Study On Extraction Technique And Immunomodulatory Effects Of Polysaccharide From Marasmius Androsaceus

The cognition of traditional Chinese medicine about fungi materia medica is based on the theoretical system of traditional Chinese medicine, because fungi materia medica has the role of regulating the function of kidney and spleen, many researchers pay more attention to it. A lot of fungi materia medica has the function of strengthening body resistance and supplementing body deficiency, it can be widely used to lassitude, burnout, cough hemoptysis, short breath and auto sweat, aversion to cold and limb cold, macronosia and deficiency, pale tongue, fine pulse and asthenia, qi deficiency, yang deficiency, blood deficiency, yin deficiency and yin and yang deficiency patients. Marasmius androsaceus (L.ex Fr.) Fr. is a traditional Chinese medicinal and edible fungus, it distributed in Jilin, Liaoning province of China, which is belonging to the basidiomycetes. This research contents: The study of extraction, purification, immunobiological activities and some of immunoregulation mechanism of Marasmius androsaceus polysaccharides (MAP). The immunomodulating activities of MAP were measured, and an active component of MAP60 was screened out .The effect of MAP60 on non-specific immunity and specific immunity of immunosupressived mice was investigated. The immunoregulation mechanism of MAP60 was studied by molecular biology technique.1. Extraction technologyObjective: To determine the optimum extraction condition of MAP. Method: Correlation studies indicated that, alcohol concentration, extraction temperature and extraction time are the main influencing factors for polysaccharides extraction. In order to know the range of these extracting conditions, single factor tests were done to analyze the influence of each factor in alcohol concentration (60%~100%), extraction temperature (50℃~90℃), extraction time (1 h~ 5 h). RSA (Response Surface Analysis) experiment design was applied in selecting the optimum combination of these extracting conditions. After a series of purification processes, water soluble MAP was classified by alcohol concentration in three component,MAP40, MAP60, MAP80. Results: By single factor tests and RSA experiment, it is ensured that the optimum extraction alcohol concentration is 90%, the extraction temperature is 80℃, the extraction time is 5 hours. Conclusion: The water soluble polysaccharide is extracted from the Marasmius androsaceus, the extracting rate of polysaccharide is the most (51.44%) at the optimum extracting condition.2. Selecting the immune activity component of MAPObjective: To select the immune activity component of MAP from MAP40, MAP60, MAP80. Method: The immune activity of MAP was investigated by the effects of MAP on proliferation of T, B, Mφlymphocyte in vitro study.Results: MAP60, MAP80 can significantly increase the proliferation of T lymphocytes cell and strongly increases proliferation of Mφ. However, all MAP component had less influence on the proliferation of B lymphocytes cells.Conclusion: MAP60, MAP80 may contain the immune activity component of MAP. Considering the cost of extracting, we selected MAP60 for further research.3. Immunocompetence of MAP60 Objective: To study the immunocompetence of MAP60.Method: Immunosuppressive model was induced by subcutaneous cyclophosphamide (Cy). The effects of MAP60 on the Cy-induced immunosuppressive mice were studied in relation to innate immunity, humoral immunity and cellular immunity parameters.Results: Cy can inhibit the non-specific immunity, cellular immunity and humoral immunity of mice. After intraperitoneal injected MAP60, the immunity of immunosupressived mice increased. While MAP60 can promote non-specific immunity such as the phagocytic function of macrophage, enhance cellular immunity such as delayed type hypersensitivity and lymphocyte transformation and enhance humoral immunity such as antibody response and secreting.Conclusion: MAP60 can antagonize the inhibition of non-specific, cellular immunity, humoral immunity in immunosuppressive mice induced by cyclophosphamide.4. Immunomodulation mechanism of MAP60Objective: To study the immunomodulation mechanism of MAP60.Method: The ability of MAP60 to stimulate interleukin-1, interleukin-2, TNF-α, INF-γrelease to peripheral blood and in culture supernatant of mice peritoneal macrophages and spleen cells were assessed by enzyme linked immunoadsorbent assay (ELISA). The gene expression of IL-2 mRNA, IFN-γmRNA, IL-1 mRNA, TNF-αmRNA were assayed by reverse transcription polymerase chain reaction (RT-PCR).Results: MAP60 can promote the secretion of interleukin-1, interleukin-2, TNF-α, IFN-γin the serum of immunosuppressive mice and promoted the secretion of IL-1 and TNF-αfrom immunosuppressive mouse peritoneal macrophages and promote the secretion of IL-2 and IFN-γfrom immunosuppressive mouse spleen cells. The level of IL-2, IFN-γ, IL-1, TNF-αin test mice were increased after peritoneal injected MAP60, MAP60 can also promote the expression of IL-2 mRNA, IFN-γmRNA, IL-1 mRNA, TNF-αmRNA in immune cells.Conclusion: It was suggested that the immune regulation effect of MAP60 may be associated with the stimulation of some important cytokine secretion and these cytokine mRNA expression at the transcription level.