Study On Antioxidant Effect Of Different Cultivars Ganoderma Lucidum And Separation,Purificationand Immune Activity Of Polysaccharides

Ganoderma is a Polyporaceae fungus medicine, "Chinese Pharmacopoeia" the provisions of Ganoderma lucidum Ganoderma lucidum (leyss. Ex Fr.) Karst. Or Ganoderma Ganoderma sinense Zhao, Xu et Zhang dry fruiting bodies. Ganoderma as one of the most prestigious traditional medicine precious medicinal herbs, medicinal history of over 2000 years. Traditional Chinese medicine, Ganoderma has the "righting, deficiency of longevity" effect, modern research shows that Ganoderma has immunomodulatory、anti-tumor、anti-aging a variety of roles, such as lowering blood fat in food, cosmetics, health food and drug development and other fields have broad prospects, containing the active ingredient Ganoderma polysaccharides、triterpenoids、 alkaloids, including Ganoderma lucidum polysaccharides (Ganoderma lucidum Polysaccharide. GLP) is one of the main active substance.In this study. water extraction and alcohol precipitation, ultrailltration fractionation, ion exchange and gel filtration separation and purification techniques such as Ganoderma lucidum polysaccharides were isolated and purified, and the use of physical and chemical properties of polysaccharides means of a preliminary study, configuration, monosaccharide composition molecular weight; Ganoderma lucidum polysaccharides in vitro preliminary evaluation of immune activity, contents include: rat spleen lymphocyte proliferation of on RAW264.7 macrophages ability to generate NO, phagocytic capacity, proliferation, and thus explore Ganoderma Contact the structural properties of the immune activity of polysaccharides between. Experiments include the following:1.Antioxidant Effects of different cultivars of Ganoderma lucidum extractEvaluated the effect of with hydroxyl radicals, superoxide anion, DPPH scavenging and tyrosinase inhibition activity, Six kinds of water-soluble and alcohol extracts of Ganoderma lucidum had a strong DPPH scavenging effect, aqueous extract of Zld had the strongest activity, IC50 was 0.03 g·l-1. Only Zld water extract and ethanol extract also had superoxide anion scavenging activity. Zlb、Z1c and Zld extract had the effect of hydroxyl radical scavenging, while aqueous extract of Zld had the strongest activity, IC50 was 7.14 g·l-1. Six kinds of alcohol extracts of Ganoderma lucidum had strong inhibition activity of tyrosinase, Zld had the strongest activity. Tyrosinase inhibition activity of water extracts of Ganoderma lucidum was weaker than alcohol extract, when the concentration of Zld was 10 g·l-1it had high activity", inhibition rate was 66.77%. Conclusion Zld is suitable as whitening and anti-aging cosmetic raw material.2.Extract、eparation and purification of Ganoderma lucidum polysaccharides2.1 Extraction of Ganoderma lucidum polysaccharidesIn this study, the traditional methods of water extraction and alcohol precipitation is used to extracted from the fruiting bodies of Ganoderma lucidum polysaccharides, considering a variety of factors to determine the extraction process of polysaccharides:solid-liquid ratio (1:20), temperature (90℃), extraction time (3h), extraction times (2 times). Utilizing the nature of the polysaccharide is not soluble in high concentrations of ethanol, and ethanol was added to a final concentration of 80% ethanol in polysaccharide extract, allowed to stand at 4℃ for 24h,then centrifuged to obtain a crude extract of Ganoderma lucidum polysaccharides.2.2 In addition to protein of polysaccharidesBecause crude extract of Ganoderma lucidum polysaccharides often unavoidable contain proteoglycan protein covalently bound to proteins and peptides as well as free, must be removed during the purification of polysaccharide protein, Sevag method used in this experiment except protein specific experimental method is:press polysaccharide 1/3 of the volume of the extract was added Sevag reagent, after standing oscillation, and finally centrifuged to remove protein layer, was repeated several times until no protein layer2.3 Dialysis of polysaccharidesSmall molecules,experimental methods to extract Ganoderma lucidum polysaccharides containing inorganic salts are:Ganoderma lucidum polysaccharide extract loaded pretreated dialysis bag,placed in the distilled water and dialyzed 72h,and then freeze-dried to obtain a crude extract of Ganoderma lucidum polysaccharides.2.4 Ganoderma lucidum polysaccharides ultrafiltration membrane filtration ratingUltrafiltration is a use of pressure-driven membrane separation process to separate the compounds of different particle sizes. Ultrafiltration can polysaccharide samples graded by size molecular preliminary rough divided into paragraphs,specific experimental methods:After lucidum polysaccharides with the right amount of powder was dissolved in distilled water,the polysaccharide solution through 0.45μm membrane filter, washed with Biomax5、10 (KD) followed by filtration, GLP Ⅰ,GLPⅡ, GLPⅢ three parts were got. This polysaccharide solution obtained by ultrafiltration,Only is a broad molecular weight distribution of the component sample, in order to obtain a more pure polysaccharide sample, further purification is needed2.5 Column chromatography purification of polysaccharidesIon-exchange chromatography and gel filtration molecular sieve is used in conjunction polysaccharides most commonly used method of purification. Ion exchange chromatography ion chromatography media based on the principle of stationary phase.depending on the components of the polysaccharide and ion adsorption packing charge different degrees different,with different polarities dissolve polar elution to achieve separation of the different components of polysaccharides. This method is applicable to a neutral polysaccharide,acidic polysaccharide purification and viscous polysaccharide in.while cellulose ion exchange resin can be removed with a polar pigment sample. In this study, DEAE-Cellulose 52 anion exchange column, the GLP Ⅰ. GLPⅡ, GLPⅢ three parts were separated and purified by column chromatography.based on samples collected GLP Ⅰ elution curve has two peaks of polysaccharides. named GLP1、GLP2; GLP Ⅱ There is a polysaccharide peak named GLP3;GLPⅢ polysaccharide has two peaks, named GLP、GLP5.Gel filtration is a schematic of a molecular sieve based on the differences of molecular weight,molecular weight of the polysaccharide.which retention time is short, it will be eluted by the eluent, small molecular weight polysaccharides on the contrary.to achieve a purified polysaccharide purposes. In this study,Sephacryl S-300 HR gel filtration chromatography to GLP1、GLP2、GLP3、GLP4、GLP5 five components further separation and purification.3 the study of the nature of structural characteristics of GLP3.1 GLP solubility and KI-I reactionThe GLP 1、GLP2、GLP3、GLP4、GLP5 separately dissolved in water^ ethanol、acetone、n-butanol、ethvl acetate and a solvent such as chloroform, and GLP1、GLP2、GLP3、GLP4、GLP5 are soluble in water, viscosity greater、insoluble in ethanol、acetone、ethyl acetate、n-butanol and chloroform and other organic solvents were found.Weigh GLP1、GLP2、GLP3、GLP4、GLP、each 1.0mg, with distilled water volume to 2.0 ml. These five kinds of polysaccharide solution were added KI-I solution, observe the color change of the solution,while using distilled water as a negative control, starch solution 0.5 mg/ml as a positive control, the results show: GLP1、GLP2、GLP3、GLP4、GLP5 iodine-potassium iodide were negative,indicating that GLP does not contain starch.3.2 Polysaccharide content was measured form GLPPhenol-sulfuric acid method is adopted to measure Polysaccharide content of GLP1、GLP2、GLP3、GLP4、GLP5, the measurement results for:Polysaccharide content of GLP1、GLP2GLP3、GLP4、GLP5 are 96.54%、97.35%、91.51%、 96.58%97..13%3.3 monosaccharide composition analysis of GLPMonosaccharide composition analysis found that, GLP mainly glucose mannose and galactose as well as their mannose、galactose、glucose is about the percentage composition of GLP 1(17%、14%、69%)、GLP2(19%、24%、57%)、 GLP3(15%、13%、72%)、 GLP4 (20%、17%、63%), GLP5 (14%、21%、65%).3.4 Molecular weight determination of GLPStandards of known molecular weight dextran to DeTran T series (T2000、T500、 T200、T70、T20) as the standard, measured by retention time HP1C-E1SD, based on the number of the standard curve and the retention time of the molecular weight dextran is lgMR=-0.183tR-7.978 (R2= 0.994)、the GLP1、GLP2、GLP3、GLP4、 GLP5 retention time substitution regression curves were calculated molecular weight 5.93、7.24,8.43、10.51、12.77KD.3.5 Infrared Spectroscopy of GLPAnalyzed by infrared spectroscopy GLP structure.a preliminary judge for β-pyranoid polysaccharides.4. GLP vitro immunization experimentsSelect the rat spleen lymphocytes and macrophages RAW264.7 preliminary evaluation of two typical models immune activity of GLP1、GLP2、GLP3、GLP4、 GLP5, specific evaluation include:effects on lymphocyte proliferation, and on RAW264.7 NO generating capacity of macrophages、phagocytic capacity and proliferation.4.1 GLP spleen lymphocyte proliferationExperimental results show that different concentrations of GLP alone in mouse lymphocytes, has mitogenic effects. When the presence of LPS or ConA, GLP can promote collaborative ConA and lipopolysaccharide spleen lymphocyte proliferation. ConA promoting lymphocyte proliferation synergistic effect is stronger than the synergistic effects of LPS. At the same time as GLP1、GLP2、GLP3、GLP4、GLP5 molecular weight increases,the proliferation of rat spleen lymphocytes are more pronounced.4.2 GLP impact on RAW264.7 macrophages ability to generate NO、phagocytic capacity、proliferationThe results showed that:different concentrations of GLP can significantly promote RAW 264.7 macrophages NO production, and showed a clear dose-dependent manner, with the GLP1、GLP2、GLP3、GLP4、GLP5 molecular weight increases, macrophages generate NO can have some enhancements:promote RAW264.7 macrophage phagocytosis, but the concentration of polysaccharides. the molecular weight of no effect on the phagocytic capacity (P> 0.05)-does not promote the value of macrophages (P> 0.05).