Purification, Identification And Immune Activity Of A New Polysaccharide From Lentinula

Mushroom,with the scientific name Lentinus edodes, belongs to basidiomycotina（Pleurotaceae） fungus. Lentinus edodes is a kind of edible and medicinal fungi and is of greatvalue in tonic foods. It is widely distributed in our country. Mushrooms contain a variety ofeffective medicinal ingredients, of which the polysaccharide is one of the main effectivecomponents. The present study of mushroom polysaccharides mainly focused on its structureand biological activity, while the structure-activity relationship and mechanism of biologicalactivity is not very clear. In this study, mushroom was used as raw materials, including theisolation and purification, structure characterization and immune activity of polysaccharides,and the synthesis of a kind of polysaccharides-calcium complex L2–Ca. The main researchresults are as follows:Polysaccharides was obtained by extraction with boiling water and precipitation withethanol. After deproteinization, alcohol precipitation, DEAE-52cellulose ion exchangecolumn chromatography, concentration, dialysis and lyophilization, four polysaccharidecomponents CL1, CL2, CL3, CL4was obtained, the highest proportion of wich was CL2.CL2was further purified by dextran gel column chromatography （Sephadex-G200）. Thepolysaccharides after further purification was named L2. The carbohydrate content of L2was97%as indicated by phenol sulfuric acid analysis with glucose as a standard. Chemical andphysical analyses indicated that L2had a molecular weight of26KDa.L2did not contain proteins and nucleic acids. It contained the stretching vibration ofO-H at3418³405cm-1, the stretching vibration of C-H at2932²927cm-1, and thestretching vibration of carboxyl at1430¹200cm-1. All of them are the characteristicabsorption peak of IR of polysaccharide. In combination with other characteristic absorptionpeak, it is determined that L2is pyran polysaccharide with no uronic acid.Gas chromatographic analysis of L2proved that the monosaccharide composition of L2contained L-arabinose, D-glucose and D-galactose, and molecular ratio of Ara: Glu: Galwere1:31:3. Periodate oxidation and Smith degradation analysis proved that L2contained1-6,1-3and1-2glycosidic bond. Their percentage were44.33%,37.45%and18.22%,respectively.Congo red test results proved that L2did not have the triple-helical conformation, whichwas closely related to activities of polysaccharides according to many literature reports.Atomic force microscopy showed the molecular chain of L2form windings, while the chain rupture and the length decreases after137℃high temperature treatment. This suggested thatconformation of L2might change after high temperature treatment and may be a one of thereasons for its deactivation after high temperature treatment.Among four crude polysaccharide fractions CL1, CL2, CL3and CL4, CL2showed themost pronounced ability to stimulate NO production in RAW264.7cell. L2increased the NO,IL-6and TNF-α production of RAW264.7cells in a dose-dependent manner with aconcertration less than250μg/mL. The study of influence of temperature and pH on L2immune activity showed that L2deactivation after treatment under137℃for30min. L2maintained a ordered chain structure in the scope of pH4.0-8.0, while the active structure wasdamaged at pH10.0.TLR2is one of the receptors of L2on RAW264.7cells and the receptors like TLR4,CR3, and Dectin^-1 can be excluded. Other immune receptors are also possible. The receptorsof L2on immune cells differed significantly from those of lentinan.The optimum conditions for the synthesis of L2-Ca was determined: the temperature was65℃, the reaction time was60min, the pH was9.0, CaCl2and L2ratio was5.5:1; Accordingto infrared absorption spectra of L2and L2-Ca, it was speculated that the calcium ion may bechelated with C-O; Compared with L2, L2–Ca can be used as calcium supplement more thanimmunomodulator.