Extraction Of Polysaccharides From Kadsura Marmorata And Its Immunomodulatory Effects On Chicken

Kadsura polysaccharides(KPS)is a type of polysaccharides compound extracted from Schisandraceae Kadsura plants. Up to date, few studies or reports focus on the extraction, chemical composition and pharmacological activities of KPS, especially reports on Kadsura marmorata was rarely. In this study, we developed an extraction method to extract crude Kadsura polysaccharides from Kadsura marmorata fruits, tested its immunity activity, separated and purified the crude polysaccharides, analysed the chemical composition of single component from KPS and evaluated its the immunity activities. An active component, KPSⅢ, was purified and selected to investigate the clinical application.To extract KPS, Kadsura marmorata fruits were treated with petroleum ether, ethyl acetate and 95% ethanol successively, and followed by extraction with water. In order to optimize extraction conditions, single factor tests, material liquid ratio (1:10~1:30), extraction temperature (60℃~100℃), extraction time (1h~5h), were performed. Center combination design(CCD) and response surface analysis (RSA) was applied to select the optimum combination for these extraction conditions. Based on the analysis from single factor tests and RSA experiments, the optimum extraction material liquid proportion was 1:28, the extraction temperature was 95℃and the extraction time was 2.4 hours. The extraction rate reached peak, 3.50 %, under the optimum extraction conditions and the sugar purity was 76.01 % measured by anthrone-vitriol colorimetric assay.Following extraction, the KPS was tested for immunomodulatory effects on chicken in vivo. Seven-days old chicken were administrated with KPS by subcutaneous injection. A series of parameters closely related to immune system were measured, such as transformation of lymphocyte (LTT), ratio of CD4+ T cells and CD8+ cells, antibody titer of Newcastle disease blood clots inhibition (ND-HI), total peripheral blood cell numbers and viscera index of spleen, thymus and bursa. The results showed that LTT from KPS high dose group (20 mg/Kg weight) was significantly higher than from astragalus polysaccharides group (APS), cytoxan group (CTX) and black control group (BCG) (P<0.01) at 7 days post immunization. The ratio of CD4+ T cells and CD8+ cells in KPS immunized group was significantly higher than CTX and BCG (P<0.01) at 21 days post immunization. The KPS immunization made viscera indexes of thymus and bursa higher than APS (P<0.05) and significantly higher than CTX and BCG (P<0.01) at 21 days post immunization. The KPS caused ND-HI antibody titer significantly higher than APS, CTX and BCG (P<0.01) at 28 days post immunization. Also, the KPS immunization increased total peripheral blood cell number while the other group did not. These data indicated that KPS could up-regulate immune responses on chicken.To obtain pure immunomodulatory KPS, the crude Kadsura polysaccharide was subjected to a series of purification processes, such as excluding proteins with trichloroacetic acid (TCA), discoloring with active carbon and classifying by 20%~80% alcohol. Then the water soluble KPS was classified into five components by different concentration of alcohol, named as KPSI, KPSⅡ, KPSⅡ, KPSⅡ, KPSV respectively. Among which, KPSⅡwas the biggest proportion, 52.31 %, and its sugar purity reached the highest, namely 95.13 %. These five components were then subjected to gradient elution with Sephadex G-25 column respectively. The gradient elution curves showed that KPSⅡ, KPSⅡwere composed by single component, while KPSI, KPSⅡand KPSVwere mixed with polysaccharides with different molecular weight. To analyze the monosugar composition, all these five components were subjected to Gas chromatography (GC) and paper chromatography (PC) analysis. GC and PC analysis results demonstrated that KPSI-1 was heteropolysaccharide consisting of Rib, Xyl and GalA in the molar ratios of 1.13: 1.04 : 0.26, KPSⅡconsisted of Fru, Glc, and a unknow sugar in the molar ratio of 0.26: 0.10: 0.11, KPSⅡconsisted of Ara, Glc, and GalA in the molar ratios of 0.22 : 0.24 : 0.09, KPSⅡ-6 consisted of Fru, Glc, GalA and a unknow sugar in the molar ratio of 0.24 : 0.09: 0.06: 0.04 and KPSV-1 consisted of Rha, Glc and GalA in the molar ratios of 0.33 : 0.15 : 0.10.Next, these five components were applied to immunize chickens to evaluate their immunomodulatory effects in vitro. To do so, T and B lymphocyte proliferations, PMφin vitro and IL-2, IFN-γ, TNF-αand NO secretion levels were measured. The results demonstrated that KPSII and KPSⅢsignificantly stimulated lymphocytes proliferation, KPSⅢand KPSIV-6 induced PMφproliferation efficiently, as well as secretion of IL-2, IFN-γ, TNF-αand NO. Taken together, the KPSIII was selected for further clinical application research.Finally, we applied the KPSⅢto do some preliminary clinical trials on chicken to figure out its commercial value. The results showed that KPSⅢsignificantly enhanced HI antibody titer of ND Losata vaccination and AGP antibody titer of IBD D78 vaccination. Additionally, KPSⅢenhances viscera index of spleen, thymus and bursa as well. KPSⅢalso improved the growth of chicken, raised feed rewards, reduced the happening of diseases and increased the survival rate of chicken. In the preventive trial, subcutaneous injection of KPSⅢat 20 mg/Kg body weight prevented the occurrence of O2 type E. coli disease effectively. The protective rate reached 75%, which was significantly higher than that of infected control group (P <0.01). In the treatment trial, mortality rate from KPS injection group was under 27.5 % which was significantly lower than that of infected control group (P <0.01) and efficient reached 82.0 %, which was significantly higher than that of infected control group (P <0.01).In conclusion, this study laid a solid ground for the further development and veterinary clinical application of kadsura polysaccharide.