The Study Of Purification,Structure Identification,Selenium Modification And Biological Activity Of Polysaccharides From Ostrea Rivularis Gould

ObjectiveOstrea rivularis Gould is a common breeding shellfish in southern coast of China.Its shell is one of the sources of Chinese traditional medicine"oyster".Its meat is delicious and rich in a variety of nutrients which with a variety of biological activity.Polysaccharides from Ostrea rivularis Gould(ORP)have antioxidant,spermatogenic,immune regulation and other activities.Thus,how to rationally and scientifically develop near oyster resources is a meaningful topic.At present,there are many reports on the study of ORP,but these studies were relatively simple.Therefore,this paper intends to study the separation,purification and characterization of ORP by using modern technology based on the Chinese medicine,and to study the selenium modification of ORP.Beside,study on antioxidant activity and mechanism of ORP in vitro.Then study its effect on cyclophosphamide induced oxidative stress in mice spermatogenesis,and explore mechanisms of ORP improve cyclophosphamide induced oxidative stress in mouse spermatogenic activity.Accumulation of information on drug research related to ORP.Laid the theoretical foundation for its development into drugs and related functional health food.Methods1.Extraction,isolation and purification of ORPUltrasonic assisted extraction was used to extract ORP.Then,the crude polysaccharides were deproteinizated by magnetic chitosan microspheres(MCM),At last,the purified polysaccharides were obtained by separated and purified by DEAE-cellulose 52 and Sephadex G100 column.2 Physicochemical properties of ORPThe content of total sugar was determined by phenol sulfuric acid method.The protein content was determined by Coomassie brilliant blue method.Sulfuric acid content was determined by BaCl2-gelatin method.The relative viscosity of polysaccharide was determined by Uygur viscosity meter.Monosaccharide composition of ORP was determined by acetylation reaction-GC.The purity and molecular weight was determined by HPLC.The functional groups and sugar ring was determined by by FT-IR.The glycosidic linkage site,glycosyl link order and branch was determined by methylation and GC-MS.NMR method was used to determine the configuration of the heterotopic carbon.The Congo red method was used to determine whether ORP chain has three spiral structures.3.Selenium modification of ORPThe selenium derivatives were obtained by the modification of BaCl2-HNO3 and Na2Se03,4.Effects of ORP on oxidative stress induced by H2O2 in TM4 cellsThe mouse testis Sertoli cells(TM4 cells)were used as experimental subjects.The oxidative stress model of TM4 cells was established by using lipopolysaccharide.The antioxidant capacity of polysaccharides and their selenated derivatives were studied.In order to study the mechanism of antioxidant activity of polysaccharides and their selenated derivatives.The expression of Nrf2 gene,the upstream regulatory factor and the downstream target protein in TM4 cells was detected by real-time fluorescence quantitative PCR and Western Blot technique.5.ORP inhibits cyclophosphamide-induced reproductive injury in mice and its mechanismBalb/c mice were injected intraperitoneally with cyclophosphamide,to establishment of cyclophosphamide-mediated oxidative stress model in male mice.Thus investigate the effects of ORPp and ORPSe on testicular tissue recovery,sperm survival rate,abnormal sperm rate.Besides,in order to study the mechanism of ORPp and ORPSe antagonizing cyclophosphamide leading to reproductive injury,the expression of Nrf2 gene,the upstream regulatory factor and the downstream target protein in TM4 cells was detected by real-time fluorescence quantitative PCR and Western Blot technique.Results1.Extraction,isolation and purification of ORP(1)The extraction rate of ORP was 9.74±0.55%.The content of protein in the crude ORP was 8.62±0.23%.(2)The crude polysaccharides were further deproteinization by MCM,and the synthesized MCM had a particle size of 2-6 ?m,the optimum protein adsorption temperature was 50 ?,and the adsorption time was 4h.The method is that the physical adsorption and adsorption process kinetics and adsorption isotherms can be fitted by Lagrangian pseudo-second order equation and the Freundlich equation,respectively.In addition,according to the adsorption equation obtained by ?G and ?Ea,MCM adsorption is spontaneous,endothermic physical adsorption reaction.(3)The crude polysaccharide was purified by DEAE-cellulose 52 and Sephadex G100 column and then obtained a purified fraction ORPP with the yield of 32.7%.2.Physicochemical properties and structure identification of ORPThe total sugar content of ORPp was 88.70%.The sulfuric acid content was 1.2%,the protein content was 0.17%,the uronic acid content was 8.59%and the relative viscosity was 1.15.The relative molecular mass is 656 kDa.ORPp consists of glucose.There was no three screw structure in ORP molecular.3.Selenium modification of ORPThe content of Se element in ORPSe was 181.9±5.6?g/g.4.Effects of ORP on Nrf2/ARE pathway in TM4 cellsBoth ORPP and ORPSe could improve the survival rate of H2O2-induced oxidative stress TM4 cells,and the cell viability was related to the dose.Indicated both of ORPp and ORPSe can improve the antioxidant capacity of TM4 cells.By real-time fluorescence quantitative PCR and Western Blot analysis,we could found that,ORPP and ORPSe can activate Nrf2/ARE pathway,increase Nrf2 and Keapl uncoupling to enter the nucleus,activate ARE elements,promote SOD,HO-1,NQ01 and other antioxidant enzymes and ? phase detoxification enzyme production,thereby enhancing TM4 Cell antioxidant capacity.5.Effects of ORP on reproductive injury induced by cyclophosphamide in male miceORPp and ORPSe can be act as inducers to enhance the expression of Nrf2 in mouse testes and activate the Nrf2/ARE signaling pathway,which is contribute to the downstream ? phase detoxification enzyme NQ01 and antioxidant enzyme HO-1 and other target gene expression.Improve the body resistance,to avoid the oxidative stress caused by cyclophosphamide damage,relieve male mice reproductive injury,improve the number of sperm,sperm survival rate,and reduce the rate of abnormal sperm.At the same dose,the spermatogenic activity of ORPSe was significantly better than that of ORPp.ConclusionIn this paper,ORP were used as experimental subjects.The separation and purification process were studied systematically and their Physicochemical properties were studied by physical and chemical methods.To study the in vitro antioxidant activity and its mechanism in vitro by establishing H2O2-induced TM4 oxidative stress model,and to establish a model of oxidative stress induced by cyclophosphamide in mice to study the spermatogenic activity and mechanism of oyster polysaccharides.In vitro antioxidant activity and its mechanism were studied by establishing H2O2-induced TM4 oxidative stress model.At last,establishment of cyclophosphamide-induced oxidative stress model to investigate the neurotrophic activity and its mechanism of ORP.It is found that the MCM material is an ideal material for deprotein in crude polysaccharides.It is suitable for the use of crude polysaccharides which need to keep active both of protein and polypeptide at the same time.Selenium modification can be inorganic selenium grafted into the polysaccharide to form organic selenium polysaccharide(ORPSe),ORPSe is a non-toxic.The antioxidant mechanism of ORPp and ORPSe in vitro should promote the antioxidant capacity of TM4 cells by activating the Nrf2/ARE signaling pathway in TM4 cells,promoting the production of antioxidant enzymes such as SOD,HO-1 and NQ01.In addition,ORPp and ORPSe could be used as inducers to enhance the expression of Nrf2,activate the Nrf2/ARE signaling pathway in mice,improve the ability of testes to resist the outside world,avoid oxidative stress injury caused by cyclophosphamide,relieve male mice reproductive injury,And antioxidant activity of ORPSSE was superior to ORPp.