Preparation And Bioactive Activity Of Polysaccharide From Lentinus Edodes Mycelia

Lentinus eddoes, belongs to Basidaiomycetes, Agaricales, Pleurotaceae, also named fragrant mushroom. As a kind of nutritive food and rare medicinal material, it is well known all over the world. The biological activities of these polysaccharides have attracted more attention because of their antioxidant, antitumor and immunomodulatory effects recently. At present, fruiting bodies and mycelium are the two main sources of fungus polysaccharides. In the last few years, a lot of studies on fruiting bodies have been reported, but little mycelium polysaccharides and its bioactivity research were reported, and mostly focused on the immunoregulatory activity. Mycelium culture has potential advantages for offering a promising alternative which is higher production, cost-effective, easy to control and without heavy metal contamination, which cause widely attention. The study was focus on the extraction process, main bioactive activity and safety evaluation. The overall goal of the studies is to provide further information for research and reasonable application of polysaccharide from Lentinus eddoes mycelium.In order to determine the optimum extraction conditions of polysaccharides from mycelia of Lentinus edodes by ultrasonic cell disruption and hot water extraction, L9(34)orthogonal test was designed to investigate the effects of the operating parameters material-to-solvent ratio, temperature, ultrasound intensity and extraction time on polysaccharide extraction. Further, the obtained extract was purified by DEAE-32 ion exchange column and Sephadex G-200 gel permeation column sequentially. To analysis the polysaccharide protect Min6 and Ins-1 from H2O2 stimulus on cellular level. DCFH-DA was used to detect Min6 and Ins-1 intracellular reactive oxygen species(ROS)level. Its antitumor activity was evaluated by 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assayand transwell assay in vitro. The Ames test, bone marrow polychromatic erythrocyte micronucleus test, chromosomal aberrations test and comet assay were used to detect the toxicity of polysaccharide from Lentinus edodes mycelium.The optimum conditions of polysaccharides extracted from Lentinus edodes mycelia were material-to-solvent ratio 1﹕15, ultrasound intensity 400 W, extraction temperature 90 ℃, and extraction time 3 h. Under the optimum conditions, the extraction yield of polysaccharides was 3.39±0.29 ‰. The polysaccharide having DPPH free radical scavenging ability was obtained and named as LMP Ⅱ. The high purity of LMP Ⅱ was demonstrated by UV spectral scanning and cellulose acetate membrane electrophoresis. HPLC results indicated that LMP Ⅱ contained mannose, arabinose, galactose, xyloseand rhamnose with a relative molar ratio of 1:0.74:3.23:1.18:10.98.The polysaccharide has the capability of scavenging superoxide anion and hydroxyl free radicals. It could inhibit the production of MAD in plasma and hydrogen peroxide-induced erythrocyte hemolysis in a dose-dependent fashion. Thus, LMP Ⅱ has a strong antioxidant potential. Through the morphological observation, polysaccharide can protect Min6 and Ins-1 from H2O2 stimulus. This suggesting that polysaccharide has a strong antioxidant potential. Using the DCFH-DA as the ROS probe, the intracellular ROS was decreased significantly in polysaccharide group, it demonstrated polysaccharide from Lentinus edodes mycelium has a ROS scavenging ability in Min6 and Ins-1.LMP Ⅱ has a remarkable inhibitory effect on the proliferation of Hep-2 cells at the concentration of 50 μg/m L, 100 μg/m L, 150 μg/m L and 200 μg/m L. Furthermore,colony formation of Hep-2 cells was reduced significantly after treatment with LMP2. Invasion of Hep-2 cells was inhibited significantly by LMP2. These results suggested that LMP2 could be explored as a potential antitumor material for laryngeal carcinoma.The LD50 of polysaccharide from Lentinus edodes mycelium by i.g. to mice is more than 20 g/kg,no toxicity existing. There are all negative results in Ames test, bone marrow polychromatic erythrocyte micronucleus tes, chromosomal aberrations test and comet assay,there are not significant differences between the negative control and the treated groups(p>0.05) respectively. The results of genetic toxicity tests are all negative. Polysaccharide from Lentinus edodes mycelium has no toxicity.The results were very important to the application of polysaccharide from Lentinus edodes mycelium and have provided experimental basis for the development of Lentinus edodes resources and application of polysaccharide. The relevant results have provided experimental basis for toxicity studies and safety evaluation of polysaccharide from Lentinus edodes mycelium.