| Sargassum pallidum(Turn.)C.Agardh,commonly known as ?big leaf seaweed?,is a kind of significant and classic edible and medicinal brown alga.S.pallidum is rich in active substances,and polysaccharides are one of the main active ingredients,which have attracted more and more attention and research interest.Pharmacological studies have shown that S.pallidum polysaccharides possess antioxidant,hypoglycemic,antitumor,sedative,and hypnotic activities.However,the structural characteristics,hypoglycemical activity and underlying action mechanisms of polysaccharides from S.pallidum are still unclear,which limit its application in food and medicine.Therefore,in the present study,the extraction,purification,physicochemical properties and structural characteristics of the polysaccharides from S.pallidum were studied.Furthermore,in vitro antioxidant,hypoglycemic activity and the mechanisms were evaluated by the combination of chemical and cell model experiments.Finally,the digestion and fermentation characteristics of S.pallidum polysaccharides and their effects on the gut microbiota were studied by the in vitro digestion and fermentation models.These results provide a theoretical basis for revealing the hypoglycemic mechanism,structure-activity relationship,and intestinal microecological regulation of S.pallidum polysaccharides.The main results were listed as follows:(1)Microwave-assisted aqueous two-phase extraction(MAATPE)technology was applied for simultaneous extraction and separation of crude S.pallidum polysaccharides(SPP).The optimal extraction parameters,physicochemical properties,and biological activities in vitro of SPPs were investigated.Results showed that the optimal extraction conditions were as follows: 21.0% ethanol(w/w),22.0% ammonium sulfate(w/w),solid-liquid ratio of 1:60g/m L,extraction time of 15 min,microwave power of 830 W,extraction temperature of 95 °C.Under the optimal conditions,the extraction yield of SPP-1 and SPP-2 was 0.75±0.04% and6.81±0.33%,respectively.In comparison,the yield of microwave-assisted water extraction polysaccharide(SPP-3)was 5.86±0.38%,and that of water extraction polysaccharide(SPP-4)was 5.31±0.25%.Chemical and instrumental analysis indicated that SPP-1,SPP-2,SPP-3 and SPP-4 had different molecular weights,monosaccharide compositions,triple helix structure and apparent morphologies.The results of in vitro activity experiments showed that the four polysaccharides had significant antioxidant and hypoglycemic activities and could be developed as potential antioxidant and hypoglycemic active substances.(2)SPPs were fractioned using DEAE-Sepharose fast-flow column and two main polysaccharide fractions(SP-P1 and SP-P2)were obtained.High performance gel permeation chromatography(HPGPC)analysis showed that SP-P1 and SP-P2 were homogeneous polysaccharides with the molecular weights of 1036.2 k Da and 144.8 k Da,respectively.Combined with monosaccharide composition,periodate oxidation-Smith degradation,methylation and 1D NMR analysis,the glycosidic types of SP-P1 were identified as?6)-?-D-Glcp-(1?,?3,4)-?-L-Fucp-(1?,?-D-Manp-(1?,?2,3,4)-?-D-Xylp-(1?,?-L-Arap-(1?,?2,4)-?-D-Xylp-(1? and ?6)-?-D-Galp-(1? with molar ratio percentages of 2.38%,34.76%,0.95%,17.62%,3.81%,14.29%,and 26.19%,respectively.Similarly,the glycosidic types of SP-P2 were identified as ?3,4)-?-L-Fucp-(1?,?-D-Manp-(1?,?2,3,6)-?-D-Galp-(1?,?3)-?-D-Xylp-(1?,?6)-?-D-Galp-(1?,?2)-?-D-Glcp NAc-(1?and ?-D-Galp NAc-(1? with the molar ratio percentges of 27.33%,1.16%,11.63%,15.12%,2.33%,12.21% and 30.23%.Combined with partial acid hydrolysis analysis and 2D NMR analysis,the backbone chains of SP-P2 were composed of ?1)-?-D-Xylp-(3?,?1,3)-?-L-Fucp-(4?,?1)-?-D-Galp-(6?,and ?1)-?-D-Glcp NAc-(2?,and the side chains were composed of ?1,3,6)-?-D-Galp-(2?,?3)-?-L-Fucp-(1,4?,?-D-Galp NAc-(1?,and ?-D-Manp-(1?.(3)The in vitro antioxidant and hypoglycemic activities and mechanisms of S.pallidum polysaccharides were investigated by the chemical and cell model experiments.Results showed that SP-P1 had the highest hydroxyl radical scavenging activity,while SP-P2 had the best ATBS radical scavenging activity.The inhibition rate of SP-P2 on ?-amylase was 60.83%at the concentration of 5.0 mg/m L.The inhibition rate of SP-P1 on ?-glucosidase reached the was 50.27±1.98% when the concentration was 2.0 mg/m L.Cell antioxidant activity(CAA)experiment showed that SP-P1 had strong antioxidant capacity in cells,and could inhibit intracellular reactive oxygen species(ROS)generation,improve ABAP induced oxidative damage Hep G2 cells within the superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)activity and decrease the malondialdehyde(MDA)content.Insulin resistance(IR)-Hep G2 cell model experiment showed that SP-P1 and SP-P2 could promote glucose consumption in IR-Hep G2 cells,enhance the activities of key glycolytic enzymes hexokinase(NK)and pyruvate kinase(HK),promote glycolysis and glycogen metabolism,and significantly increase the glycogen content of cells.Mechanistic investigation indicated SP-P2 could significantly up-regulate the expression levels of IRS-1,GS,PI3 K and GLUT4 genes in IR-Hep G2 cells.It was further speculated that SP-P2 might regulate glucose synthesis and metabolism in Type 2 diabetes by participating in the traditional IRS-1/PI3K/Akt signaling pathway,thus exerting hypoglycemic activity.(4)In vitro simulated digestion and fermentation experiments were conducted to study the structure activity changes of SPP and their effects on gut microflora.Results showed that there were differences in each component in the simulated in vitro saliva,gastric and intestinal digestion.The molecular weights of SPP decreased gradually.The release of fucose mainly happened in the saliva digestion,the release of glucose mainly in the gastric and intestinal digestion,and the release of galacturonic acid was mainly in saliva digestion.After gastric digestion,the DPPH and hydroxyl free radical scavenging abilities were significantly enhanced,and the ?-amylase and ?-glucosidase inhibitory activities were also enhanced.Galacturonic acid changed significantly with the release of free monosaccharide in the fermentation process.Total short chain fatty acids(SCFAs)increased after fermentation of SPP,among which iso-butyric acid,iso-valeric acid and n-valeric acid were largely increased,and galactose,glucose and mannose produced the highest SCFAs.In addition,SPP could improve the diversity of intestinal microbial community,especially in the early fermentation stage.After fermentation,the Bifidobacterium adolescentis,Bacteroides dorei and Lactobacillus plantarum were remarkably promoted.Therefore,SPP had a beneficial effect on the health of intestinal flora by regulating the composition and abundance of beneficial flora in the fermentation process. |
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