Structural Analysis And Biological Activity Of A Jelly-like Polysaccharide From Lentinus Edodes

Mushrooms have been known for their nutritional and culinary values as well as viewed as tonics. They have been used as medicines by humans over decades up to now. In many Asian countries, they are considered as functional food which can provide health benefits beyond the traditional nutrients they contain. Phylum Basidiomycota, constitute of very diverse groups of mushrooms in which, most of them have macroscopic fruiting. Lentinus edodes (Berk.) Sing.,(Shiitake, Jap; Xiang-gu; Chn.) is a wood-inhabiting basidiomycetes, which belongs to the family Polyporaceae under order Agaricale. This mushroom is one among the most popular species of Fungi because of its constitutive bioactive compounds such as, high molecular weight β-glucans (such as Lentinan), proteins/peptide bound polysaccharides, among others; which were proven to have antitumor effects as well as immunostimulating potentials.Several studies have been conducted on extraction, purification and structural elucidation of water-extractable polysaccharides from fruiting bodies of Lentinus edodes. However, few or none of them involved isolation and structural characterization of solubilized polysaccharide fractions from freeze-thawing process of water-extracted crude polysaccharide. In this research, structural characterization of solubilized polysaccharide fractions from freeze-thawing process was investigated. Some of the polysaccharides fractions obtained were assayed for anti-proliferative activity in vitro by using HCT-116and HT-29human colorectal cancer cell lines.The fruiting bodies of Lentinus edodes were extracted successfully with hot water, precipitated with ethanol80%(v/v) and deproteined by using sevag method, to obtain a crude polysaccharide WPLE. Upon submission of WPLE to the freeze-thawing process, a high polydispersed Jelly-like polysaccharide I-WPLE was obtained. On solubilization of this polysaccharide (with2%aq. NaOH), neutralizing with50%acetic acid, and submitting to freeze-thawing process followed by centrifuging at10,000r.p.m for20min, a water soluble fraction I-WPLE-S emerged. It exhibited a wide range of molecular weight distribution from4kDa to>1000kDa. I-WPLE-S was found a neutral polysaccharide with galactose (7.14%), glucose (86.52%) and mannose (3.37%). In addition, chromatography techniques were used for further isolation and purification of fraction I-WPLE-S in which, on applying to Sepharose CL-6B preparative column, four fractions (I- WPLE-S1, I-WPLE-S2, I-WPLE-S3and I-WPLE-S4) were collected. The four fractions were further purified on Sepharose CL-6B analytical gel-column and showed single peaks. The homogeneity and molecular weight of fractions I-WPLE-S1and I-WPLE-S2were assessed by GPC using Sepharose CL-6B and both showed single symmetric peaks with polydispersity index (Mw/Mn) of less than1.5and Mw estimation of>500kDa (I-WPLE-S2) and>1000kDa (I-WPLE-S1). The homogeneity and molecular weight distributions of fraction I-WPLE-S3and I-WPLE-S4were estimated by HPGPC linked with GPC of TSK-gel G-3000PWxl, and both gives narrow and symmetric peaks by HPGPC with the estimated Mw of12.74kDa and4.21kDa respectively, with a polydispersity index of<1.5.The HPLC analysis showed that; fraction I-WPLE-Sl consisted of Gal(3.77%), Glc (94.84%) and Man (5.16%), while I-WPLE-S2constitute of only Glc (92.40%) and Man (7.60%); I-WPLE-S3contains Gal(22.36%), Glc (65.81%) and Man (11.84%); and I-WPLE-S4has only Gal(6.14%) and Glc (93.86%). However, structural analyses done on fraction I-WPLE-S2and I-WPLE-S4, using NMR and FT-IT revealed that; I-WPLE-S2consisting of (1â†'3)-β-D-Glcp main chain with single residues of D-Mannose attached to the C-6of the D-glucose on the main chain; while I-WPLE-S4consisting of (1â†'6)-β-D-Glcp back bone with single residues of D-galactose probably side attached to the C-6of the glucose of (1â†'6)-β-D-Glcp main chain. Structure of I-WPLE-S1, based on the FT-IR and HPLC was possibly a B-D-Glucan; and I-WPLE-S3was a heteropolysaccharide possibly, a mannogalactoglucan.Moreover, the in vitro assay of anti-proliferative effect of fractions I-WPLE-S3and I-WPLE-S4on human colorectal cancer HCT-116and HT-29cell lines showed that, the polysaccharides did not have anti-proliferative effect on HCT-116tumor cells line, but they showed slight significant differences compared to the control, at5mg/mL concentration on HT-29tumor cells line after72hr. of incubation, at37℃in5%CO2incubator. I-WPLE-S3showed25%significant difference of the effect, while I-WPLE-S4showed14%difference from the control.