Fruiting Bodies Of Lentinus Edodes Research Articles

Ultrasound assisted extraction of polysaccharides from Lentinus edodes and its anti-hepatitis B activity in vitro

The aim of this study was to optimize the extraction process of polysaccharides from the fruiting bodies of Lentinus edodes and investigate its anti-hepatitis B virus activity. The extracting parameters including ultrasonic power (240–320W), extraction temperature (40–60°C) and extraction time (15–25min) was optimized by using three-variable-three-level Box-Behnken design based on the single-factor experiments. Data analysis results showed that the optimal conditions for extracting LEPs were an extraction temperature of 45°C, extraction time of 21min and ultrasonic power of 290W. Under these optimal conditions, the experimental yield of LEPs was 9.75%, a 1.62-fold increase compared with conventional heat water extraction (HWE). In addition, crude polysaccharides were purified to obtain two fractions (LEP-1 and LEP-2). Chemical analysis showed that these components were rich in glucose, arabinose and mannose. Furthermore, HepG2.2.15 cells were used as in vitro models to evaluate their anti-hepatitis B virus (HBV) activity. The results suggest that LEPs possesses potent anti-HBV activity in vitro.

A Lentinus edodes polysaccharide induces mitochondrial-mediated apoptosis in human cervical carcinoma HeLa cells

In this study, a homogeneous polysaccharide (LEP1) with an average molecular weight of 53kDa was successfully purified from the fruiting bodies of Lentinus edodes and its anticancer efficacy on human cervical carcinoma HeLa cells in vitro and associated possible molecular mechanism were also evaluated. MTT assay showed that LEP1 exhibited a dose-dependent inhibitory effect on the proliferation of HeLa cells and caused apoptotic death. Our present findings provided the first evidence that LEP1 induced the apoptosis of HeLa cells via a mitochondria dependent pathway, as indicated by an increase in Bax/Bcl-2 ratio, a loss of mitochondrial membrane potential (Δym), the release of cytochrome c from the mitochondria to the cytosol, activation of caspase-9 and caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP) in HeLa cells. These combined results unequivocally indicated that the involvement of mitochondria-mediated signaling pathway in LEP1-induced apoptosis and strongly provided experimental evidence for the use of LEP1 as a potential therapeutic agent in the prevention and treatment of human cervical carcinoma.

In vivo anticancer and immunomodulating activities of mannogalactoglucan-type polysaccharides from Lentinus edodes (Berkeley) Singer.

There is considerable interest in the potential of mushrooms in modulating the immune system and/or suppressing tumor growth. Among the studied bioactive compounds in mushrooms, polysaccharides are the most important. Nontoxic fungal polysaccharides have a more important role in immunomodulating and antitumor activities which are related to their effects to act of immune effecter cells such as lymphocytes, macrophages, dendritic cells, and natural killer cells involved in the innate and adaptive immunity. Two mannogalactoglucan-type polysaccharides (WPLE-N-2 and WPLE-A0.5-2), purified from the fruiting bodies of Lentinus edodes, were evaluated for their effects on the cellular immune response of Sarcoma 180 (S-180)-bearing mice. Mice were treated with 100 mg/kg body weight of the polysaccharides for 10 days. Significant tumor regressions of the polysaccharide groups’ mice were observed compared to the control group. These polysaccharides could induce an increase in nitrite oxide (NO) production in peritoneal macrophages, significantly increase macrophage phagocytosis of tumor-bearing mice and augment concanavalin (ConA) and lipopolysaccharide (LPS)-induced splenocytes proliferation. Our results indicated that immunomodulating activity occurred through host mediation in response to lymphocyte proliferation, macrophage phagocytosis and induction of NO production while the antitumor activity occurred through direct cytotoxicity. Our findings suggest that mannogalactoglucan-type polysaccharides from L. edodes can be explored as novel potential immunostimulants. Our research provides essential data to a better understanding of L. edodes bioactive compounds, especially polysaccharides. Our results also confirm the key role of β-linkages in the antitumor and immunomodulating effects of polysaccharides.

Induction of apoptosis in S180 tumour bearing mice by polysaccharide from Lentinus edodes via mitochondria apoptotic pathway

JLNT, a purified polysaccharide with the structure of β-(1→3)-D-glucose main chains and β-(1→6)-D-glucose side chains and with an average molecular weight of 605.4 kDa, was extracted and isolated from the fruitbodies of Lentinus edodes, which has immunoregulatory activity and anti-tumour effects. In the present study, we found that JLNT possessed direct anti-tumour effects on MCF-7 and S180 cells and apoptosis -inducing activity in S180 tumour bearing mice. Therefore, we investigated the mechanisms of JLNT-induced apoptosis. Results showed that JLNT increased the expression of p53, enhanced the Bax/Bcl-2 ratios by up-regulating Bax and down-regulating Bcl-2, promoted the expression of Smac, leading to the activation of caspase-3 and finally the initiation of cell apoptosis in S180 tumour bearing mice. Taken together, these findings provide the experimental evidence that JLNT could induce apoptosis of S180 cells via mitochondria pathways.

Determination of the Triple Helical Chain Conformation of β-Glucan by Facile and Reliable Triple-Detector Size Exclusion Chromatography

Triple helical polysaccharides (t-polysaccharides) are easily gelated in water, resulting in difficult fractionation, leading to the complex and time-consuming chain conformational characterization. Moreover, the fractionation is not always successful due to the coexistence of individual chains and aggregates. In this work, we developed a facile and reliable method to rapidly and accurately characterize the chain conformation of t-polysaccharide without fractionation needed in traditional conformation characterization. A triple helical β-1,3-glucan (t-β-1,3-glucan), extracted from the fruiting bodies of Lentinus edodes, was identified to consist of a β-1,3-glucan with two β-1,6-D-glucopyranoside branchings for every five β-1,3-glucopyranoside linear linkages by one- and two-dimensional NMR and GC-MS analysis. The chain conformations of the t-β-glucan in aqueous solution and in DMSO were successfully characterized by a combination of size exclusion chromatography (SEC), multiangle static light scattering, a differential refractometer, and a capillary viscosity detector (triple-detector SEC). The results revealed that the predominate species of the t-β-glucan in a 0.15 M NaCl aqueous solution existed as a triple helical conformation with high chain stiffness, and a few aggregates (4%) coexisted here. The Mark-Houwink and ⟨S(2)⟩(1/2) versus M(w) equations of individual triple helical chains and aggregates were obtained simultaneously, and the results confirmed again the coexistence of two kinds of chain conformations. The fractal dimension indicated that the aggregate in the aqueous solution was a kind of reversible microgel with a 3D network structure. Furthermore, the chain morphology of the t-β-glucan in aqueous solution was observed directly by transmission electron microscopy and atomic force microscopy to support the worm-like chain for the individuals and 3D network for the aggregates. The triple-detector SEC technology was facile and reliable for the system with two fractions of different chain conformation, and the test time required was only 1/30 of what the traditional method needed.

Purification, structural elucidation and antitumor activity of a novel mannogalactoglucan from the fruiting bodies of Lentinus edodes

A mannogalactoglucan, named LE-MGG, was isolated from the basidiocarps of Lentinus edodes by hot water-extraction, ethanol precipitation anion exchange chromatography, and further purified by gel-permeation chromatography (GPC). Its structural features were investigated by high performance liquid chromatography (HPLC), high performance gel-permeation chromatography (HPGPC), methylation analysis, periodate oxidation-Smith degradation, and by IR and NMR spectroscopy, including two-dimensional (2D) NMR. HPLC analysis revealed that LE-MGG contained mannose–galactose–glucose in the molar ratio of 10:18:72. GPC and HPGPC showed that LE-MGG was a homogeneous fraction (d=1.34) and its molecular weight was estimated to be 18kDa. Chemical and spectroscopic studies indicated that LE-MGG consists of (1→6)-, (1→4)- and (1→3)-linked β-d-glucopyranosyl residues, (1→6)-linked α-d-galactopyranosyl residues, (1→3,6)- and (1→2,4)-linked α-d-mannopyranosyl residues and terminal residues of β-d-glucopyranosyl. Cytotoxicity assay showed that LE-MGG presented higher antitumor activities against S-180 cell with a dose-dependent manner, and exhibited lower cytotoxicity to carcinoma HCT-116 and HT-29 cells. Our studies showed also that LE-MGG presented antitumor bioactivities on Sarcoma 180 solid tumor cell implanted in Kunming mice. This finding suggests that mannogalactoglucan should be explored as potential antitumor agents and could be potentially applied as a natural antitumor drug.

β-Glucan from Lentinus edodes Inhibits Nitric Oxide and Tumor Necrosis Factor-α Production and Phosphorylation of Mitogen-activated Protein Kinases in Lipopolysaccharide-stimulated Murine RAW 264.7 Macrophages

Lentinan (LNT), a β-glucan from the fruiting bodies of Lentinus edodes, is well known to have immunomodulatory activity. NO and TNF-α are associated with many inflammatory diseases. In this study, we investigated the effects of LNT extracted by sonication (LNT-S) on the NO and TNF-α production in LPS-stimulated murine RAW 264.7 macrophages. The results suggested that treatment with LNT-S not only resulted in the striking inhibition of TNF-α and NO production in LPS-activated macrophage RAW 264.7 cells, but also the protein expression of inducible NOS (iNOS) and the gene expression of iNOS mRNA and TNF-α mRNA. It is surprising that LNT-S enhanced LPS-induced NF-κB p65 nuclear translocation and NF-κB luciferase activity, but severely inhibited the phosphorylation of JNK1/2 and ERK1/2. The neutralizing antibodies of anti-Dectin-1 and anti-TLR2 hardly affected the inhibition of NO production. All of these results suggested that the suppression of LPS-induced NO and TNF-α production was at least partially attributable to the inhibition of JNK1/2 and ERK1/2 activation. This work discovered a promising molecule to control the diseases associated with overproduction of NO and TNF-α.

Antioxidant activities of polysaccharides from Lentinus edodes and their significance for disease prevention

The crude polysaccharide (LEP) was extracted by hot water from the fruiting bodies of Lentinus edodes, and further purified by DEAE-cellulose and Sepharose CL-6B chromatography, giving three polysaccharide fractions coded as LEPA1, LEPB1 and LEPC1. In this study, their chemical and physical characteristics of polysaccharide fractions and antioxidant capacities, including scavenging activity against hydroxyl radicals, superoxide radicals and Fe2+-chelating ability, were valuated. The results showed that LEPC1 exhibited significantly antioxidant activity at a concentration-dependent manner. Therefore these results indicated that the water-extractable polysaccharide fraction was a potent antioxidant and could be developed to be new health medicine for fighting against various human diseases.

Chain structures of glucans from Lentinus edodes and their effects on NO production from RAW 264.7 macrophages

Abstract Four glucans (LNT-H, LNT-S, LNT-E, and LNT-B) were successively isolated from the fruiting bodies of Lentinus edodes by hot-water, sonication, enzyme, and NaOH. The chemical structures were characterized by GC, FTIR, and 13 C NMR, which demonstrated that the four samples are (1 → 3)-β- d -glucans with (1 → 6)-glucopyranoside side groups having different molecular weight. The results of viscometry and atomic force microscopy (AFM) proved that LNT-H, LNT-S, LNT-E, and LNT-B exist as stiff triple helices in water and random coil in dimethyl sulfoxide (DMSO). The morphology of the original triple helix was almost dominated by linear shape, but the renatured LNT-B contains some branched structures in AFM image. Among the four glucans, LNT-H, LNT-E, and LNT-B are clearly contaminated by endotoxin, while LNT-S significantly exhibits nitric oxide (NO) inhibition effect in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner. Therefore, LNT-S is probably used as a promising molecule which can be helpful to the diseases associated with NO overproduction. This is the first primary report dealing with the suppression of LPS-induced NO from macrophage RAW 264.7 cells by Lentinan.

Immunomodulatory β-Glucan from Lentinus edodes Activates Mitogen-activated Protein Kinases and Nuclear Factor-κB in Murine RAW 264.7 Macrophages

Lentinan, a cell wall β-glucan from the fruiting bodies of Lentinus edodes, is well known to be a biological defense modifier, but the signal transduction pathway(s) induced by Lentinan have not been elucidated. In this study, we extracted Lentinan (LNT-S) by ultrasonication from Lentinus edodes and report that, in murine RAW 264.7 macrophages, LNT-S glucan activated NF-κB p65 and triggered its nuclear translocation as determined by Western blotting. Moreover, LNT-S enhanced NF-κB-luciferase activity in the Dual-Luciferase gene system assay. Its upstream signaling molecules, MAPKs such as ERK1/2 and JNK1/2, were shown to be activated by assessing the level of phosphorylation in a time- and concentration-dependent manner, but its downstream proinflammatory enzyme, inducible NOS, was not observed. The data evaluated using a TNF-α ELISA kit and Griess reagent further demonstrated that no proinflammatory mediators such as TNF-α and NO were produced by LNT-S stimulation in RAW 264.7 cells. In contrast, LPS significantly induced inducible NOS expression and increased NO and TNF-α production, which are associated with activation of the NF-κB p65/p50 heterodimer complex. It is possible that LNT-S did not activate NF-κB p65/p50, and the activation of NF-κB p65 was not sufficient to stimulate cytokine production. These data demonstrate that LNT-S glucan carries out its immunomodulating activity by activating MAPK signaling pathways without secretion of TNF-α and NO.

Antitumor Activities ofO-Sulfonated Derivatives of (1→3)-α-D-Glucan from DifferentLentinus edodes

Four water-insoluble (1-->3)-alpha-D-glucans, coded L-II1, L-II2, L-II3 and L-II4, with different molecular weights were isolated from four kinds of fruiting bodies of Lentinus Edodes. The four alpha-D-glucans were O-sulfonated to obtain derivatives (SL-II) having degrees of substitution (DS) from 0.9 to 2.1 respectively. The structure of the samples was analyzed by infrared spectra, elemental analysis, and 13C NMR. The weight-average molecular weight (Mw), radii of gyration (<s2>z1/2) and intrinsic viscosity ([eta]) of the native alpha-D-glucans and O-sulfonated derivatives were measured by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in 0.2 M aqueous NaCl and in dimethyl sulfoxide (DMSO) containing 0.25 M LiCl at 25 degrees C respectively. The Mw values of the O-sulfonated derivatives were much lower than those of the native alpha-D-glucans. The experimental results indicate that the O-sulfonated derivatives are water-soluble and exist as an expanded flexible chain in aqueous solution owing to intramolecular hydrogen bonding or interaction between charge groups. The in vivo and in vitro antitumor activities of the native alpha-D-glucans and their O-sulfonated derivatives against solid tumor Sarcoma 180 cells were evaluated and compared. Interestingly, all of the O-sulfonated derivatives exhibited higher antitumor activities than those of the native glucans. The results reveal that the effect of O-sulfonation of the alpha-D-glucan on the improvement of their antitumor activities was considerable.

Effects of molecular structure on antitumor activities of (1→3)- β- d-glucans from different Lentinus Edodes

Four (1→3)- β- d-glucans coded as L-I 1, L-I 2, L-I 3 and L-I 4 with high molecular weight (1.47×10 6–1.67×10 6) were isolated from four kinds of fruiting bodies of Lentinus Edodes with different strains by extracting with NaOH/NaBH 4, then precipitating with 1 M acetic acid. The results from element analysis indicated that the polysaccharides contained 4.6–15.2 wt% proteins. The polysaccharides were treated with acetone to remove the bound protein, coded as LNP, and by ultrasonic irradiation to degrade molecular weight, coded as LU. The weight-average molecular weight M w, radii of gyration < s 2> z 1/2 and intrinsic viscosity [ η] of the samples in aqueous 0.2 M NaCl/0.005 M NaOH and in dimethyl sulfoxide (DMSO) were measured by size exclusion chromatography combined with laser light scattering and viscometry at 25 oC, respectively. The M w drop by a factor of 3 from in aqueous solution to in DMSO, and M w dependence of < s 2> z 1/2 revealed that the β-glucan exists as triple-helical conformation in aqueous NaCl and as single flexible chains in DMSO. The single chain samples were prepared in DMSO, coded as LSC. The assay in vivo and in vitro antitumor activities against Sarcoma 180 (S-180) solid tumor for the polysaccharides showed that the native triple helical (1→3)- β- d-glucans containing protein exhibited obvious antitumor bioactivities (in vivo highest inhibition ratio ξ reached to 70.0% for L-I 3), whereas that ( ξ=2.2%) of LSC having only single flexible chains significantly decreased. Moreover, the antitumor activities of the LNP, LU and LSC samples are lower than those of the native ones, suggesting that the antitumor activities of polysaccharide were related with their conformation, molecular weight and content of the bound protein. The triple helical conformation plays an important role in enhancement of the antitumor activities.

Amino acid sequence of a nuclease (nuclease Le1) from Lentinus edodes.

The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5'-GMP. To obtain the basic information on the mechanism of production of 5'-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide. Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.

Fungus fruit body lytic enzyme from a myxomycete, Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).

Convenient and effective methods for in vitro cultivation of mycelium and fruiting bodies of Lentinus edodes

Three strains were selected from 18 commercial strains of the shiitake mushroom, Lentinus edodes , on the basis of rate and density of mycelial growth. Comparison of biomass production between stationary and shake flask cultures was strain-dependent, strain Le-465 producing higher yields from shaken than stationary cultures while no differences were recorded in Le-11 and Le-103. Among 5 media tested for mycelial growth yield, a molasses medium gave the highest biomass after 14 d, but by day 28 yield from that medium was the second lowest. By day 28, a chemically defined medium was the best for biomass yield. Homogenization of mycelia provided an excellent uniform source of inoculum for replicate trials. Homogenization of the inoculum increased the yield of mycelium by day 28, when fruiting started. Chemically-defined media were best for fruiting of L. edodes . There was not much difference in fruiting performance between cultures incubated at a uniform 25 °C and those incubated in a 14/22° cycle. Supplementation of flask cultures with vermiculite promoted growth and fruiting. In flask cultures kept in a normal incubator a cotton stopper, as opposed to one made of plastic foam, was most suitable for growth and fruiting. Inclusion of a carbon dioxide trap was not necessary. The best fruiting in vitro was obtained by inoculation on to solid supports, like a wire mesh or agar, in crystallizing dishes which were kept within a plastic chamber whose air-flow and humidity could be regulated with air-vents and an air-pump. Under such conditions, vermiculite addition to the media did not enhance fruiting.

Purification and characterization of a base non-specific and adenylic acid preferring ribonuclease from the fruit bodies of Lentinus edodes.

Purification and characterization of a base non-specific and adenylic acid preferring ribonuclease from the fruit bodies of Lentinus edodes.

Purification and Characterization of a Nuclease from Lentinus edodes.

An endonuclease with 3'-nucleotidase activity (nuclease Le1) was purified from fruit bodies of Lentinus edodes in a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent molecular weight of nuclease Le1 was about 27000. The nuclease was inactivated in the presence of ethylenediaminetetraacetic acid (EDTA) and reactivated by the addition of Zn2+. Hydrolysis of poly U by the nuclease showed many intermediate size oligomers prior to the formation of 5'-uridine monophosphate (UMP). Therefore, it was concluded that nuclease Le1 was a Zn(2+)-endonuclease similar to P1-nuclease from Penicillium citrinum. The nuclease was very sensitive to ionic strength, but pH-profiles of the hydrolysis of four 3'-nucleotides were very similar to those of P1 nuclease from P. citrinum.

Mannitol Metabolism in Lentinus edodes , the Shiitake Mushroom

Mannitol metabolism was evaluated in fruiting bodies of Lentinus edodes. Cell extracts were prepared from fruiting bodies, and key enzymes involved in mannitol metabolism were assayed, including hexokinase, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, and fructose-6-phosphatase. Mannitol dehydrogenase, fructose-6-phosphatase, mannitol-1-phosphatase, and hexokinase activities were found in extracts of fruiting bodies. However, mannitol-1-phosphate dehydrogenase activity was not detected. Mycelial cultures were grown in an enriched liquid medium, and enzymes of the mannitol cycle were assayed in cell extracts of rapidly growing cells. Mannitol-1-phosphate dehydrogenase activity was also not found in mycelial extracts. Hence, evidence for a complete mannitol cycle both in vegetative mycelia and during mushroom development was lacking. The pathway of mannitol synthesis in L. edodes appears to utilize fructose as an intermediate.

Molecular species of cerebrosides in fruiting bodies of Lentinus edodes and their biological activity

Cerebroside fraction was obtained from fresh fruiting bodies of Lentinus edodes and separated into ten molecular species by reverse-phase high-performance liquid chromatography. The species were identified by GLC, GC-MS and NMR. Their component glycosides and sphingoids were uniformly glucose and (4 E,8 E)-9-methyl-4,8-sphingadienine, respectively. The component fatty acids were 2-hydroxy acids with the carbon chain length of 16, 15, 14, 18, 24, 17, 25, 26, 22 and 23 (from major to minor). The cerebrosides with the C 14–18 fatty acids showed strong fruiting-inducing activity in Schizophyllum commune. Those with the C 22 and C 23 ones had one-eighth and one-sixteenth of the activity, respectively, and those with C 24–26 had no detectable activity. 22 and 23 must be the carbon chain lengths of the component fatty acid of the sphingolipids critical for expression of biological activity.

Substrate preference of .GAMMA.-glutamyltransferase from caps of fruiting bodies of Lentinus edodes.

Substrate preference of .GAMMA.-glutamyltransferase from caps of fruiting bodies of Lentinus edodes.