Rapid screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Polyporus umbellatus and mechanism of action in the treatment of gout.

INTRODUCTION: Studies show that Polyporus umbellatus has some pharmacological effects in enhancing immunity and against gout. OBJECTIVES: We aimed to establish new techniques for extraction, biological activity screening, and preparation of xanthine oxidase inhibitors (XODIs) from P. umbellatus. METHODS: First, the extraction of P. umbellatus was investigated using the back propagation (BP) neural network genetic algorithm mathematical regression model, and the extraction variables were optimised to maximise P. umbellatus yield. Second, XODIs were rapidly screened using ultrafiltration, and the change of XOD activity was tested by enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on XOD. Meanwhile, the potential anti-gout effects of the obtained active substances were verified using molecular docking, molecular dynamics simulations, and network pharmacology analysis. Finally, with activity screening as guide, a high-speed countercurrent chromatography (HSCCC) method combined with consecutive injection and two-phase solvent system preparation using the UNIFAC mathematical model was successfully developed for separation and purification of XODIs, and the XODIs were identified using MS and NMR. RESULTS: The results verified that polyporusterone A, polyporusterone B, ergosta-4,6,8(14),22-tetraen-3-one, and ergosta-7,22-dien-3-one of P. umbellatus exhibited high biological affinity towards XOD. Their structures have been further identified by NMR, indicating that the method is effective and applicable for rapid screening and identification of XODIs. CONCLUSION: This study provides new ideas for the search for natural XODIs active ingredients, and the study provide valuable support for the further development of functional foods with potential therapeutic benefits.

Quantitative Analysis of Polysaccharide Composition in Polyporus umbellatus by HPLC-ESI-TOF-MS.

Polyporus umbellatus is a well-known and important medicinal fungus in Asia. Its polysaccharides possess interesting bioactivities such as antitumor, antioxidant, hepatoprotective and immunomodulatory effects. A qualitative and quantitative method has been established for the analysis of 12 monosaccharides comprising polysaccharides of Polyporus umbellatus based on high-performance liquid chromatography coupled with electrospray ionization-ion trap-time of flight-mass spectrometry. The hydrolysis conditions of the polysaccharides were optimized by orthogonal design. The results of optimized hydrolysis were as follows: neutral sugars and uronic acids 4 mol/L trifluoroacetic acid (TFA), 6 h, 120 °C; and amino sugars 3 mol/L TFA, 3 h, 100 °C. The resulting monosaccharides derivatized with 1-phenyl-3-methyl-5-pyrazolone have been well separated and analyzed by the established method. Identification of the monosaccharides was carried out by analyzing the mass spectral behaviors and chromatography characteristics of 1-phenyl-3-methyl-5-pyrazolone labeled monosaccharides. The results showed that polysaccharides in Polyporus umbellatus were composed of mannose, glucosamine, rhamnose, ribose, lyxose, erythrose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and fucose. Quantitative recoveries of these monosaccharides in the samples were in the range of 96.10-103.70%. This method is simple, accurate, and sensitive for the identification and quantification of monosaccharides, and can be applied to the quality control of Polyporusumbellatus as a natural medicine.

[Study on polysaccharide content and monosaccharide composition of Polyporus umbellatus from different production areas].

In order to provide scientific basics for exploitation and sufficient application of Polyporus umbellatus resources and study the monosaccharide composition of P. umbellatus polysaccharides,the anthrone-sulfuric acid method was applied to compare polysaccharide content of P. umbellatus from 17 producing areas. The monosaccharides were derived by 1-phenyl-3-methyl-5-pyrazolone( PMP) and the derivatives were identified by UPLC-MS/MS and the content of each monosaccharide component was determined simultaneously. The results demonstrated that there was a certain difference in total polysaccharide content of P. umbellatus from different regions,and the content of total P. umbellatus polysaccharide from Shaanxi province and Sichuan province( 1. 15% and 1. 90%) was relatively higher than that of others areas. Polysaccharides from P. umbellatus was mainly composed of eight monosaccharides,including glucose,glucuronic acid,galactose,ribose,xylose,arabinose,mannose and fucose. The contents of glucose( 17. 65 mg·g-1) was higher than others. The ribose was the lowest( 0. 13 mg·g-1). In addition,fructose,rhamnose and galacturonic acid were also detected in some samples. Furthermore,the results of cluster analysis( CA) and principal component analysis( PCA) indicated that totally 17 batches of P. umbellatus polysaccharide could be classified into three clusters,samples collected from Wuchang in Heilongjiang province were clustered into one group separately. The study can provide a basis for rational utilization of P. umbellatus resources,and also implies the sequence of monosaccharide linking and pharmacological activity of P. umbellatus polysaccharides.

[Correlative analysis advance of chemical constituents of Polyporus umbellatus and Armillaria mellea].

Medicinal Polyporus umbellatus is the dry sclerotia of P. umbellatus, with the effect of diuresis; Armillaria mellea is a parasitic fungus which can infect plants up to 300 genera, with sedative, anticonvulsant and some other biological activities. As the medicinal value of P. umbellatus and A. mellea is increasingly wide concerned, the market quantity demanded of them is gradually increased and the demand outstrips the supply. The symbiotic A. mellea and P. umbellatus are both the medicinal and edible fungi with diverse activities, including hypoglycemic action, improve immunity and antitumor and so on. The growth of the sclerotia forming from the mycelium of P. umbellatus is related to the infection of the symbiotic A. mellea and their secondary products. In this study, by comparing the chemical constituents of the mycelium and sclerotia of P. umbellatus and A. mellea, we found that they all produced steroids and nitrogen-containing heterocycles. The sclerotia of P. umbellatus and A. mellea also produced triterpenes secondary metabolites. In addition, the mycelium and infected sclerotia of P. umbellatus mainly produced different steroids, and the sclerotia produced some other special secondary metabolites, such as long-chain fatty acids, ceramides, phenol and so on. By analyzing above all kinds of differences, speculated that these may be caused by the infection of the symbiotic A. mellea which mainly produced sesquiterpenes, diterpenes and other secondary metabolites. The contents and types of compounds of P. umbellatus and A. mellea are closely related to their symbiosis and reproduction, therefore, many symbiosis mechanisms should be found by utilizing more molecular biology technology to elucidate this complex symbiotic infection and provide scientific basis for improving the yield and quality of P. umbellatus and A. mellea.

[Molecular cloning and characterization of two oxidative stress related genes from medicinal fungus Polyporus umbellatus].

OBJECTIVE: To clone the NADPH gene (PuNOX) and Glyoxal oxidase gene (PuGLOX) from a medicinal fungus Polyporus umbellatus, and to carry out the bioinformatic analysis. METHODS: We used the Rapid Amplification of cDNA ends (RACE) technique to obtain the full length cDNA of these two genes. We used a series of bioinformatic tools to characterize physiochemical properties of the two deduced protein. The analyses of multiple alignment and phylogenetic trees were performed using Bioeditor and MEGA 5.0 softwares. RESULTS: The entire cDNA of PuNOX and PuGLOX were 1674 bp, 1723 bp in length and encoded a 557-amino acid protein and 515-amino acid protein with a molecular weight of 63.845 kDa and 55.891 kDa and the isoelectric point of 5.58 and 4.82, respectively. PuNOX had high identities (74 to 80%) with NADPH peroxidase from other fungus. From the evolutionary tree, PuNOX was closely related to that of Pleurotus ostreatus. PuGLOX had high identities (> 50%) with Glyoxal oxidases from various fungus. Phylogenetic tree analysis suggested that PuGLOX was closely related to that of Phanerochaete chrysosporium. CONCLUSION: Molecular characterization of the two oxidative stress related genes will be useful for further functional determination of the genes involved in the sclerotium development of Polyporus umbellatus.

Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells.

BACKGROUND: Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. METHODS: Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. RESULTS: In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. CONCLUSIONS: In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an increase in [Ca2+]i, P38 activation, and ROS generation via NADPH oxidase and mitochondria.

[Physicochemical properties of medicinal fungus Polyporus umbellatus sclerotial exudate].

This study was conducted to investigate the physicochemical properties of Polyporus umbellatus sclerotial exudate. Morphological characteristics of the sclerotia and its exudate were observed during different stages of sclerotial formation. The pH of the exudate was detected at different time during cultivation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of P. umbellatus sclerotial exudate during cultivating time. Additionally, the protein content was measured by means of BCA protein assay. Furthermore, CAT content was detected using ultraviolet absorption method. That the protein content of the exudate and CAT specific activity rose gradually during the passage of the cultivating time indicated a high level of oxidative stress during P. umbellatus sclerotial exudate formation. The results showed that the pH of the exudate increased gradually and then dropped down during sclerotial formation. That the pH of the exudate maintained the acidity state during the cultivation indirectly indicated that acidic environment would help sclerotial formation. The exudate produced gradually and was absorbed by the sclerotia itself.

Inhibitory effects of Korean indigenous plants on tyrosinase and melanogenesis.

To search for new depigmenting cosmetic ingredients from Korean herbal extracts of traditional Korean medicines (TKMs), we screened about 17 TKM extracts collected in the Republic of Korea. Samples were prepared from the natural plants, including medicinal plants such as Chrysanthemum indicum (flower), using methanol, methylene chloride, ethyl acetate (EtOAc), n-butyl alcohol, and water as the extraction and/or the partitioning solvents. We then tested their inhibitory effects on melanogenesis by using in vitro tyrosinase inhibition assay, in vitro l-3,4-dihydroxy-indole-2-carboxylic acid (l-DOPA) auto-oxidation assay, and B16 melanoma cells. In addition, cytotoxicity testing (NR50 and MTT50) was conducted to evaluate safety. From the results of these assays, four fractions with good efficacy and low toxicity were selected among them, including EtOAc fraction of Smilax china (rhizome), Paeonia lactiflora (root), and Polyporus umbellatus (sclerotium), and BuOH fraction of Evodia officinalis (fruit). In the inhibition assay of intracellular tyrosinase activity and melanogenesis in B16 melanoma cell line, the four plant fractions showed dose-dependent inhibitory effects, and the EtOAc fraction of P. lactiflora showed the highest activity among the four fractions. The EtOAc fraction of P. lactiflora was found to be the most effective substrate.

Extraction, characterization, and anti-nonalcoholic steatohepatitis activity of a (1,3) (1,6)-ß-D-glucan from the Polyporus umbellatus (Pers.) Fries.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by inflammation and hepatic steatosis that may coincide with fibrotic activity. To date, no pharmacological agents have been approved for NASH treatment. Here, a homogeneous (1,3),(1,6)-ß-D-glucan (PUP-W-1, Mw: 41.07 kDa) was successfully purified from Polyporus umbellatus (Pers.) Fries sclerotia and characterized. The analysis showed that the PUP-W-1 backbone consisted of a repeating chain of eight →3)-ß-D-Glcp-(1 â†’ units, with branched chains of four ß-D-Glcp residues, joined by repeating 1,6-linkage units at the O-6 position of the backbone. The pharmacological effects of PUP-W-1 treatment in the context of NASH pathogenesis were explored using a methionine choline-deficient (MCD) diet-induced murine steatohepatitis model. The MCD model mice exhibited pronounced steatohepatitis, inflammatory activity, steatosis, stellate cell activation, and mild fibrotic activity. Treatment of the mice for three weeks with PUP-W-1 prevented the development of NASH due to the suppression of inflammation, lipid accumulation, and fibrosis. As suggested by these findings, PUP-W-1 may hold promise as a natural drug candidate or precursor for the treatment of NASH.

Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

BACKGROUND: Mushrooms have been used in Asia as traditional foods and medicines for a long time. Ergosta-4,6,8(14),22-tetraen-3-one (ergone) is one of the well-known bioactive steroids, which exists widely in various medicinal fungi such as Polyporus umbellatus, Russula cyanoxantha, and Cordyceps sinensis. Ergone has been demonstrated to possess cytotoxic activity. However, the molecular mechanisms by which ergone exerts its cytotoxic activity are currently unknown. METHODS: In the present study, ergone possessed a remarkable anti-proliferative activity toward human hepatocellular carcinoma HepG2 cells. We assayed the cell cycle by flow cytometry using PI staining; investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V/PI staining; observed the nuclear fragmentation by Hoechst 33258 staining and studied the protein expression of Bax, Bcl-2, p-53, procaspase-3, -8, -9, PARP and cleaved PARP by Western blotting analysis. RESULTS: Cells treated with ergone showed typical markers of apoptosis: G2/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. Furthermore, PARP-cleavage; activation of caspase-3, -8, -9; up-regulation of Bax and down-regulation of Bcl-2 were observed in HepG2 cells treated with ergone, which show that both the intrinsic and extrinsic apoptotic pathways are involved in ergone-induced apoptosis in HepG2 cells. Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in HepG2 cells in a caspase-dependent manner. GENERAL SIGNIFICANCE: In this study, we reported for the first time that ergone-induced apoptosis through activating the caspase. These results would be useful for the further utilization of many medicinal fungi in cancer treatment.

Structure elucidation and immunological function analysis of a novel ß-glucan from the fruit bodies of Polyporus umbellatus (Pers.) Fries.

ß-Glucans derived from various sources such as yeast cell walls and medicinal mushrooms are considered as valuable biological response modifiers for their ability to enhance the activity of immune cells, aid in wound healing and help prevent infections. We herein characterize the structure of a novel water-soluble polysaccharide (Zhuling polysaccharide, ZPS) from the fruit bodies of medicinal mushroom Polyporus umbellatus and investigate its immunobiological function. ZPS has a molecular mass of 2.27 x 10(3) kDa and contains >90% d-glucose as its monosaccharide constituent. On the basis of partial acid hydrolysis, methylation analysis, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy and the ideal repeating unit of ZPS is established: (1 → 6, 1 → 4)-linked ß-d-glucopyranosyl backbone, substituted at O-3 position of (1 → 6)-linked ß-d-glucopyranosyl by (1 → 3)-linked ß-d-glucopyranosyl branches. ZPS consists of approximately 2930 repeating units, each contains a side chain of no more than three residues in length. Functionally, ZPS is a potent activator of B cells, macrophages and dendritic cells. Depletion of ZPS branches causes a substantial reduction in its ability not only to activate B cells in vitro but also to elicit specific IgM production in vivo. Virtually all healthy human subjects possess high-titer circulating antibodies against ZPS backbone, suggesting that ZPS epitope is shared by environmental antigens capable of eliciting adaptive humoral responses in the population.

Phytochemistry and bioactivities of the main constituents of Polyporus umbellatus (Pers.) Fries.

BACKGROUND: Edible fungi resources have good application prospects in the research and development of food, medicine, and health products. Polyporus umbellatus (Pers.) Fries, as a precious edible and medicinal fungus, has long been used by Chinese medicine to treat urinary systems and related kidney diseases. PURPOSE: In recent years, researchers have discovered and isolated a variety of active compounds from P. umbellatus. Modern phytochemical and pharmacological experiments showed that the crude extract of P. umbellatus had many biological functions and could be widely used in the fields of food, pharmaceutical and cosmetics. This paper summarizes the active components of P. umbellatus, through elaborating its mechanism of action, further clarify the action substances, in order to improve the utilization rate of P. umbellatus, promote the development and application of P. umbellatus in food, pharmaceutical and cosmetics industry. METHODS: In this paper, the literatures related to P. umbellatus were summarized and classified by "China National Knowledge Instructure (CNKI)", "Google Scholar" and "Web of Science". Compared with other articles, this work systematically sorted out all the active substances with clear structures in P. umbellatus. On this basis, combined with the chemical composition of P. umbellatus, its functional efficacy was expounded, and the effects of different types of active substances in P. umbellatus were further presented. RESULTS: The main chemical constituents of P. umbellatus include polysaccharide and sterol, and the secondary compounds include fatty acids, phenols and other small molecules. These active substances endowed P. umbellatus anti-cancer, antibacterial, diuretic, antioxidant, enhance immune system, promote hair growth and other pharmacological activities, which has been verified many times in vivo and in vitro experiments. CONCLUSION: Modern in vitro or in vivo pharmacological experiments and clinical practice for the efficacy of P. umbellatus provides a strong support, and the separation of compounds in P. umbellatus has also deepened people's understanding of this traditional Chinese medicine, greatly promoted the development and application of P. umbellatus. However, the complex active substances of poring also hinder the research of P. umbellatus to some extent, and the mechanism of action and potential synergistic or antagonistic effect of the mixture of various active ingredients have not been clearly analyzed. How to use the bioactivity-guided separation strategy to identify more bioactive components and analyze the molecular mechanism of the main active components have become the main problems of P. umbellatus research, but also provides a direction for the further study of it.

Hot water extract of the sclerotium of Polyporus rhinocerus Cooke enhances the immune functions of murine macrophages.

Treatment of hot water extract of the sclerotium of Polyporus rhinocerus (PRW) with murine macrophages including RAW 264.7 cell line and primary macrophages (PMs) could enhance their functional activities. These include a significant up-regulation of pinocytosis; an increase in the production of reactive oxygen species (ROS) and nitric oxide (NO); an increase in tumor necrosis factor alpha (TNF-alpha) production and inducible nitric oxide synthase (iNOS) expression in both RAW 264.7 cells and PMs. Cell surface receptors for yeast-derived beta-glucan, including Dectin-1, CR3, and TLR2, were determined by flow cytometry, and the expression of Dectin-1+ cells on the cell surface decreased in the responses of PMs to PRW. PRW increased phosphorylation of IkappaBalpha, which could trigger the nuclear factor kappa B (NF-kappaB) signal pathway for macrophage activation in RAW 264.7 cells. Therefore, the immunomodulatory effect of PRW could be mediated by macrophage activation via the NF-kappaB signal pathway.

Polysaccharides from Polyporus umbellatus: A review on their extraction, modification, structure, and bioactivities.

Polyporus umbellatus (Pers.) Fries, a well-known medicinal fungus, has been reported to exhibit important functions of diuresis and dampness infiltration in traditional Chinese Medicine. Accumulating evidences have demonstrated that the P. umbellatus polysaccharides (PUPs) are the main and representative pharmacologically active ingredients and display multiple bioactivities both in vivo and in vitro methods, such as those of antioxidant, immunomodulatory, antitumor, anti-proliferative and hepatoprotective. Besides, many PUPs have been isolated from the different sources of P. umbellatus, including sclerotia, fruiting body, mycelia and fermentation liquid of this fungus. The purpose of the present review is to comprehensively and systematically reorganize the available information related to the extraction, purification, modification, structure characterization and to discuss diverse biological activities of PUPs to support their potential application value in pharmaceuticals field, functional foods and cosmetics areas. In addition, new invaluable insights on the future research with PUPs have also been proposed in the important areas of structural characterization and pharmacological activities.

Homogeneous polyporus polysaccharide inhibits bladder cancer by polarizing macrophages to M1 subtype in tumor microenvironment.

BACKGROUND: Polyporus polysaccharide (PPS), an active ingredient of traditional Chinese medicinal Polyporus umbellatus, has multiple biological functions, such as anti-cancer, immune-regulating and hepatoprotective activities. The purpose of this study was to investigate the mechanism of homogeneous polyporus polysaccharide (HPP) activated macrophages in the treatment of bladder cancer. METHODS: 100 ng/mL Phorbol myristate acetate (PMA) was used to induce THP-1 human leukemic cells as a macrophage model. Then macrophages derived from THP-1 were treated with different concentrations of HPP (1, 10 and 100 µg/mL). Flow cytometry and RT-PCR were used to detected the expression of CD16, CD23, CD86, CD40 and interleukin (IL)-Iß, iNOS mRNA. ELISA was used to test the change of IL-1ß and TNF-α in macrophage after the treatment with HPP. The conditioned medium from HPP-polarized macrophages was used to detect the effect of activated macrophages on bladder cancer. MTT assay, 5-ethynyl-2'-deoxyuridine assay, flow cytometry, Transwell assay, and Western blot analysis were used to detect the effects of polarized macrophages on the viability, proliferation, apoptosis, and migration of bladder cancer cells. Western blot was also used to analysis the change of JAK2/NF-κB pathway protein. RESULTS: HPP promoted the expression of pro-inflammatory factors, such as IL-Iß, TNF-α and iNOS, and surface molecules CD86, CD16, CD23, and CD40 in macrophages and then polarized macrophages to M1 type. Results demonstrated that activated macrophages inhibited the proliferation of bladder cancer cells, regulated their apoptosis, and inhibited migration and epithelial-mesenchymal transformation (EMT). JAK2/NF-κB pathways were downregulated in the anti-bladder cancer process of activated macrophages. CONCLUSION: The findings indicated that HPP inhibited the proliferation and progression of bladder cancer by the polarization of macrophages to M1 type, and JAK2/NF-κB pathway was downregulated in the process of anti-bladder cancer.

Structural studies of immunomodulatory (1 → 3)-, (1 → 4)-α glucan from an edible mushroom Polyporus grammocephalus.

A water soluble polysaccharide (PGPS) with molecular weight ~ 1.4 × 105 Da was isolated by alkali treatment from an edible mushroom Polyporus grammocephalus and purified by gel chromatography using sepharose-6B column. Monosaccharide analysis revealed that PGPS was made up of glucose only. PGPS contained (1 â†’ 3)-α-D-Glcp and (1 â†’ 4)-α-D-Glcp moieties in a molar ratio of nearly 1:2. Through a series of chemical and spectroscopic (1D/2D NMR) investigations, the repeating unit of the glucan was established as: →3)-α-D-Glcp(1 â†’ [4)-α-D-Glcp(1]2→ This α-glucan was observed to stimulate some prime components of immune system, namely, macrophages, splenocytes, and thymocytes.

Polysaccharide purified from Polyporus umbellatus (Per) Fr induces the activation and maturation of murine bone-derived dendritic cells via toll-like receptor 4.

In this study, we report that a polysaccharide isolated from a Chinese medicinal herb, Zhu Ling (the sclerotium of Polyporus umbellatus (Per) Fr), induces phenotypic and functional maturation of murine bone-derived dendritic cells (BMDCs). Treatment of BMDCs with Polyporus polysaccharide (PPS) resulted in enhanced cell-surface expression of CD86, as well as enhanced production of both interleukin (IL)-12 p40 and IL-10 in a dose-dependent manner. In addition, treatment of BMDCs with PPS resulted in increased T cell-stimulatory capacity and decreased phagocytic ability. PPS-induced production of IL-12 p40 was inhibited by monoclonal antibodies to Toll-like receptor 4 (TLR4). Flow cytometric analysis showed that fluorescence-labeled PPS (f-PPS) bound specifically to BMDCs. This binding was blocked by both unlabeled PPS and anti-TLR4, but not by anti-TLR2 and anti-CR3 monoclonal antibodies. Taken together, our data show that PPS promotes the activation and maturation of murine BMDCs via TLR4.

The inhibitory effect of ergosterol, a bioactive constituent of a traditional Japanese herbal medicine saireito on the activity of mucosal-type mast cells.

Mucosal inflammation in ulcerative colitis (UC) is presumed to be regulated primarily by type 2 T helper cell immune responses and mucosal mast cells in the colon are thought to play an important role in the pathogenesis of the mucosal inflammation. Saireito, a Japanese herbal medicine of standardized quality, originating from traditional Chinese medicine (Kampo medicine), is composed of two different Kampo medicines (shosaikoto and goreisan) and is often used for UC in Japan. In this study, we examined the direct effects of these Kampo medicines and their constituents on the antigen-induced degranulation of mucosal-type mast cells. Mucosal-type murine bone marrow-derived mast cells (mBMMCs) were pretreated by these drugs for 24 h, and immunoglobulin E (IgE) receptor-triggered degranulation of mBMMCs was assessed by beta-hexosaminidase release. Goreisan showed inhibitory effects on degranulation of mBMMCs in a dose-dependent manner. Among the five constituent medicinal herbs of goreisan, Poria and Polyporus had the inhibitory effects on mBMMCs. Ergosterol, a principal and common component of Poria and Polyporus, also suppressed the degranulation of mBMMCs. Our results provide a molecular basis to explain a portion of the beneficial therapeutic properties of saireito on UC.

Cytotoxic steroids from Polyporus umbellatus.

The steroids ergone (1), (22E, 24R)-ergosta-7,22-dien-3ß-ol (2), 5α,8α-epidioxy-(22E,24R) -ergosta-6,22-dien-3ß-ol (3), ergosta-6,22-dien-3ß,5α,6ß-triol (4), and polyporusterone B (5) were isolated from Polyporus umbellatus by bioassay-guided approach. They showed potent anticancer activity against HepG2 cells. Ergone displayed remarkable anticancer activity against HepG2, Hep-2, and Hela cancer cells, of which HepG2 cells were the most sensitive. Furthermore, the cytotoxic effects of ergone on normal human cells (HUVEC) were smaller than on cancer cells. The results showed that ergone had more selective cytotoxic activity against cancer cells than against normal cells.

Quantitative HPLC method and pharmacokinetic studies of ergosta-4,6,8(14),22-tetraen-3-one, a natural product with diuretic activity from Polyporus umbellatus.

A simple and specific HPLC method with dual wavelength UV detection for the determination of ergosta-4,6,8(14),22-tetraen-3-one (ergone) in rat plasma was developed and proved to be efficient. The method used ergosterol as internal standard (IS). Following a single-step protein precipitation, the analyte and IS were separated on an Inertsil ODS-3 column with a mobile phase containing methanol-water (99:1, v/v) at a flow rate of 1 mL/min. The analytes were detected by using UV detection at wavelength of 350 (ergone) and 283 (IS) nm, respectively. The calibration curve was linear over the range of 0.1-2.0 µg/mL and the lower limit of quantification was 0.1 µg/mL. The intra-day and inter-day precision studies showed good reproducibility with RSD less than 8.5%. The intra-day and inter-day accuracy ranged from 95.6 to 104%. Mean extraction recovery was above 95% at the low, medium and high concentrations. The present HPLC-UV method was simple and reliable. The method described herein had been successfully applied for the pharmacokinetic studies in male SD rats after administration of 20 mg/kg dose of solution of ergone.