Systematical Analysis Of The Polysaccharides From Lentinus Edodes (Berkeley) Pegler And Their Antitumor Properties

Lentinus edodes (L. edodes) is the first cultivated edible mushroom in the world and the second most widely used Traditional Chinese Medicine (TCM) mushroom in global market. Its importance is attributed to both its nutritional value and medical application. Some bioactive compounds of L. edodes including phenolic compounds, ergosterol, selenium, eritadenine, vitamins, as well as calcium and minerals have been isolated and characterized. Recently, polysaccharides extracted from fruiting bodies, mycelia and sclerotia of L. edodes have been shown to have numerous activities including antitumor. immunomodulatory, antioxidant, anti-inflammatory, antiviral, hepatoprotective, hypocholesterolemia, hypoglycemic, etc. The present research study aim is an investigation of chemical structures, biological activity and structure-activity relationship of polysaccharides, other than those reported, extracted from the fruiting bodies of L. edodes.The dried and milled fruiting bodies was submitted to aqueous extraction (cold and hot) and alkaline extraction (1M NaOH). The aqueous extracts were submitted to ethanol precipitation (95%EtOH), alkaline extract was first neutralized with hydrochloric acid (3M HC1), dialyzed with membranes of Mw cut-off3500Da and then submitted to ethanol precipitation. Three crude polysaccharides were obtained namely CW-LEP (59%Glc.22%Gal.15%Man.1%Fuc,1%Rha and2%GlcA), WPLE (77%Glc.11%Gal.7%Man,1%Fuc.2%Rha and2%GlcA) and APLE (88%Glc,3%Gal.7%Man and2%Rha) from cold, hot and alkaline extractions, respectively.CW-LEP, obtained from cold water extraction, was fractionated and purifiedby GPC, using Sepharose CL-6B, Sephadex G-200and Sephadex G-75columns, into six fractions namely:CW-LEP-I-a (60%Glc,17%Gal.19%Man,2%GlcA and1%Fuc), CW-LEP-I-b (50%Glc,16%Gal,27%Man.2%GlcA and4%Fuc). CW-LEP-Ⅱ-a (42%Glc.14%Gal.39%Man.4%GlcA and1%Fuc).CW-LEP-Ⅱ-b (66%Glc.7%Gal,21%Man and5%GicA), CW-LEP-Ⅲ-a (48%Glc,31%Gal,15%Man,3%GlcA and3%Fuc) and CW-LEP-Ⅲ-b (61%Glc,23%Gal.11%Man.4%GlcA and1%Fuc). Their Mw were estimated to be>2000kDa,584.8kDa,383.2kDa,77.0kDa.18.3kDa and5.9kDa, respectively. Thus, CW-LEP-Ⅰ-a might be a galactomannoglucan-type polysaccharide, CW-LEP-Ⅰ-b a fucogalactomannoglucan-type polysaccharide, CW-LEP-Ⅱ-a and CW-LEP-Ⅱ-bglucuronogalactomannoglucan-type polysaccharides. CW-LEP-Ⅲ-a a fucomannogalactoglucan-type polysaccharide and CW-LEP-Ⅲ-b a glucuronomannogalactoglucan-type polysaccharide.WPLE. obtained from hot water extraction, was submitted successivelv to anion-exchange chromatography on a DEAE-cellulose column with stepwise elution (water.0.5M NaCl and1M NaCl), freeze-thawing process and gradient ethanol precipitation (30%,50%,70%and90%). After these processes different polysaccharide fractions were obtained, and then were purified and characterized.WPLE-N, the water-eluted fraction, was heterogeneous and composed of Glc-Gal-Man in the molar ratio of79:15:6:and it was purified into three fractions of WPLE-N-1. WPLE-N-2and WPLE-N-3on Sepharose CL-6B column. GPC and HPGPC showed that these three fractions are homogeneous and their Mw were estimated to be757.5kDa,20.9kDa and4.7kDa, respectively. HPLC, methylation analysis, periodate oxidation-Smith degradation, FT-IR and NMR spectroscopy, including2D NMR (HMBC and HMQC) indicated that WPLE-N-1was a glucan-type polysaccharide consisted of (1→-6)-β-D-Glcp residues; while WPLE-N-2and WPLE-N-3were mannogalactoglucan-type polysaccharides containing (1→6)-,(1→4)-and (1→3)-a-D-Glcp residues,(1→6)-a-D-Galp,(1→3.6)-and (1→2,4)-α-D-Manp residues and terminal residues of D-Glcp residues. WPLE-A0.5, the0.5M NaCl-eluted fraction, was also heterogeneous and composed of Glc-Gal-Man-Fuc-GlcA in the molar ratio of49:31:15:4:1; and it was fractionated on a Sepharose CL-6B. Its main fraction WPLE-A0.5-2was homogeneous with a Mw of18kDa. Chemical and spectroscopic studies indicated that WPLE-AO.5-2was a mannogalactoglucan-type polysaccharide consisted of (1→6)-,(1→4)-and (1→3)-linked β-D-Glcp residues,(1→6)-liked a-D-Galp residues,(1→3,6)-and (1→2,4)-linked a-D-Manp residues and terminal residues of β-D-Glcp.S-WPLE, the soluble fraction from freeze-thawing process, was heterogeneous and composed of Glc (48.3%), Gal (24.0%), Man (20.1%), Fuc (2.8%), GlcA (2.0%), Rha (2.5%) and Xyl (0.2%). S-WPLE was then fractionated and purified by SEC, using Sepharose CL-6B, Sephadex G-100and Sephadex G-75columns, into six homogeneous fractions namely S-WPLE-Ⅰ-a, S-WPLE-Ⅰ-b, S-WPLE-Ⅱ-a, S-WPLE-Ⅱ-b, S-WPLE-Ⅲ-a and S-WPLE-Ⅲ-b and they were composed of Glc-Gal-Man in the molar ratio of98:1:1,91:5:4,88:4:8,74:16:10,79:14:7and70:14:11, respectively. Their Mw were estimated to be>2000kDa,755.32kDa,121.5kDa,23.6kDa,15.2kDa and7.5kDa. respectively. Methylation analysis experiments revealed that S-WPLE-Ⅰ-a contained (1→3)-,(1→4)-,(1→6)-,(1→3,6)-D-Glcp residues and terminal residues of D-Glcp; S-WPLE-I-b consisted of (1→3)-,(1→4)-.(1→6)-and (1→3.6)-D-Glcpresidues.(1-6)-D-Galp residues,(1→6)-linked D-Manp residues and terminal D-Glcp residues; S-WPLE-Ⅱ-a and S-WPLE-Ⅱ-b were constituted of (1→3)-,(1→4)-,(1->6)-and (1→3,6)-D-Glcp residues,(1→6)-D-Galp residues.(1→2,4)-D-Manp residues and terminal residues of D-Glcp at different ratios; S-WPLE-Ⅲ-a comprised (1→3)-,(1→4)-,(1→6)-and (1→3.6)-D-Glcpresidues,(1→-6)-D-Galp residues,(1→6)-,(1→2.4)-D-Manp residues and terminal D-Glcp residues:while S-WPLE-Ⅲ-b contained (1→3)-,(1→6)-and (1→3,6)-D-Glcp residues,(1→4)-D-Galp residues,(1→3,6),(1→2,4)-D-Manp residues and tenninal D-Glcp residues. It could thus be concluded that S-WPLE-Ⅰ-a and S-WPLE-Ⅰ-b were glucan-type polysaccharides. S-WPLE-Ⅱ-a was a galactomannoglucan-type polysaccharide, and S-WPLE-Ⅱ-b. S-WPLE-Ⅲ-a and S-WPLE-Ⅲ-b were mannogalactoglucan-type polysaccharides.The fractionation of WPLE by gradient EtOH precipitation with30%.50%.70%and90%yielded to four fractions of WPLE-E30. WPLE-E50. WPLE-E70and WPLE-E90. respectively. WPLE-E30and WPLE-E50were mainly constituted of Glc (97%and96%, respectively). WPLE-E70and WPLE-E90were composed of Glc-Gal-Man-GlcA-Fuc in the molar ratio of72:15:9:3:1and80:6:11:3:0, respectively.WPLE-E50was fractionated into three fractions on Sepharose CL-6B column, namely WPLE-E50-Ⅰ (96%Glc,2%Gal,2%Man). WPLE-E50-Ⅱ-a (86%Glc,3%Gal,11%Man) and WPLE-E50-Ⅱ-b (82%Glc,10%Gal,6%Man,2%Fuc). Based on these above HPLC results, it could thus be concluded that WPLE-E50-Iwas a glucan-type polysaccharide. WPLE-E50-Ⅱ-a a galactomannoglucan-type polysaccharide and WPLE-E50-Ⅱ-ba mannogalactoglucan-type polysaccharide. Their M(?)’ were>2000kDa,297.4kDa and9.1kDa, respectively.wPLE-E70, after fractionation and purification on Sepharose CL-6B and Sephadex G-75columns, gave six fractions of WPLE-E70-Ⅰ-a, WPLE-E70-Ⅰ-b (71%Glc.12%Gal.14%Man,2%GlcA,1%Fuc), WPLE-E70-Ⅱ-a (72%Glc,12%Gal,12%Man.2%GlcA,1%Fuc), WPLE-E70-Ⅱ-b (84%Glc,5%Gal,6%Man,4%GlcA.1%Fuc), WPLE-E70-Ⅲ-a (70%Glc,18%Gal,6%Man.3%GlcA,3%Fuc) and WPLE-E70-Ⅲ-b (74%Glc,14%Gal.6%Man,3%GlcA,3%Fuc). It could thus be concluded that WPLE-E70-Ⅰ-b and WPLE-E70-Ⅱ-a were galactomannoglucan-type polysaccharides, WPLE-E70-Ⅱ-b was a glucuronogalactomannoglucan-typepolysaccharide; WPLE-E70-Ⅲ-a and WPLE-E70-Ⅲ-b might be glucuronomannogalactoglucan-type polysaccharides. WPLE-E70-I-b, WPLE-E70-Ⅱ-a. WPLE-E70-Ⅱ-b. WPLE-E70-Ⅲ-a and WPLE-E70-Ⅲ-b gave each a single and symmetrical peak on HPGPC. Their M(?)’ were475.4kDa,400.0kDa,95.1kDa,17.3kDa and6.8kDa, respectively estimated also by HPGPC using standard dextrans of known molecular weight. The peak of WPLE-E70-Ⅰ-a was not a symmetrical one and its M(?)’ was estimated to be1159.5kDa.WPLE-E90fractionation and purification on Sepharose CL-6B, Sephadex G-150and Sephadex G-50yielded in six fractions of WPLE-E90-Ⅰ-a (76%Glc,3%Gal,19%Man,2%GlcA), WPLE-E90-Ⅰ-b (52%Glc.25%Gal,21%Man,2%GlcA). WPLE-E90-Ⅱ-a (73%Glc.2%Gal,22%Man.3%GlcA). WPLE-E90-Ⅱ-b (82%Glc,4%Gal.11%Man,3%GlcA). WPLE-E90-Ⅲ-a (82%Glc,9%Gal7%Man,2%Fuc) and WPLE-E90-Ⅲ-b (75%Glc,13%Gal,8%Man.1%GlcA.3%Fuc). Thus. WPLE-E90-Ⅰ-a.WPLE-E90-Ⅱ-a and WPLE-E90-Ⅱ-b might be glucuronogalactomannoglucan-typepolysaccharides, WPLE-E90-Ⅰ-b and WPLE-E90-Ⅲ-aglucuronomannogalactoglucan-type polysaccharides. and WPLE-E90-Ⅲ-b a fucomannogalactoglucan-type polysaccharide. WPLE-E90-Ⅰ-a. WPLE-E90-Ⅰ-b, WPLE-E90-Ⅱ-a. WPLE-E90-Ⅱ-D. WPLE-E90-Ⅲ-a and WPLE-E90-Ⅲ-b each presented a single and symmetrical peak on HPGPC suggesting that they are homogeneous fractions and based on the equation determined by linear regression from different standard dextrans of known Mw. their Mw were estimated to be442.6kDa.289.3kDa.135.6kDa.43.7kDa.8.4kDa and4.2kDa. respectivelv.APLE. obtained from alkaline extraction, was submitted to freeze-thawing process yielding to two fractions of I-APLE composed mainly of Glc (97%) and S-APLE constituted of Glc (91%). Gal (5%) and Man (4%). S-APLE, the soluble fraction from freeze-thawing process, was heterogeneous and was then fractionated and purified by SEC, using Sepharose CL-6B column, into four homogeneous fractions namely S-APLE-Ⅰ-a (96%Glc.4%Man). S-APLE-Ⅱ-b (93%Glc.7%Gal), S-APLE-Ⅲ (84%Glc.11%Gal.5%Man) and S-APLE-Ⅳ (71%Glc,16%Gal,13%Man) and their Mw were estimated to be20.4kDa.68.5kDa,230kDa and2120kDa. respectively. Thus, S-APLE-Ⅰ-a could be considered as mannoglucan-type polysaccharide, S-APLE-Ⅱ-b as galactoglucan-type polysaccharide. S-APLE-Ⅲ and S-APLE-Ⅳ as mannogalactoglucan-types polysaccharides. In total, we have obtained34polysaccharide fractions including5glucan-types,1galactoglucan-type,4mannoglucan-types,8galactomannoglucan-typesand16mannogalactoglucan-types.On a preliminary bioactivity test (cytotoxicity assay). WPLE-N-1. WPLE-N-2. WPLE-N-3, WPLE-A05-2, S-WPLE. S-WPLE-Ⅰ-a. S-WPLE-Ⅰ-b, S-WPLE-Ⅱ-a, S-WPLE-Ⅱ-b. S-WPLE-Ⅲ-a and S-WPLE-Ⅲ-b, at different concentrations (1,2,5mg/mL), exhibited antitumor activity against Sarcoma S-180, Carcinoma HCT-116and HT-29in vitro with a dose-dependent manner. High Mw polysaccharides appeared to be more effective than those of low Mw. In addition, these polysaccharides presented selectively higher antitumor activities against suspended cell (Sarcoma S-180) than adherent ones (HCT-116and HT-29carcinoma cells). The role of Gal, Man and bound protein in the anti-proliferative effect should also be considered.The results of the in vivo antitumor activity assay of WPLE-N-1, WPLE-N-2and WPLE-A0.5-2against Sarcoma180solid tumor grown in Kunming mice at the dose of100mg/kg for10days showed that WPLE-N-2and WPLE-A0.5-2, which are mannogalactoglucan-type polysaccharides, exhibited a higher inhibition than WPLE-N-1which is a glucan-type polysaccharide. Furthermore, in vivo bioassay test indicated that WPLE-N-1,WPLE-N-2and WPLE-A0.5-2could remarkably increase the phagocytosis of macrophages. induce the secretion of nitric oxide, enhance T and B lymphocyte proliferations, and augment the tumor weight regression.Thus, the analysis and evaluation of Lentinus edodes polysaccharides provides a potential opportunity for the discovery of novel therapeutic agents that exhibit beneficial antitumor and immunomodulatory properties. Our results suggest then that mannogalactoglucan-type polysaccharides should be explored as potential antitumor and immunomodulatory agents, utilized as tumor cell growth inhibitors for food and pharmaceutical industries and could be potentially applied as natural antitumor drug.This research contributed to a better understanding of L. edodes compounds, especially polysaccharides, and provided some essential data for successfully interpretation of the bioactivities of its extracts.