactose= 0.15:2.74:0.64:1.00:0.49:1.11,mannose:glucose:galactose= 1.00:11.18:0.96,and rhamnose:xylose:fucose:mannose:glucose:galactose=0.17: 2.53:0.61:1.00:0.46:0.92,respectively.The infrared absorption spectroscopy(IR) of SPP-2-3 and SPP-3-2 showed a character of口-glycosidic bond.Periodate oxidation,Smith degradation and methylation analysis of SPP-2-3 showed that mannose were linked mainly by(1→3) glycosidic bond.In addition to(1→3) glycosidic bond in glucose and galactose,there was also(1→6) glycosidic bond.Xylose was linked mainly by(1→2) glycosidic bond.In addition to(1→3) glycosidic bond in fucose,there was also(1→4) glycosidic bond.For SPP-3-2,mannose and galactose were linked mainly by(1→3) glycosidic bond.Glucose was linked by(1→6) or(1→2) glycosidic bond.Fucose and L-xylose was linked by(1→2) or(1→4) glycosidic bond.