Studies On Subcritical Water Extraction,Characterization And Immune Activities Of Polysaccharides From Sagittaria Sagittifolia

Sagittaria sagittifolia,belonging to Alismataceae Sagittaria family,is a perennial herb cultivated widely in China.It has been reported that Sagittaria sagittifolia,as a medicine and food dual-purpose plant,not only has a high nutritional value,but also has a variety of biological activities such as anti-oxidation,detoxification,hypoglycemic,anti-tumor,and immune regulation.Polysaccharides are the main active substance of Sagittaria sagittifolia.However,up to now there are few studies on polysaccharides of Sagittaria sagittifolia,and there is no report on the extraction of Sagittaria sagittifolia polysaccharides using subcritical water.Based on this,this paper proposes to establish a subcritical water extraction technique for polysaccharides of Sagittaria sagittifolia and then separate and purifie them.The structure and solution behaviors of Sagittaria sagittifolia polysaccharides are characterized by modern analysis and spectroscopic techniques.At the same time,the polysaccharides of Sagittaria sagittifolia are in vitro immunized.The study aims to provide key technologies for the efficient preparation of Sagittaria sagittifolia polysaccharides and provide theoretical basis and reference for the development and application of Sagittaria sagittifolia resources.The main research contents and results are as follows:1.Subcritical water extraction and antioxidant activity of Sagittaria sagittifolia polysaccharides?1?A subcritical water extraction method for Sagittaria sagittifolia polysaccharides was established.Combined with the results of single factor experiments,an orthogonal test of the extraction process of Sagittaria sagittifolia polysaccharides was optimized as the following:pH was 7,extraction temperature was 170°C,extraction time was 16 min and the liquid-solid ratio was 30:1?mL:g?.Under this condition,the extraction yield of Sagittaria sagittifolia polysaccharides was?24.57±0.49?%.Compared with traditional hot water extraction,subcritical water extraction not only shortened the extraction time,but also increased the extraction yield by 10.87%.?2?Three antioxidant determinations?reducing power,DPPH·scavenging rate and ABTS⁺·scavenging rate?were used to evaluate the antioxidative capacity of Sagittaria sagittifolia polysaccharides.The results showed that both of the crude polysaccharides?1-5 mg/m L?extracted by subcritical water?SWE-SSP?or by traditional hot water?HWE-SSP?had strong antioxidative capacity in vitro in a dose-dependent manner.2.Separation,purification and structural characterization of Sagittaria sagittifolia polysaccharides?1?The subcritical water extraction was respectively dealt with alcohol precipitation,deproteinization and dialysis,then the relative high-purity Sagittaria sagittifolia crude polysaccharide?SSP?was separated and purified by anion exchange and Sephadex column chromatography to obtain two polysaccharide fractions of SSP-W1 and SSP-S1.The polysaccharide contents of SSP-W1 and SSP-S1 were 94.61%and 88.82%,respectively.The weight-average molecular weights?Mw?of SSP-W1 and SSP-S1 were 62.03 kDa and 15.20 kDa,respectively.The uronic acid contents of SSP-W1 and SSP-S1 were 3.67%and 2.18%,respectively.?2?SSP-W1 was composed of three monosaccharides of D-arabinose,D-glucose,D-galactose and with a molar ratio of 1:341.126:28.689.SSP-W1 had no nucleic acid,protein,pigments and other ingredients.SSP-W1 was an?-configuration pyranose.The main chain and the branched chain were mainly composed of glucose.There was no triple helix structure,which mainly existed in a disordered structure.The surface topography was folded and curled,which was characterized by agglomerative distribution,uneven surface topography and a relatively compact structure.It was a molecular aggregate formed by the intertwining of multiple molecular chains.?3?SSP-S1 was composed of four monosaccharides of L-rhamnose,D-arabinose,D-glucose,D-galactose and with a molar ratio of 1:2.883:59.812:6.795.SSP-S1 was free of nucleic acids,proteins,pigments and other ingredients.The main chain and the branched chain were mainly composed of glucose.It has sulfate-O-SO₃-H and?-bond types.There was no triple helix structure,which mainly existed in a disordered structure.It was in the form of a sheet,smooth surface,relatively flat and tight,and SSP-S1existed in the form of a single chain and was uniformly dispersed.3.Immune activity of Sagittaria sagittifolia polysaccharidesMouse RAW264.7 macrophages and human hepatoma HepG2 cells were used as in vitro cell models.MTT assay,Neutral Red,and Western blot were used to detect the effect of proliferation activity,phagocytic index and NO and immune cytokine secretion of Sagittaria sagittifolia polysaccharides on macrophages.Effect of RAW264.7macrophages induced by polysaccharides on the proliferation of HepG2 cells was studied.The results showed that SWE-SSP,SSP-W1 and SSP-S1 group could promote the proliferation of macrophages?P<0.01?compared with the normal group under the concentration of 50-200?g/m L.At the concentration of 200?g/m L,SWE-SSP,SSP-W1and SSP-S1 group could enhance the phagocytosis of macrophages significantly?P<0.01?.Among them,compared with the normal group,the SSP-S1 group increased by 145.50%,which was equivalent to the LPS group.At the concentration of 100-200?g/m L,SWE-SSP and SSP-S1 could significantly promote the secretion of NO,TNF-??cytokines of Th1?and IL-10?cytokines of Th2?to coordinate the expression and regulation of various cytokines in the body to achieve macrophage immune response.SWE-SSP,SSP-W1 and SSP-S1 could inhibit HepG2 proliferation indirectly by stimulating cytokine secretion from macrophages.At the concentration of 200?g/m L,the inhibition rate of SSP-S1 group reached to 16.55%.