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A polysaccharide (CP2-S), consisting of glucose with a weight average molecular weight of 5.9 × 10 6 , was purified from the fruit bodies of Cordyceps militaris . In this work, the corresponding structure and anti-tumor activity in vivo were investigated. Methylation and NMR analysis revealed that CP2-S was composed of a →4)-α-D-Glc p -(1→ backbone with partial substitution occurring at O -6 by T-linked α-D-Glc p in every ten residues, which has not been reported in previous reports. In vivo anti-tumor experiments showed that CP2-S could inhibit the growth of Lewis lung carcinoma in mice. Tumor inhibition rates were 17.8%, 24.5%, and 29.5% at dosages of 12.5, 50, and 100 mg/kg/d, respectively. Compared with the cisplatin group, mice treated with CP2-S exhibited a significant increase in spleen index (increased 22.7-42.4%) and thymus index (increased 47.7-36.8%). Additionally, serum levels of IgM and IgG in tumor-bearing mice increased by approximately 6.11~10.75-folds and 1.31~1.38-folds, respectively. These findings prove that CP2-S significantly inhibited the growth of Lewis lung carcinoma through immune-enhancing activity in mice.

The herbal medicine Polygonatum cyrtonema is highly regarded in China for its medicinal and dietary properties. However, further research is needed to elucidate the structure of its polysaccharide and understand how it promotes human health by modulating the gut microbiota. This study aims to investigate a homogeneous polysaccharide (PCP95-1-1) from Polygonatum cyrtonema and assess its susceptibility to digestion as well as its utilization by intestinal microbiota. The results confirmed that PCP95-1-1 is an agavin-type fructan, which possesses two fructose chains, namely β-(2 → 6) and β-(2 → 1) fructosyl-fructose, attached to the sucrose core, and has branches of β-D-Fruf residues. Moreover, PCP95-1-1 demonstrated resistance to digestion and maintained its reducing sugar content throughout the digestive system, indicating it could reach the gut without being digested. In vitro fermentation of PCP95-1-1 significantly decreased the pH value (p < 0.05) while notably increasing the production of short-chain fatty acids (SCFAs), confirming its utilization by human gut microbiota. Additionally, PCP95-1-1 exhibited a significant ability (p < 0.05) to beneficial bacteria such as Megamonas and Bifidobacterium, while reducing the presence of facultative or conditional pathogens such as Escherichia-Shigella and Klebsiella at the genus level. Consequently, PCP95-1-1 has the potential to positively influence physical well-being by modulating the gut microbiota environment and can be developed as a functional food., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Cordyceps militaris is a medicinal mushroom and has been extensively used as a traditional medicine in East Asia. After the chrysalis seeds are matured and harvested, the spent substrate of C. militaris still contains active ingredients but is usually discarded as waste. This study aimed to determine the antioxidant and anti-inflammatory activities of C. militaris spent substrate extract and its inhibitory activity on the Malassezia commensal yeasts that can cause dandruff and seborrheic dermatitis. Active substances in the spent substrate of C. militaris were extracted using a hot water extraction method and were used for the determination of antioxidant activity by measuring their ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radicals, hydrogen peroxide, and superoxide anions. The ability to inhibit Malassezia was analyzed using the broth microdilution method, and the reparative effect on oxidative damage in HaCaT cells was measured using in vitro cell analysis. Respiratory burst evaluation was used to determine the anti-inflammatory capacity of extracts. Analysis of the Malassezia-inhibiting activity of the extracts showed that the minimum inhibitory concentration was 6.25 mg/mL. The half maximal inhibitory concentration (IC50) values of DPPH, O2-, H2O2 and OH- were 3.845 mg/mL, 2.673 mg/mL, 0.037 mg/mL and 0.046 mg/mL, respectively. In the concentration range of 2 to 50%, the extract was non-toxic to cells and was able to protect HaCaT cells from H2O2 damage. When the volume fraction of the extract was 20.96%, its anti-inflammatory ability reached 50%. These results demonstrated that the extract may be a safe and efficacious source for pharmaceutical or cosmetic applications, with Malassezia-inhibiting, antioxidant and anti-inflammatory activities., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To investigate the influence of molecular weight ( M w ) on the anti-inflammatory activity of β-D-glucan from Ganoderma lucidum , ultrasonic irradiation was applied to treat the β-D-glucan (GLP, 2.42 × 10 6 g/mol) solution to obtain two degraded fractions with molecular weight of 6.53 × 10 5 g/mol (GLPC) and 3.49 × 10 4 g/mol (GLPN). Structural analysis proved that the degraded fractions possessed similar repeated units with the original β-D-glucan. The in vitro anti-inflammatory activity studies showed that all fractions could significantly inhibit LPS-induced expression of cytokines including TNF-α, IL-8, MIF and MCP-1 in Caco-2 cells at certain concentrations. Moreover, GLPC and GLPN exhibited better anti-inflammatory activity than GLPC. The intestinal anti-inflammatory activity evaluated by dextran sulfate sodium (DSS)-induced colitis mice model showed that intragastric administration of GLPN (lower M w fraction) could significantly recover inflamed tissues of mice. Compared with GLP and GLPC, GLPN exhibited stronger ability to inhibit the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). The results revealed that M w of β-D-glucan influenced its anti-inflammatory activity and decreasing of M w would improve the activity, which provided evidence for the potential use of β-D-glucan from G. lucidum as anti-colitis ingredients., Competing Interests: Author JZho was employed by Shanghai Baixin Bio-Tech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Tang, Feng, Liu, Tang, Yan, Zhou, Liu, Zhou and Zhang.)

Porous carbohydrate materials such as tobacco shreds readily absorb moisture and become damp during processing, storage, and consumption (smoking). Traditional humectants have the ability of moisture retention but moisture-proofing is poor. Polygonatum cyrtonema Hua polysaccharide (PCP 85-1-1) was separated by fractional precipitation and was purified by anion exchange and gel permeation chromatography. The average molecular weight (Mw) of PCP 85-1-1 was 2.88 × 10 3 Da. The monosaccharide composition implied that PCP 85-1-1 consisted of fucose, glucose, and fructose, and the molar ratio was 22.73:33.63:43.65. When 2% PCP 85-1-1 was added to tobacco shreds, the ability of moisture retention and moisture-proofing were significantly enhanced. The moisture retention index (MRI) and moisture-proofing index (MPI) increased from 1.95 and 1.67 to 2.11 and 2.14, respectively. Additionally, the effects of PCP 85-1-1 on the aroma and taste of tobacco shreds were evaluated by electronic tongue and gas chromatography-mass spectrometry (GC-MS). These results indicated that PCP 85-1-1 had the characteristics of preventing water absorption under high relative humidity and moisturizing under dry conditions. The problem that traditional humectants are poorly moisture-proof was solved. PCP 85-1-1 can be utilized as a natural humectant on porous carbohydrates, which provides a reference for its development and utilization.

To systemically understand the biosynthetic pathways of bioactive substances, including triterpenoids and polysaccharides, in Ganoderma lucidum, the correlation between substrate degradation and carbohydrate and triterpenoid metabolism during growth was analyzed by combining changes in metabolite content and changes in related enzyme expression in G. lucidum over 5 growth phases. Changes in low-polarity triterpenoid content were correlated with changes in glucose and mannitol contents in fruiting bodies. Additionally, changes in medium-polarity triterpenoid content were correlated with changes in the lignocellulose content of the substrate and with the glucose, trehalose, and mannitol contents of fruiting bodies. Weighted gene coexpression network analysis (WGCNA) indicated that changes in trehalose and polyol contents were related to carbohydrate catabolism and polysaccharide synthesis. Changes in triterpenoid content were related to expression of the carbohydrate catabolic enzymes laccase, cellulase, hemicellulase, and polysaccharide synthase and to the expression of several cytochrome P450 monooxygenases (CYPs). It was concluded that the products of cellulose and hemicellulose degradation participate in polyol, trehalose, and polysaccharide synthesis during initial fruiting body formation. These carbohydrates accumulate in the early phase of fruiting body formation and are utilized when the fruiting bodies mature and a large number of spores are ejected. An increase in carbohydrate metabolism provides additional precursors for the synthesis of triterpenoids. IMPORTANCE Most studies of G. lucidum have focused on its medicinal function and on the mechanism of its activity, whereas the physiological metabolism and synthesis of bioactive substances during the growth of this species have been less studied. Therefore, theoretical guidance for cultivation methods to increase the production of bioactive compounds remains lacking. This study integrated changes in the lignocellulose, carbohydrate, and triterpenoid contents of G. lucidum with enzyme expression from transcriptomics data using WGCNA. The findings helped us better understand the connections between substrate utilization and the synthesis of polysaccharides and triterpenoids during the cultivation cycle of G. lucidum . The results of WGCNA suggest that the synthesis of triterpenoids can be enhanced not only through regulating the expression of enzymes in the triterpenoid pathway, but also through regulating carbohydrate metabolism and substrate degradation. This study provides a potential approach and identifies enzymes that can be targeted to regulate lignocellulose degradation and accelerate the accumulation of bioactive substances by regulating substrate degradation in G. lucidum .

Two polysaccharide fractions (GLSB50 and GLSB70) with total sugar content of 82.07 wt% and 53.79 wt%, respectively, were obtained from the water extracts of unbroken Ganoderma lucidum spores by sequential ethanol precipitation treatment. Compared with GLSB70, GLSB50 exhibited better activity on stimulation of humoral immune responses in immunosuppressed mice. A novel β-D-glucan (GLSB50A-III-1) with weight average molecular weight (M w ) of 1.93 × 10 5  g/mol was purified from GLSB50 through chromatography separation. The exponent α value of Mark-Houwink-Sakurada equation was calculated to be 0.13, indicating that GLSB50A-III-1 presented globular spheres conformation in aqueous solution. Structural analysis showed that GLSB50A-III-1 mainly consisted of (1 → 3), (1 → 4), (1 → 6)-linked β-d-glucose residues in the backbone, with two single β-D-Glcp attached at O-6 of β-(1 → 3) and β-(1 → 4)-linked residues separately as side chains. The repeat unit of GLSB50A-III-1 was deduced as follows., (Copyright © 2021. Published by Elsevier B.V.)

Ganoderma lucidum mycelia are rich in active substances such as triterpenoids and sterols. However, reports on the development of effective submerged fermentation processes are lacking and the resulting total triterpene and sterol yield is still quite low. In this study, a new G. lucidum strain G0017 mycelium isolated by screening was studied in a 3-L fermenter to investigate the effect of aeration rate in liquid submerged fermentation production of triterpenoids and sterols. By fitting the specific mycelial growth rate and the specific production rate of the triterpenoid and sterol model, an effective multistage aeration rate control process for triterpenoid and sterol fermentation production was developed. This process was validated and proven in 3-L and 50-L fermenters. The resulting yields of triterpenoids and sterols were 3.34 and 3.46 g/L, respectively, which were 69.54% and 75.63% higher than the fixed aeration rate of 1.50 volume of air per volume of liquid per minute. This optimized fermentation production process conceivably could be applied to larger-scale industrial production and perhaps also to improve liquid submerged fermentation processes with relevant edible and medicinal mushrooms.

The study aimed at detecting 13 organic acids (oxalic acid, maleic acid, citric acid, tartaric acid, malic acid, quinic acid, succinic acid, fumaric acid, formic acid, acetic acid, propionic acid, isobutyric acid, and butyric acid) by establishing a high-performance liquid chromatography (HPLC). The analysis was performed using two sugar columns, i.e., SH1011 column and KC-811 column. The optimal conditions were as follows: 4 mmol/L HClO 4 solution as the eluent with UV-visible detector (210 nm), a flow rate of 1 mL/min at the temperature of 60°C, and the injection volume at 10  μ L. The results showed that all the calibration curves had excellent linearity ( R 2 > 0.9991) within the test ranges. The RSD values of the thirteen analytes were lower than 2.94% at three levels, the recoveries were 91.9%-102.0%, the limit of detection (LOD) was between 0.05 and 10.63  μ g/mL, and the quantification (LOQ) was between 0.10 and 19.53  μ g/mL. Finally, the proposed methodology was successfully applied for the analysis of organic acids in the culture medium of edible fungi. In conclusion, the study findings proved that the method was sensitive, accurate, reproducible, and could be readily applied to analyze the organic acids in the samples., Competing Interests: No conflicts of interest prevail among researchers., (Copyright © 2020 Changxia Yu et al.)

In the present study, regioselective sulfation of β-glucan (GLP) from Ganoderma lucidum were firstly established by using 4,4'-dimethoxytrityl chloride and hexamethyldisilazane as protecting precursor. 2,4,6-O-sulfated, 6-O-sulfated and 2,4-O-sulfated GLP derivatives were prepared and the molecular weights (M w ) of derivatives were determined to range from 0.94 × 10 4 to 6.27 × 10 4  g/mol, while the degrees of sulfation (DS) were calculated to vary from 0.83 to 1.74. The regioselective sulfation of GLP was confirmed by FT-IR, 13 C NMR spectroscopy and methylation analysis. Results indicated that the sulfated substitution sites were predominantly at C-6 in 6-O-sulfated GLP (S 6-O GLP) and C-4 in 2,4-O-sulfated GLP (S 2,4-O GLP), respectively. Clotting assays (APTT, PT and TT) in vitro showed that sulfate groups were essential for anticoagulant activity and S 6-O GLP exhibited much higher than others. Meanwhile, sulfated GLP with higher DS and M w showed stronger anticoagulant activity in the case of the same condition., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The mushroom today known as Ganoderma lingzhi has been used for centuries in the countries of Eastern Asia as a very important medicinal mushroom. It prefers growing on rotten wood of broadleaf trees and is mainly distributed in the tropics and subtropics. Its relative G. lucidum occurs naturally almost all the Earth, and it colonizes mostly oak and beech trees in Central Europe. G. lingzhi and G. lucidum are similar species. To obtain the qualitative parameters of G. lingzhi and G. lucidum, several strains (five G. lingzhi strains and five G. lucidum ones) were chosen and cultivated in both Slovakia and China, using wood chip (beech and oak) substrate and liquid fermentation method, respectively. It was found that there were more low-polarity triterpenes in G. lucidum, while G. lingzhi contained more high-polarity triterpenes. Beech substrate was more suitable for the accumulation of triterpenes in solid cultivation for both strains of G. lucidum and G. lingzhi. Strain C4 of G. lingzhi and strain K2 of G. lucidum contained higher triterpenes in either mycelium or fruiting bodies. Data in this study can help to identify these two species and bring a great benefit to the production of bioactive compounds of G. lucidum from Slovakia.

The naturally occurring amino acid ergothioneine (EGT) has excellent free radical scavenging ability, which was not different to ascorbic acid. The IC50 values for EGT scavenging hydrogen peroxide, hydroxyl radicals, and superoxide anions were 11.65 ± 0.31, 70.31 ± 1.59, and 160.44 ± 0.32 μg/mL, respectively. The EGT concentration in different species of mushrooms was significantly different (p < 0.05), but it was not significantly related to the ability of the mushrooms to scavenge reactive oxygen species (p > 0.05). After isolating EGT from mushrooms, we demonstrated that the antioxidant ability of EGT accounts for about 25% of the total antioxidant ability of the extract. We studied the stability of EGT and found that it has excellent light, thermal, and acid-base stability. However, the presence of Cu2+ decreased the concentration of EGT. Unlike EGT, the thermal stability of the EGT extracted from Pleurotus citrinopileatus (PEGT) was not as good as EGT, while long-term high-temperature heating caused a decrease in the concentration of PEGT. The results of our study provide a basis for further investigating EGT from mushrooms for research and development.

Cordyceps militaris are widely cultivated in China for an important raw material for health foods. CM-H0810 is a C. militaris strain used in the production of C. militaris in Shanghai, the surrounding areas of Shanghai, and Guangdong province in China. We evaluated the effect of culture time on the bioactive components in the fruit bodies of C. militaris CM-H0810 to provide scientific references for production of C. militaris fruit bodies with good quality. The results showed that the polysaccharide contents increased gradually during 35-45 d, but it declined with the prolongation of culture time. The highest polysaccharide content was 3.46% at 45 d. With the prolongation of culture time the cordycepin content gradually increased; the highest cordycepin content was 3.57 μg/mg at 60 d, which increased 321% compared to that at 35 d. Contrary to cordycepin, the adenosine content declined gradually, with the highest content of 1.86 μg/mg at 35 d and the lowest content of 1.48 μg/mg at 60 d. Our study indicates that it is necessary to select suitable harvest times in view of different compounds that are desirable to obtain in high quantities.

Ganoderma, named lingzhi in China, has been used for centuries as drug and nutraceutical to treat diseases. Based on our research and other literatures, the chapter summarizes the progress of preparation, structural features and properties, bioactivities of Ganoderma polysaccharides. The aim is to provide a comprehensive source of information for researchers and consumers of Ganoderma, so they can better understand Ganoderma polysaccharides and their biological activities. In addition, more clinical studies should be carried out to meet the criteria for new drug development, and more convincing scientific data should be provided. In addition, on the basis of a large number of studies on Ganoderma polysaccharides, we suggest that more clinical studies should be carried out so that Ganoderma can be better recognized and applied all over the world.

Lentinus edodes fruiting bodies are rich in active substances such as polysaccharides and eritadenine. Patients with gout, however, should avoid or severely limit their intake of foods containing large amounts of purine. In this study we quantitatively analyzed the polysaccharide and purine compounds dissolved from L. edodes fruiting bodies during cleaning, soaking, and cooking. Eritadenine, adenosine, guanosine, guanosine monophosphate, adenosine monophosphate, xanthine, and adenine dissolved from L. edodes fruiting bodies during cleaning with tap water; their dissolution rates ranged between 3.77% and 24.30%. Dissolution rates of polysaccharide and purine compounds in L. edodes fruiting bodies increased linearly with increases in the duration of soaking and cooking, and adding acetic acid or NaHCO3 in the soaking or cooking solutions significantly either inhibited or promoted their dissolution rates. On the basis of these experimental results, we offer science-based suggestions for reasonable treatment of L. edodes fruiting bodies before eating for both patients with gout and healthy people.

A water soluble Nostoc sphaeroids kütz polysaccharide (NSKP) was extracted and purified by precipitating hot-water extract from Nostoc sphaeroids kütz with 40% (v/v) ethanol. The yield was 40.10% (w/w), and weight average molecular weight (M w ) was determined as 1.31 × 10 5 g/mol. Cell culture study showed that NSKP could stimulate the NO release from RAW264.7 macrophages. The ρ value (R g /R h ) of NSKP was calculated as 1.50, indicating flexible random coils conformation of NSKP in aqueous solution. According to methylation analysis, and 1D & 2D NMR spectra, the molecular structure of polysaccharide NSKP was deduced as below., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ganoderic acid S, ganoderic acid T and ganoderal B are the main bioactive triterpenes of Ganoderma lucidum. In this study, mycelia of G. lucidum were obtained by two-stage fermentation and then extracted by ethanol and petroleum ether sequentially to obtain crude triterpenes. The crude sample was further purified by recycling high-speed counter-current chromatography with n-hexane-ethyl acetate-methanol-water (7:12:11:5, v/v/v/v) as the optimized two-phase solvent system. A 16.4 mg aliquot of ganoderol B with a purity of 90.4% was separated from 300 mg of the crude sample in a single run. After employing the recycling elution mode of HSCCC with n-hexane-ethyl acetate-methanol-water (6:10:8:4.5, v/v/v/v) for five cycles, 25.7 mg ganoderic acid T and 3.7 mg ganoderic acid S with purities of 97.8 and 83.0%, respectively, were obtained. The purities of three compounds were determined by high-performance liquid chromatography and their chemical structures were identified by NMR and MS data., (Copyright © 2018 John Wiley & Sons, Ltd.)

β-d-glucan (GLP20) isolated from Ganoderma lucidum fruiting bodies was successfully fractionated into five fractions with different weight-average molecular weights (M w ) through ultrasonic irradiation. The M w , radius of gyration (R g ), hydrodynamic radius (R h ) and intrinsic viscosity ([η]) of these fractions in phosphate buffered saline (PBS) aqueous solution were determined using HPSEC-MALLS-RI-VS system. The results indicated that β-d-glucan displayed rigid chain conformations with ρ values lager than 2.06 for the five fractions. By applying the polymer solution theory, the exponent (ν and α) values of R g  = kM w ν and [η] = kM w α were calculated as 0.72 and 1.05, respectively, which confirmed the rigid chain conformation of glucans with M w from 2.9 × 10 5 to 2.42 × 10 6  g·mol -1 . According to the known theory for chains, the molar mass per unit contour length M L , persistence length q and contour length h per main-chain glucose residue were estimated to be 2150 nm -1 , 128 nm and 0.30 nm, respectively, which indicated that the glucan existed as triple-helical chains in PBS aqueous solution. Bioactivity study of glucan fractions on lymphocytes and THP-1 macrophages demonstrated that all these fractions could stimulate lymphocyte proliferation, promote macrophages to form pseudopodia, and enhance the levels of inflammatory cytokines IL-6 and TNF-α. Meanwhile, the glucan fractions with M w higher than 1.82 × 10 6  g·mol -1 exhibited better activity on enhancing the release of inflammatory cytokines., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ganoderma lucidum is a medicinal mushroom that is well known for its ability to enhance human health, and products made from this fungus have been highly profitable. The substrate-degrading ability of G. lucidum could be related to its growth. CAZy proteins were more abundant in its genome than in the other white rot fungi models. Among these CAZy proteins, changes in lignocellulolytic enzymes during growth have not been well studied. Using genomic, transcriptomic and secretomic analyses, this study focuses on the lignocellulolytic enzymes of G. lucidum strain G0119 to determine which of these degradative enzymes contribute to its growth. From the genome sequencing data, genes belonging to CAZy protein families, especially genes involved in lignocellulose degradation, were investigated. The gene expression, protein abundance and enzymatic activity of lignocellulolytic enzymes in mycelia over a growth cycle were analysed. The overall expression cellulase was higher than that of hemicellulase and lignin-modifying enzymes, particularly during the development of fruiting bodies. The cellulase and hemicellulase abundances and activities increased after the fruiting bodies matured, when basidiospores were produced in massive quantities till the end of the growth cycle. Additionally, the protein abundances of the lignin-modifying enzymes and the expression of their corresponding genes, including laccases and lignin-degrading heme peroxidases, were highest when the mycelia fully spread in the compost bag. Type I cellobiohydrolase was observed to be the most abundant extracellular lignocellulolytic enzyme produced by the G. lucidum strain G0119. The AA2 family haem peroxidases were the dominant lignin-modifying enzyme expressed during the mycelial growth phase, and several laccases might play roles during the formation of the primordium. This study provides insight into the changes in the lignocellulose degradation ability of G. lucidum during its growth and will facilitate the discovery of new approaches to accelerate the growth of G. lucidum in culture., Competing Interests: The authors have declared that no competing interests exist.

Sulfated polysaccharides exhibit various biological properties, including anti-coagulant, anti-oxidant, anti-viral, anti-cancer, anti-inflammatory and immune regulatory activities. In the present study, the anti-inflammatory properties of GLPss58, a sulfated polysaccharide from Ganoderma lucidum formed by chemical sulfation, were investigated. We found that GLPss58 inhibited L-selectin/sTyr-sLeX binding significantly, blocked the binding of anti-l-selectin antibodies to L-selectin on the surface of human peripheral blood lymphocytes, and inhibited the secondary lymphoid tissue chemokine-induced chemotactic invasion of HPBLs. In vivo studies in mice showed that lymphocyte homing from peripheral blood to spleen and lymph nodes was significantly inhibited by GLPss58. Furthermore, GLPss58 also inhibited the activation of complement systems and blocked the binding of TNF-α and IFN-γ to their antibodies. These results indicate that GLPss58 is able to inhibit not only the L-selectin-mediated inflammation, but also the complement system- and cytokines mediated-inflammation. Our results suggest that GLPss58 is a favorable potential anti-inflammatory agent., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We analyzed the changes in triterpenes and soluble polysaccharides in Ganoderma lucidum strain G0119 during 4 growth phases in 3 regions of the fruiting bodies using reversed-phase high-performance liquid chromatography, and we also analyzed the soluble polysaccharides using high-performance size-exclusion chroma-tography-multiple-angle laser-light scattering refractive index analysis. The strong polar triterpenes decreased while weak polar triterpenes increased during the growth cycle of G. lucidum. The highest contents of ganoderic acid B, ganoderic acid A, and ganoderenic acid B were detected in the stipe during phase II, and ganoderic acid S, ganoderic acid T, and ganoderiol B peaked in the base during phase IV. The total content of soluble polysaccharides in samples decreased after the primordium developed into a fruiting body. Two high-molecular-weight fractions were detected in the soluble polysaccharide samples: α-l,4-glucan and β-l,3-glucan, respectively. They were primarily distributed in the pileus during phase II, and both decreased after this phase. These results led us to select a more suitable growth phase and region for harvesting to obtain extracts with higher contents of triterpenes and soluble polysaccharides.

Due to the difficulty and high cost for the preparation of triterpenes, one single standard for the simultaneous determination of multi-components (SSDMC) with high performance liquid chromatography (HPLC) is an advanced solution for multi-component analysis. Experiments were carried out to investigate the feasibility of SSDMC for the analysis of Ganoderma triterpenes, with external standard method (ESM) compared, and the samples of Ganoderma were classified by the content of Ganoderma triterpenes. The analysis was performed by using a Fortis Speed Core-C18 column (150mm×4.6mm I.D., 2.6μm) at gradient elution of 0.01% glacial acetic acid-water (V/V) and acetonitrile with diode array detection (252nm), at a flow rate of 1mL/min. The results showed that all calibration curves had good linearity (r 2 >0.9999) within test ranges. The LOD and LOQ were lower than 2.52ng and 6.43ng, respectively. The RSD for intra-day and inter-day of the seventeen analytes were less than 3.12% at three levels, and the recoveries were 91.4-103.0%. The contents of other 16 triterpenes were determined with ganoderic acid A by SSDMC, which showed that there were few differences compared with the results obtained by ESM. Moreover, the classification of 25 different species and strains of Ganoderma by using the content of triterpenes intuitively reflected the distinction among Ganoderma. In summary, the developed method could be readily utilized as a method of quality evaluation for Ganoderma triterpenes., (Copyright © 2017. Published by Elsevier B.V.)

A water soluble β-glucan (GLSWA-I) with a weight average molecular weight ∼1.57×10 5 g/mol, isolated from the aqueous extract of the broken cellular wall Ganoderma lucidum spores, was purified by anion-exchange and gel-permeation chromatography. The immunological activity test in vivo showed that GLSWA-I could significantly promote dinitrochlorobenzene (DNCB) induced delayed-type ear swelling in mice. Based on the monosaccharides composition analysis, methylation analysis, IR, 1D and 2D NMR spectroscopy, the repeating unit of polysaccharide GLSWA-I was elucidated as follows., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: TLC bioautography for tyrosinase inhibitors has made recent progress; however, an assay with a relative low consumption of enzyme and quantitative capability would greatly advance the efficacy of related TLC bioautographic assays., Objective: An improved TLC bioautographic assay for detecting tyrosinase inhibitors was developed and validated in this study., Methods: L-DOPA (better water-solubility than L-tyrosine) was used as the substrate instead of reported L-tyrosine. The effects of enzyme and substrate concentrations, reaction temperatures and times, and pH values of the reaction system as well as different plate types on the TLC bioautographic assay were optimised. The quantitative analysis was conducted by densitometric scanning of spot areas, and expressed as the relative tyrosinase inhibitory capacity (RTIC) using a positive control (kojic acid) equivalent., Results: The limit of detection (LOD) of this assay was 1.0 ng for kojic acid. This assay has acceptable accuracy (101.73-102.90%), intra- and inter-day, and intra- and inter-plate precisions [relative standard deviation (RSD), less than 7.0%], and ruggedness (RSD, less than 3.5%). The consumption of enzyme (75 U/mL) is relatively low. Two tyrosinase inhibitory compounds including naringenin and 1-O-β-D-glucopyranosyl-4-allylbenzene have been isolated from Rhodiola sacra guided by this TLC bioautographic assay., Conclusion: Our improved assay is a relatively low-cost, sensitive, and quantitative method compared to the reported TLC bioautographic assays. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Molecular weight (Mw) distributions of polysaccharides from the fruiting bodies of different Ganoderma lucidum strains and G. sinense were investigated and compared using high-pressure size exclusion chromatography/multiangle laser light scattering/refractive index analysis. Results showed that there were big differences in the Mw distributions and characteristics of polysaccharides from 2 species of Ganoderma. All tested G. lucidum materials exhibited similar polysaccharide distributions and similar characteristics for each fraction. The fraction with highest Mw (peak 1) was identified as β-(1→3)-linked D-glucan with (1→6)-β-D-glucopyranosyl side branches. G. sinense fruiting bodies did not include the β-D-glucan when compared with G. lucidum. A high-pressure size exclusion chromatography method was developed and applied to determine the amount of high-Mw β-D-glucan in G. lucidum fruiting bodies. Results indicated that there was no obvious relationship between β-D-glucan content and the genetic similarity of G. lucidum. The strain labeled "Longzhi no. 2" was determined to possess the largest amount of β-D-glucan: 8.2 mg/mL based on the dry weight of fruiting bodies. The β-D-glucan content in the hot water extract of Longzhi no. 2 reached 17.05%. For the "Hunong no. 1" strain, the β-D-glucan content in log-cultivated fruiting bodies was much higher than that in bag-cultivated ones. This method could be used to improve quality control of polysaccharides in G. lucidum.

In this study, cordycepin, N(6)-(2-hydroxyethyl)-adenosine (HEA) and adenosine from the fruiting bodies of Cordyceps militaris were separated by using macroporous resin NKA-II adsorption. The parameters of static adsorption were tested and the optimized conditions were as follow: the total adsorption time was 12h, 50% ethanol was used for desorption and the desorption time was 9h. The crude sample that was prepared by macroporous resin NKA-II contained 3.4% cordycepin, 3.7% HEA and 4.9% adenosine. Then the crude sample was further purified by recycling high-speed counter-current chromatography (HSCCC) with ethyl acetate, n-butanol, 1.5% aqueous ammonium hydroxide (1:4:5, v/v/v) as the optimized two-phase solvent system. Three nucleosides including 15.6mg of cordycepin, 16.9mg of HEA and 23.2mg of adenosine were obtained from 500mg of crude sample in one-step separation. The purities of three compounds were 98.5, 98.3 and 98.0%, respectively, as determined by high performance liquid chromatography., (Copyright © 2016. Published by Elsevier B.V.)

Introduction: Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay., Objective: To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study., Methods: The new TLC bioautographic assay was based on reaction of lipase with β-naphthyl myristate and the subsequent formation of the purple dye between β-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay., Results: The β-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01 ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07-105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64-4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8-4.9%)., Conclusion: The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors., (Copyright © 2015 John Wiley & Sons, Ltd.)

Three polysaccharide fractions from fruiting bodies of Coprinus comatus-CC30, CC60, and CC80-are obtained by water extraction and ethanol precipitation with ethanol percentages of 30%, 60%, and 80%, respectively. The hypoglycemic activity of the three fractions was tested based on mice with alloxan-induced diabetes. Results indicate that fraction CC60 is the most effective fraction in water extract from C. comatus; it can remarkably reduce the blood glucose concentration in 120 min at a dosage of 1000 mg/kg administered orally. It also presents a long-term hypoglycemic effect during 21 days of injection at the same dosage. This polysaccharide fraction provide a novel path to improve the treatment currently used for patients with diabetes. The data on mice spleen lymphocyte proliferation and protein tyrosine phosphatase 1B (PTP1B)-inhibiting activity of fractions indicate that the hypoglycemic activity of CC60 is possibly activated through immune stimulation, not PTP1B inhibition.

A purified polysaccharide coded as GLP20 was obtained by precipitating a hot-water extract from Ganoderma lucidum fruiting bodies with 20% (V/V) ethanol. Its total carbohydrate content was 95.9%. Structural analysis showed that GLP20 was a β-(1→3)-linked d-glucan with a (1→6)-β-d-glucopyranosyl side-branching unit on every third residue. Cell culture study revealed that GLP20 can significantly increase NO production of RAW264.7 macrophages. The analysis of light scattering and high performance size exclusion chromatography (HPSEC) showed that the molecular weight and polydispersity of GLP20 was 3.75 × 10(6)Da and 1.36, respectively. GLP20 had a rigid chain conformation in aqueous solution. A conformation transition occurred in the alkaline solution with NaOH concentration larger than 0.15M. The transition from ordered structure to single chain happened when GLP20 was heated above 135°C in water solution and was irreversible as demonstrated by differential scanning calorimetry (DSC). GLP20 existed as random coils in DMSO., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

TAPA1 was previously isolated from Tremella aurantialba fruiting bodies. In this paper, an acetylated derivative (TAPA1-ac) and a deactylated derivative (TAPA1-deac) of TAPA1 were prepared, and their characterization and immunostimulating activities were reported. Acetylation and deacetylation were found to occur actually by FT-IR and NMR spectra, together with calculational results. The degree of substitution (DS) of acetyl groups in TAPA1-ac was 0.23 and the content was 5.82%, which was higher than those of TAPA1 (0.03% and 0.70%, respectively) and TAPA1-deac (all were zero). Compared with TAPA1, TAPA1-ac showed significant immunostimulation effects on mouse spleen lymphocytes (MSLs) proliferation and nitric oxide (NO) production by macrophages RAW264.7, whereas TAPA1-deac showed markedly lower effects. These findings seemed to suggest that immunostimulating activities, including MSLs stimulation activity and NO production potency, might relate to the DS and content of acetyl groups, indicating that acetylation of TAPA1 was an effective way of enhancing immuno-stimulating activities., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A novel polysaccharide (CP2-S) was purified from Cordyceps militaris fruit bodies by hot water extraction, ethanol precipitation, DEAE-Sepharose Fast Flow and Sephacryl S-400 high-resolution chromatography. The polysaccharide had a molecular weight of 5.938 × 10(6) g/mol and was mainly composed of glucose. CP2-S had carbohydrate content estimated to be 100% using the phenol-sulfuric acid method. Immunostimulating experiments in vitro indicated that CP2-S could stimulate nitric oxide production, phagocytosis, respiratory burst activity, and secretion of interleukin-1β and interleukin-2 of macrophages, suggesting that this water-soluble polysaccharide from the fruit body of C. militaris is a natural immunostimulating polysaccharide with potential for further application.

To obtain a low-molecular-weight polysaccharide with immuno-enhancing activity, hot water extract of Ganoderma lucidum fruit bodies was separated by membrane ultrafiltration, anion exchange, and gel filtration chromatography, and the immunological activities of fractions were assessed on the basis of nitric oxide production by RAW 264.7 macrophages. A novel polysaccharide (TB3-2-2) was successfully isolated and purified. TB3-2-2 is a homogeneous polysaccharide, with a relative molecular weight of 5.11 × 103 Da, identified by high-performance liquid chromatography and was composed of galactose and glucose in a molar ratio of 2:3 determined by high-performance anion exchange chromatography. TB3-2-2 had a carbohydrate content of 99%, as measured using the phenol-sulfuric acid method. Proliferation of mouse spleen lymphocytes and the expression level of interleukin-6 was significantly increased by TB3-2-2. Results indicate that the low-molecular-weight polysaccharide with immunological activity in G. lucidum is worthy of further research and development.

A novel small-molecule polysaccharide with a molecular mass of 2.6 kDa, was isolated from the culinary-medicinal Maitake mushroom Grifola frondosa. GFPS is composed of fucose, rhamnose, galactose, glucose, and mannose; galactose, glucose, and mannose were the dominant monosaccharides. Absorption peaks at 1077 cm-1, 1024 cm-1, and 873 cm-1, as revealed by infrared spectrum, suggesting that GFPS consists of pyranoside. GFPS significantly enhanced the production of nitric oxide and secretion of cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-δ) from macrophages in vitro. These results indicate that this novel small-molecule polysaccharide might be beneficial for immune defense.

A new meroterpene, psoracorylifol F (1), was isolated from Psoralea corylifolia fruits, along with two known meroterpenes (2 and 3), guided by TLC bioautography against O(2)(-) radicals. The structure of 1 was elucidated by means of NMR, HRESIMS, and X-ray crystallographic analysis. All the three metroterpenes possessed potential inhibitory activity against LPS-induced NO production in RAW 264.7 cells with IC(50) values ranging from 7.71 to 27.63μM., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A method of detecting carbohydrates (fucose, trehalose, mannitol, arabitol, mannose, glucose, galactose, fructose, and ribose) by high-performance anion chromatography-pulsed amperometric detection (HAPEC-PAD) was established. The conditions are: CarboPac MA1 column, NaOH as the eluent, temperature 30°C, Au working electrode, Ag/AgCl reference electrode, and flow rate 0.4 mL/min. These nine analytes, which yielded high resolution by this method, could be detected in 40 minutes. Mushrooms were tested and good precision, stability, and reproducibility were achieved. This method is suitable for mushroom samples and could support research and development on sugar and sugar alcohol, which contains special effects.

Oridonin, a diterpenoid isolated from Rabdosia rubescens (Hemsl.) Hara, inhibited the growth of human tumor cell lines SW620 (colon), MCF-7 (breast) and K562 (bone marrow), and induced significant levels of apoptosis in SW620. Morphological changes indicative of cell apoptosis were observed after the cells were exposed to oridonin for 24 h. Growth inhibition was associated with G1 phase arrest, and with time- and dose-dependent increases in caspase-3 activity. We therefore conclude that oridonin inhibits the proliferation of SW620 cells by induction of apoptosis via the activation of caspase-3. Our data suggest that oridonin may have significant potential as an anti-colorectal adenocarcinoma agent.

The medicinal mushroom Ganoderma lucidum (G. lucidum) has been used for the treatment of various diseases, and is known for the immune-enhancing activity of its polysaccharide. However, little is known about another of its major constituents, triterpene. This study investigated the anticancer mechanism of a triterpene-enriched extract from G. lucidum. The triterpene-enriched extract, GLAI, was prepared from fruiting bodies of G. lucidum by sequential hot water extraction, removal of ethanol-insoluble polysaccharides and gel-filtration chromatography. The mechanisms of GLAI-induced apoptosis on SW620 human colorectal adenocarcinoma cells were investigated. Tumor cell lines in vitro were treated with different concentrations of GLAI. Cell proliferation was measured by the Alamar blue assay, morphology of cell apoptosis was observed, cell apoptosis was detected by flow cytometry (FCM) and caspase-3 activity was detected by Caspase-3 cellular activity assay. The results showed that GLAI inhibited the growth of different tumor cells and caused significant apoptosis in a dose-dependent manner. Marked morphological changes of cell apoptosis were observed after the cells had been exposed to GLAI for 24 h. The Caspase-3 assay results showed that the activity of the caspase-3 enzyme increased in both a time- and dose-dependent manner, whereas GLAI resulted in the down-regulation of Bcl-2 gene expression at the mRNA level and XIAP protein production at the protein level. Conversely, GLAI up-regulates the expression of the apoptosis enhancer Bax gene and p53 protein. These findings suggest that the triterpenes contained in G. lucidum are potential anticancer agents.

Ethanolic extracts of fruit bodies of 5 species (8 strains) of the genus Phellinus, and isolated fractions derived from 1 of these extracts (Ph. baumii PB-10), were evaluated for antioxidant activity, inhibitory effects on the growth of human tumor cells, and the capacity to protect PC12 cells against H2O2-induced oxidative damage. Extracts of all 8 strains of Phellinus spp. exhibited antioxidant activity and protected PC12 cells against oxidative damage at different magnitudes of potency. The strongest antioxidant activity was exhibited by extracts of Ph. baumii PB-10, with recorded IC50 values for superoxide radical and hydrogen peroxide scavenging activity of 3.76 microg/mL and 4.24 microg/mL, respectively. Radical-scavenging activity and protection levels against H2O2-induced damage to PC12 cells were highly correlated with the flavonoid content of the extracts and isolated fractions. All the extracts inhibited L1210, SW620, and MCF-7 tumor cell proliferation at 200 microg/mL concentrations, but inhibition was not correlated with the flavone content of the test samples and was clearly dependent upon the presence of other, as yet, unidentified components. Our data indicate that fruit bodies of species of the genus Phellinus represent a potentially valuable source of natural antioxidants of relevance to both the health and food industries.

The immunomodulatory effect of GLIS (Lingzhi or Reishi medicinal mushroom Ganoderma lucidum immunomodulating substance) on macrophages has been investigated as part of ongoing research into the anticancer properties of this mushroom. Proliferation of bone marrow macrophages (BMMs) was enhanced by GLIS in a dose-dependent manner. Microscopic examination revealed that numerous GLIS-treated BMMs were enlarged and formed pseudopodia. Exposure of BMMs to GLIS resulted in significant increases in NO production, induction of cellular respiratory burst activity, and increased levels of IL-1β, IL-6, IL-12p35, IL-12p40, IL-18, and TNF-α gene expression and levels of TNF-α, IL-1β, and IL-12 secretion. Our data indicate that GLIS activates the immune system by modulating cytokine production.

Aims: Ganoderma lucidum, a traditional Chinese medicine, is well known as a modulator of functions of the immune system as well as an anti-tumour agent. However, its active compounds and their molecular mechanisms of action are not well established. GLIS, a proteoglycan isolated from the fruiting body of G. lucidum, stimulates directly the activation of B lymphocytes. In this work, the immunoactivation capacities of GLIS as well as its anti-tumour effect were investigated in vitro and in vivo., Main Methods: Tumour-bearing mice were prepared by inoculation of mouse sarcoma S180 cells into BALB/c mice. Lymphocytes and bone marrow-derived macrophages were isolated from spleen and tibia/femurs, respectively. After stimulation with GLIS different immune responses of these cells were analysed. Anti-tumour effect of GLIS was determined., Key Findings: After treatment with GLIS, spleen-derived B lymphocytes from tumour-bearing mice became activated, proliferated and produced large amounts of immunoglobulins. Bone marrow-derived macrophages from tumour-bearing mice also became activated after exposure to GLIS, and they produced important immunomodulatory substances, such as IL-1β, TNF-α and reactive nitrogen intermediates, like NO. GLIS markedly increased phagocytosis of macrophages, and very importantly, it markedly raised the macrophage-mediated tumour cytotoxicity. Treatment of mice with GLIS caused an inhibition of mouse sarcoma S180 tumour growth by 60% in vivo., Significance: These results indicate that GLIS exhibits a capacity to increase remarkably both humoral and cellular immune activities of tumour-bearing mice and inhibits tumour growth significantly. The anti-tumour effect of GLIS results from its capacity to increase the host's immune activity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Fibroblast migration plays an important role in the normal wound healing process; however, dysregulated cell migration may contribute to the progressive formation of fibrotic lesions in the diseased condition. To examine the role of focal-adhesion-kinase (FAK)-related non-kinase (FRNK) in regulation of fibrotic lung fibroblast migration, we examined cell migration, FRNK expression, and activation of focal adhesion kinase (FAK) and Rho GTPase (Rho and Rac) in primary lung fibroblasts derived from both idiopathic pulmonary fibrosis (IPF) patients and normal human controls. Fibrotic (IPF) lung fibroblasts have increased cell migration when compared to control human lung fibroblasts. FRNK expression is significantly reduced in IPF lung fibroblasts, while activation of FAK, Rho and Rac is increased in IPF lung fibroblasts. Endogenous FRNK expression is inversely correlated with FAK activation and cell migration rate in IPF lung fibroblasts. Forced exogenous FRNK expression abrogates the increased cell migration, and blocked the activation of FAK and Rho GTPase (Rho and Rac), in IPF lung fibroblasts. These data for the first time provide evidence that downregulation of endogenous FRNK plays a role in promoting cell migration through FAK and Rho GTPase in fibrotic IPF lung fibroblasts., (Copyright 2010 Elsevier Inc. All rights reserved.)

Osthole, a natural coumarin derivative, is extracted from the fruit of Cnidium monnieri Cusson. Breast cancer is one of the most commonly diagnosed cancers and the leading cause of death in women. Recent studies have shown that Osthole has anti-tumor activity. However, the effects of Osthole on the migration and invasion of cancer cells have not yet been reported. Here, we found that Osthole is effective in inhibiting the migration and invasion of breast cancer cells by wound healing and transwell assays. Luciferase and zymography assays revealed that Osthole effectively inhibits matrix metalloproteinase-2 promoter and enzyme activity, which might be one of the causes that lead to the inhibition of migration and invasion by Osthole. This is the first report on the inhibitory function of Osthole in migration and invasion in breast cancer cells. Our findings indicate a need for further evaluation of Osthole in breast cancer chemotherapy and chemoprevention.

A novel acidic heteropolysaccharide (TAPA1) was purified from hot water extracts of Tremella aurantialba fruiting bodies by DEAE-Sepharose Fast Flow and Sephacryl S-500 High-Resolution Chromatography. The heteropolysaccharide had a molecular weight of ca. 1.35x10(6)Da, and a carbohydrate content estimated to be approximately 98.7% by the phenol-sulfuric acid method. It was composed mainly of d-mannose, d-xylose, and d-glucuronic acid in the ratio of ca. 5:4:1, along with trace amounts of d-galacturonic acid and d-glucose. Monosaccharide compositional analysis and GC-MS of methylated derivatives, combined with (1)H and (13)C NMR spectroscopies (including COSY, TOCSY, NOESY, HSQC, and HMBC spectra), revealed TAPA1 to consist of an alpha-(1-->3)-linked mannopyranosyl backbone, partially substituted at position 4 with xylose side chains, and at position 2 with side chains composed of either xylose, mannose, and glucuronic acid or of xylose and mannose. Bioactivity testing showed that TAPA1 stimulated the proliferation of mouse spleen lymphocytes in vitro.

PISP1, a heteropolysaccharide isolated from fruiting bodies of Phellinus igniarius by hot aqueous extraction and purified by DEAE-Sepharose anion-exchange and gel filtration chromatography, is composed of fucose, galactose, mannose, and 3-O-Me-galactose in a ratio of 1:2:1:2. Methylation, monosaccharide analysis, and NMR studies (1H NMR, 13C NMR, COSY-45 degrees , TOCSY, ROESY, HSQC, and HMBC) revealed that PISP1 had a backbone consisting of 1,6-disubstituted-3-O-Me-alpha-D-galactopyranosyl residue, 1,3,6-trisubstituted-alpha-D-manopyranosyl residue, 1,4-disubstituted-alpha-D-galactopyranosyl residue, and 1,2-disubstituted-alpha-D-galactopyranosyl residue, and had a 1-substituted-alpha-L-fucopyranosyl terminal attached to O-3 of a manopyranosyl residue. Preliminary bioactivity tests conducted in vitro revealed that PISP1 stimulated the proliferation of mouse spleen lymphocytes.

A water-soluble glycopeptide (GLPCW-II) was isolated from the fruiting bodies of Ganoderma lucidum by DEAE-Sepharose Fast-Flow and Sephacryl S-300 High Resolution Chromatography. The glycopeptide had a molecular weight of 1.2x10(4)Da (determined by HPLC), and consisted of approximately 90% carbohydrate and approximately 8% protein as determined using the phenol-sulfuric acid method and the BCA protein assay reagent kit, respectively. The polysaccharide moiety was composed mainly of D-Glc, L-Fuc, and D-Gal in the ratio of 1.00:1.09:4.09. To facilitate structure-activity studies, the structure of the GLPCW-II polysaccharide moiety was elucidated using 1H and 13C NMR spectroscopy including COSY, TOCSY, HMBC, HSQC, and ROESY, combined with GC-MS of methylated derivatives, and shown to consist of repeating units with the following structure: [Formula: see text].

The immunomodulatory effect of Ganoderma lucidum immunomodulating substance (GLIS) on macrophages has been investigated as part of on-going research into the anti-cancer properties of Ganoderma lucidum. Proliferation of bone marrow macrophages (BMMs) was enhanced by GLIS in a dose-dependent manner. Microscopic examination revealed that numerous GLIS-treated RAW264.7 macrophages were enlarged and formed pseudopodia. Exposure of RAW264.7 macrophages to GLIS resulted in significant increases in NO production, induction of cellular respiratory burst activity, and increased levels of IL-1beta, IL-12p35 and IL-12p40 gene expression. Our data indicate that GLIS activates the immune system by modulating cytokine production.

PIP60-1, a novel heteropolysaccharide isolated from fruiting bodies of the medicinal fungus, Phellinus igniarius, has a molecular weight of 1.71 x 10(4)Da and is composed of L-fucose, D-glucose, D-mannose, D-galactose and 3-O-Me-D-galactose in a ratio of 1:1:1:2:1. A structural investigation of PIP60-1 carried out using sugar and methylation analyses, combined with (1)H and (13)C NMR spectroscopy, including COSY, TOCSY, NOESY, HSQC and HMBC experiments, established the repeating unit of the polysaccharide as the following: [structure: see text]

A water-soluble fucogalactan (CMP3), with a molecular mass of 1.03 x 10(4) Da as determined by high-performance size-exclusion chromatography (HPSEC), was obtained from the crude intracellular polysaccharide of Coprinus comatus mycelium. Its chemical structure was characterized by sugar and methylation analysis along with 1H and 13C NMR spectroscopy, including NOESY and HMBC experiments for linkage and sequence analysis. The polysaccharide is composed of a pentasaccharide repeating unit with the following structure: [structure:see text].

A bioactive fraction (GLIS) was isolated from the fruiting body of the fungus Ganoderma lucidum using successive chromatographic steps. GLIS is a proteoglycan and has a carbohydrate: protein ratio of 11.5 : 1. The carbohydrate portion is composed of seven different monosaccharides, predominantly D-glucose, D-galactose and D-mannose in the molar ratio of 3.0 : 1 : 1.GLIS stimulated the proliferation of mouse spleen lymphocytes, resulting in a three to four-fold increase in the percentage of B cells. GLIS also activated mouse spleen lymphocytes, and most of the activated cells were B cells. The B cells were enlarged, expressed CD71 and CD25 on the cell surface, and showed an increase in the secretion of immunoglobulin. Lymphocytes also showed a slightly increased production of IL-2, whereas the secretion of IL-4 was not influenced by GLIS. Furthermore, GLIS did not influence the intracellular Ca2+ concentration of lymphocytes, but it enhanced the expression of protein kinase C alpha and protein kinase C gamma in B cells. According to our results GLIS is a new B cell-stimulating factor.