Study On Purification, Immunomodulating And Antioxidant Activities Of Momordica Charantia Polysaccharide

Momordica charantia is a common vegetable in people living and also an important functional food ingredients. Studies have shown that Momordica charantia could improve immunity, reduce blood sugar, antimicrobial and antioxidant effects, and clearly pointed out that the momordica charantia polysaccharide (MCP) is one of the main functional active components. Previous reports on MCP mainly focuses on lowering blood glucose, while the active reseach of antioxidant and immune aspects were weak, especially in the preparation of homogeneous fraetions, so it was impossible to learned their mechanism, and dose-effect relationship, structure-activity relationship between active component and its pharmacological effects. This dissertation is focused on the study of antioxidant and immune activity of MCP in vivo and in vitro, track of antioxidant and immune active fraction isolated from crude polyaccharide, structural analysis of active fraction. Aimed to provide a theoretical for the further development of drugs and functional foods of MCP. The main findings are as follows:1. Optimization of extraction condition of MCP:Based on the single factors experiment, the optimum parameters of cellulase assisted ultrasonic wave of momordica charantia polysaccharide (MCP) were ensured by the method of orthogonally rotational combination design and the regression equation was established. The results indicated that the optimal extraction technology was to extract at ultrasound power 390W, cellulase amount 3500 U/g (enzyme activity≥200 U/mg), ratio of solid to liquid 1:38, extracting time 40 min, pH value 5, temperature 56℃,then the yield of crude momordica charantia polysaccharide is 21.1%, respectively higher 7.8%,13.5% and 7.7% comparaing the specific heat of flooding,ultrasonic wave and cellulase.2. Evaluation of the immunomodulatory activity of MCP:lymphocytes were obtained from healthy mice spleen and cultivated with different concentrate MCP in 96 wells for improved lymphopoiesis test, reslutes showed that MCP ranging from 60μg/mL to 100μg/mL could remarkly promot the spleen cell proliferation in vitro by rejecting or assisting ConA treated in a dose-dependent manner (P<0.01); Immunosuppressed mouse models were established by intraperitoneal injections with cyclophosphamide (CY) followed by daily intragastric administration of different dose MCP. After the treatments, the mice were examined for phagocytotic function of the macrophages, serum IgM level following exposure to chicken red blood cells, the lymphocyte transformation rate, natural killer cells activity and immune organ weight index. Reslutes showed that treatment of the immunosuppressed mice with MCP at the daily dose of 150 mg/(kg-d) and 300 mg/(kg-d) was able to antagonize CY ide-induced immunosuppression and significantly increased their phagocytotic function of the macrophages, serum IgM level following exposure to chicken red blood cells, the lymphocyte transformation rate, natural killer cells activity and immune organ weight index (P<0.05); the later increased all the test level above of immuno suppressive mice to the same level of health mice (P<0.05).3 Evaluation of the immunomodulatory activity of MCP:The antioxidant activity of extracts from MCP was assayed by three methods, DPPH, ABTS radical scavenging and ferric reducing (FRAP) assay, the reslutes showed that IC50 value of DPPH and ABTS radical scavenging activity is respectively 571.8μg/mL,737μg/mL, and MCP could eliminate radical at a higher percentage of 90% with content 1.6mg/mL; FRAP value of MCP is 0.187μmolFeSO4/mg, the positive correlation (P<0.01) existed between reductive capacity and concentration. The antioxidant activity Immunosuppressive mouse model was established by CY and the level of oxygen radical genesis enzyme and free radical in vivo was observed, results showed that a single treatment of CY could increase the level of XOD, NOS in spleen and the level of XOD, MPO, NOS in thymus (P<0.05), decrease activity of T-AOC, T-SOD activity in serum, content of GSH in the thymus (P<0.05); while MCP could antagonize prominently the decreasing of the activity of XOD, MPO in spleen and thymus induced by CY, significantly increase the level of T-AOC, T-SOD, GSH, decrease the content of MDA and make all the test index above up to normal levels (P<0.05), but had no effect on NOS.4 Purification and structural analysis of active components of MCP:6 purified polysaccharides were isolated from crude polyaccharide by deproteinization, dialysis and column chromatography on DEAE-52 cellulose, The mouse spleen cells proliferation stimulating effects and the activity of ABTS radical scavenging in vitro of each fraction at various concentrations were assayed, and two active fraction of immunomodulation and antioxidant, MCP3, MCP6,were obtained and structural analysised. The results showed that:the two active fraction were all heteropolysaccharide and didn't contain protein and starch; MCP3 consisted of glucose, galactose, arabinose, mannose, xylose, rhamnose and its relative molecular weight was 2.00×104;MCP6 consisted of glucose, mannose, galactose, xylose, arabinose and its relative molecular weight was 1.35×104; MCP3, MCP6 had adsorption bands characteric of polysaccharides, contained pyranoid rings and two kinds of link methods of a-glycoside linkage andβ-glycoside linkage.Innovation:(1) To establish the extraction technology of cellulase assisted ultrasonic wave of MCP; (2) To confirm that MCP have strong immunomodulation and antioxidant activity; (3) There were two components of immunomodulation and antioxidant activity that were solated from the MCP and identified that they were all heteropolysaccharide with low molecular weight and had adsorption bands characteric of polysaccharides with pyranoid rings.