Preparation,physicochemical Properties And Hepatoprotective Activities Of Polysaccharides From Corbicula Fluminea Juice

Liver injury is one of the common diseases of the liver,and persistent liver injury will gradually evolve into hepatic fibrosis,cirrhosis and finally liver cancer,thus seriously threatening human life and health.Corbicula fluminea juice(CFJ)is an industrial by-products produced during the boiling of C.fluminea,which contains abundant nutrient substances such as protein,amino acids and polysaccharides.Meanwhile,C.fluminea is regarded as a good product for protecting liver in China.However,CFJ as an industrial by-products,which is often discharged as outlet water,which caused waste of resources and environmental pollution.Polysaccharides as one of the major bioactive components of CFJ exhibit a broad spectrum of physiological functions,such as anti-oxidation,anti-tumor,improving immunity and protecting liver.Therefore,in this project,the polysaccharides(CFJPs)were extracted from CFJ by using three-phase partitioning,ultrasonic-assisted extraction and hot water extraction methods,and their physicochemical properties,structural characteristics,in vitro antioxidant activity and cellular immune activity were comparatively investigated,and the highly bioactive CFJP was established.Moreover,a homogenous CFJP fraction was obtained by using ammonium sulfate precipitation method,and its structural feature and in vitro biological activity were studied.On this basis,the in vivo hepatoprotective effect of the homogenous CFJP fraction was evaluated through a CCl₄-induced live injury mice model.The main results are as follows:(1)Three CFJPs(CFJP-T,CFJP-U and CFJP-W)were extracted from CFJ by using three-phase partitioning,ultrasonic-assisted extraction and hot water extraction methods.The results showed that CFJP-T had higher extraction yield(60.92%),total sugar content(91.05%),uronic acid content(8.55%)and lower protein content(7.15%)than CFJP-U and CFJP-W.The three CFJPs were all acidic heteropolysaccharides,which were composed of fucose,glucosamine,glucose(Glc),mannitose,fructose and galacturonic acid with different molar ratios.Glc is the main monosaccharide in the three CFJPs,the percentage content of which was more than90%.The three CFJPs had similar preliminary chemical structures and different molecular weights and molecular weight distributions.Scanning electron microscope observation showed that different extraction methods had important effects on the surface morphologies and molecular sizes of the three CFJPs.In vitro antioxidant and cellular immune activities of the CFJPs were comparatively evaluated.The results showed that the three CFJPs exhibited good antioxidant activity in vitro in dose-dependent manners,but presented different differences in cell immune activity.CFJP-T possessed better DPPH and hydroxyl radical scavenging ability than CFJP-W and CFJP-U.There was no significant difference in NO release between CFJP-U and CFJP-W,which was not dose-dependent,while the cellular immune activity of CFJP-T was higher and dose-dependent than the others.(2)CFJP-T was separated by using(NH₄)₂SO₄precipitation method,and a homogenous polysaccharide component CFJP-60 was obtained when the mass fraction of(NH₄)₂SO₄was 60%.The structural feature of the CFJP-60 was identified by combining chemical and instrumental analyses.In vitro antioxidant activity and cellular immunity activity of the CFJP-60 were also determined.The results showed that the CFJP-60 was predominantly composed of Glc with a molecular weight of1.40×10⁷Da.Its possible repeating structural units are as follows:The scavenging rates of DPPH and hydroxyl radicals of CFJP-60 were 41.93%and 61.71%at 5 mg/m L,respectively;and the release amount of NO in cellular immunity was 3.63?mol at 500?g/m L,further revealing that its excellent antioxidant and cellular immume activities in vitro.(3)In vivo hepatoprotecitve effect of the CFJP-60 and its impact on intestinal microflora was investigated by using a CCl₄-induced acute liver injury in mice.The results showed that,compared with the model control group(MC),the supplements with CFJP-60(200,400,800 mg/kg body weight[BW])not only effectively and dose-dependently reduced the weight loss and organ injures,but also alleviated hepatic function and serum lipid metabolism.Meanwhile,CFJP-60 can also reduce the metastasis of pathogenic bacteria and the spread of inflammation by regulating the relative abundance of beneficial(Ruminococcaceae,Lactobacillaceae,Firmicutes and Bacteroidete)and harmful bacteria(Proteobacteria)in the intestinal microflora,prevent cell apoptosis,enhance the intestinal barrier function,regulate the disturbance of intestinal microflora and alleviate liver injury,so as to achieve the role of liver protection.