The Separation And Extraction, Biological Activities Of Polysaccharides From Shaoxing Rice Wine And Their Effects On Intestinal Microflora

So far the consumption of Chinese rice wine has been transformed from sensory preferenceto nutritional requirements. And the salient features of Chinese rice wine such as low alcoholcontent, nutritious and healthy values has given rise to people’s concerns. Recently, research onbioactive substances of Chinese rice wine has focused on amino acid, polyphenols andpolypeptides and others, but there is very little report about bioactive polysaccharides.Thus, in this paper, we studied the techniques of extraction precipiation and purificationof polysaccharide from Shaoxing rice wine. And the structure of polysaccharide was analyzedby the primary analysis. Furthermore, the effects of polysaccharides from Shaoxing rice wineon immune regulation, tumor inhibition and their function to regulate alteration of intestinalflora were investigated. The main results are as following:(1) With the results of single factors, response surface method was used to optimize thetechnological condition of the precipitation method for the yield of polysaccharides fromShaoxing rice wine. The results showed that the maximum experimental value the concentrationratio of Shaoxing rice wine3.0, the ethanol concentration82%(v/v), the precipitation time19.0h, the precipitation temperature3.4°C. Under the optimum conditions, the yield ofpolysaccharides from Shaoxing rice wine could reach30.49g/L.(2) The antioxidative activity of polysaccharides from Shaoxing rice wine in vitro wasinvestigated. It was found that the radical scavenging capacity of polysaccharides fromShaoxing rice wine increased with the increasing of polysaccharide concentration. Thepolysaccharides from Shaoxing rice wine at1.0mg/mL scavenged the DPPH, hydrogenperoxide, superoxide ion at92%,53%and86%, respectively.(3) Following the isolation of the complex by Sephadex G75, the sample was furtherpurified by DEAE-Sepharose FF chromatography. Then, a novel polysaccharide CRWP1wasobtained. It was showed as a single symmetrical peak on high-performance gel-permeationchromatography (HPGPC) and the average molecular weight was7850Da. Furthermore, thestructure of CRWP1was identified by high performance anion exchange chromatography withpulsed amperometric detector (HPAEC-PAD), ultraviolet spectrum (UV), fourier transforminfrared spectrometry (FTIR) and NMR. The results showed that CRWP1was a heteroglycan,which was composed mainly of glucose, arabinose, xylose, and a small quantity of fucose,galactose and mannose. There was one glycosidic configuration (α) in structure ofpolysaccharides from Shaoxing rice wine, and the glycosidic bond was galactopyranose type.It was also revealed that the structure of polysaccharides from Shaoxing rice wine contained1,3glycosidic bonds as the main chain and1,4glycosidic bonds as branched chain.(4) Animal experiment was utilized to investigate the immunologic enhancement ofpolysaccharide CRWP1. It was found that CRWP1could improve the weights of immuneorgans of immunodeficient mice. Also, CRWP1could significantly improve the phagocytosisof macrophages in mice induced by cyclophosphamide and promote the proliferarion oflymphocyte from spleen. Meanwhile, the results showed that CRWP1in vitro could promotethe production of serum cytokines (IL-6、IFN-γ、TNF-α), the contents of immunoglobulin and alexin in immunodeficient mice. This suggests that CRWP1can improve immune functionof immunodeficient mice.(5) The anti-tumor effects of CRWP1were detected by establishing the mice-transplantedtumor models with injecting the S180ascites tumor. It was found that CRWP1could improvethe weights of immune organs of tumor-S180tumor-bearing mice. Also, CRWP1couldsignificantly inhibit the growth of S180solid tumor. The anti-tumor rate among15mg/kg.d CY+50mg/kg.d CRWP1,25mg/kg.d CY+50mg/kg.d CRWP1,35mg/kg.d CY+50mg/kg.dCRWP1were62.04%,78.78%and84.49%, respectively. It suggested that the combinationtreatment with CY and CRWP1has a synergistic effects on tumor inhibition.(6) The expression of Ki-67, Cyclin D1, Bax and Bcl-2protein in the S180tumor weredetected by immunohistochemical techniques to investigate the anti-tumor mechanism ofCRWP1. The results showed that one of the mechanisms of anti-tumor might be that CRWP1could accelerate the apoptosis of tumor cell, inhibit the cell cycle and tumor tissue growth bydown-regulating the expression of Ki-67, Cyclin D1, and Bcl-2protein and up-regulating theexpression of Bax protein.(7) The effects of CRWP1on intestinal microflora in mice were investigated by the anti-digestibility of CRWP1against artificial human gastric acid and α-amylase, the proliferationeffect. It was found that the maximal hydrolysis degree of CRWP1in the artificial human gastricacid and α-amylase were2.67%and4.98%respectively, suggesting that CRWP1had relativelyhigh stability and digestive tolerance. In addition, it was found that CRWP1could stimulate theproliferation of probiotics such as Bifidobacterium and Lactobacillus, inhibit the proliferationof Enterobacter, Enterococcus and Clostridium perfringens. Furthermore, CRWP1hadprebiotics efficacy by stimulating the production of short-chain fatty acid, acetic acid, butyricacid and lactic acid.