Ameliorative effect of an acidic polysaccharide from Phellinus linteus on ulcerative colitis in a DSS-induced mouse model.

Phellinus linteus, a rare medicinal fungus, displays strong antitumor and anti-inflammatory activities because of its active metabolites, particularly polysaccharides. We investigated effects of P. linteus acidic polysaccharide (PLAP) on amelioration of dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in a mouse model, and associated mechanisms. PLAP treatment alleviated major UC symptoms (weight loss, reduced food intake, increased disease activity index), and ameliorated histopathological colon tissue damage, reduced levels of pro-inflammatory factors (TNF-α, IL-6, IL-1ß), enhanced anti-inflammatory factor IL-10 level, reduced levels of oxidative stress-related enzymes iNOS and MPO, and enhanced expression of tight junction proteins (ZO-1, occludin, claudin-1). qPCR analysis revealed that PLAP downregulated phosphorylation levels of p65 and p38 and transcriptional level of TLR-4. High-throughput sequencing showed that PLAP restored gut microbiota diversity and species abundances in the UC model, and gas chromatographic analysis showed that it increased levels of beneficial short-chain fatty acids. Our findings indicate that PLAP has strong potential for development as an anti-UC agent based on its reduction of inflammation and oxidative stress levels, modulation of gut microbiota composition, and promotion of normal intestinal barrier function.

Phellinus linteus polysaccharides: A review on their preparation, structure-activity relationships, and drug delivery systems.

Phellinus linteus polysaccharides exhibit antitumor, immunomodulatory, anti-inflammatory, and antioxidant properties, mitigate insulin resistance, and enhance the diversity and abundance of gut microbiota. However, the bioactivities of P. linteus polysaccharides vary owing to the complex structure, thereby, limiting their application. Various processing strategies have been employed to modify them for improving the functional properties and yield. Herein, we compare the primary modes of extraction and purification employed to improve the yield and purity, review the structure-activity relationships, and discuss the application of P. linteus polysaccharides using nano-carriers for the encapsulation and delivery of various drugs to improve bioactivity. The limitations and future perspectives are also discussed. Exploring the bioactivity, structure-activity relationship, processing methods, and delivery routes of P. linteus polysaccharides will facilitate the development of functional foods and dietary supplements rich in P. linteus polysaccharides.

Clostridium butyricum and Chitooligosaccharides in Synbiotic Combination Ameliorate Symptoms in a DSS-Induced Ulcerative Colitis Mouse Model by Modulating Gut Microbiota and Enhancing Intestinal Barrier Function.

Effects of Clostridium butyricum and chitooligosaccharides (COS), singly and in synbiotic combination, were evaluated in a C57BL/6 mouse model of dextran sulfate (DSS)-induced acute ulcerative colitis (UC). Treatment with C. butyricum and/or COS ameliorated UC symptoms in vivo, and the strongest effects were observed for the combination in terms of reduced mortality rates and disease activity indices, increased body weight and colon length, and improved histological features. The C. butyricum and COS combination achieved the following: (i) regulated levels of inflammation-related cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1ß [IL-1ß], IL-6, IL-10) and had a stronger anti-inflammatory effect than either component alone, based on inhibition of Toll-like receptor 4 (TLR-4)/NF-κB/MAPK signaling pathway activation; (ii) enhanced intestinal barrier function by restoring levels of tight junction proteins (occludin, claudin-1, ZO-1) and MUC2; (iii) increased abundance and diversity of beneficial bacteria (gut microbiota) and reduced levels of pathogenic bacteria; and (iv) enhanced production of short-chain fatty acids. Our findings indicate that the synbiotic C. butyricum and COS combination has strong potential as a therapeutic adjuvant for UC. IMPORTANCE Ulcerative colitis (UC), an idiopathic intestinal disease characterized by continuous remission/relapse inflammatory cycles in the colonic mucosal layer, has strong adverse effects on patients' quality of life and considerable costs for health care systems. Probiotics, prebiotics, and synbiotics are regarded as potential therapeutic agents for UC, in terms of safety and efficacy. In this study, we present detailed evaluation of effects in a DSS-induced UC mouse model of a synbiotic composed of Clostridium butyricum and COS (molecular weight [MW], 2,500 Da). We found that synergistic (synbiotic) action of the C. butyricum and COS combination is more effective than either factor alone for prevention and/or therapy of UC by regulating gut microbiota and intestinal barrier function. Our findings indicate that C. butyricum and COS in combination has strong potential for development as anti-UC therapeutic drugs or adjuvant agents in pharmaceutical, food, and livestock industries. Highlights include the following. (i) The C. butyricum and COS combination ameliorated clinical UC symptoms and improved colonic morphology. (ii) The C. butyricum and COS combination displayed strong anti-inflammatory and antioxidant effects. (iii) The C. butyricum and COS combination enhanced expression of tight junction proteins. (iv) The C. butyricum and COS combination inhibited the TRL-4/NF-κB/MAPK signaling pathway. (v) The C. butyricum and COS combination modulated gut microbiota abundance and composition.

Carbon Source Affects Synthesis, Structures, and Activities of Mycelial Polysaccharides from Medicinal Fungus Inonotus obliquus.

The effects of various carbon sources on mycelial growth and polysaccharide synthesis of the medicinal fungus Inonotus obliquus in liquid fermentation were investigated. After 12-d fermentation, mycelial biomass, polysaccharide yield, and polysaccharide content were significantly higher in Glc+Lac group (glucose and lactose used as combined carbon source) than in other groups. Crude polysaccharides (CIOPs) and the derivative neutral polysaccharides (NIOPs) were obtained from mycelia fermented using Glc, fructose (Fru), Lac, or Glc+Lac as carbon source. Molecular weights of four NIOPs (termed as NIOPG, NIOPF, NIOPL, and NIOPGL) were respectively 780.90, 1105.00, 25.32, and 10.28 kDa. Monosaccharide composition analyses revealed that NIOPs were composed of Glc, Man, and Gal at different molar ratios. The NIOPs were classified as α-type heteropolysaccharides with 1→2, 1→3, 1→4, 1→6 linkages in differing proportions. In in vitro cell proliferation assays, viability of RAW264.7 macrophages was more strongly enhanced by NIOPL or NIOPGL than by NIOPG or NIOPF, and proliferation of HeLa or S180 tumor cells was more strongly inhibited by NIOPG or NIOPGL than by NIOPF or NIOPL, indicating that immune-enhancing and anti-tumor activities of NIOPs were substantially affected by carbon source. qRT-PCR analysis revealed that expression levels of phosphoglucose isomerase (PGI) and UDP-Glc 4-epimerase (UGE), two key genes involved in polysaccharide synthesis, varied depending on carbon source. Our findings, taken together, clearly demonstrate that carbon source plays an essential role in determining structure and activities of I. obliquus polysaccharides by regulating expression of key genes in polysaccharide biosynthetic pathway.

Phellinus linteus polysaccharide extract improves insulin resistance by regulating gut microbiota composition.

The hypoglycemic effect of Phellinus linteus polysaccharide extract (PLPE) has been documented in several previous studies, but the functional interactions among PLPE, gut microbiota, and the hypoglycemic effect remain unclear. We examined the regulatory effect of PLPE on gut microbiota, and the molecular mechanism underlying improvement of insulin resistance, using a type 2 diabetic rat model. Here, 24 male Sprague-Dawley rats were randomly divided into four groups that were subjected to intervention of saline (normal and model control group), metformin (120 mg/kg.bw), and PLPE (600 mg/kg.bw) by oral administration. After 8 weeks of treatment, PLPE increased levels of short-chain fatty acids (SCFAs) by enhancing abundance of SCFA-producing bacteria. SCFAs maintained intestinal barrier function and reduced lipopolysaccharides content in blood, thereby helping to reduce systemic inflammation and reverse insulin resistance. Our findings suggest that PLPE (in which polysaccharides are the major component) has potential application as a prebiotic for regulating gut microbiota composition in diabetic patients.

Mycelial polysaccharides of Lentinus edodes (shiitake mushroom) in submerged culture exert immunoenhancing effect on macrophage cells via MAPK pathway.

A crude polysaccharide fraction (termed cLEP) and two derived fractions (termed LEP1 and LEP2) from Lentinus edodes mycelia were purified and characterized. LEP1/-2 were classified as α-type heteropolysaccharides with 1 → 2, 1 → 3, 1 → 4, 1 → 6 linkages. Their monosaccharide components were respectively Gal, Glc, Man, Ara, Fuc, and Rha (molar ratio 10.17:9.75:9.01:1.61:1.26:1), and Glc, Man, Fuc, Rha, and Gal (molar ratio 5.18:4.69:2.85:1.43:1). In vitro culture experiments with macrophage RAW264.7 cells showed no cytotoxic effects of the polysaccharides. Phagocytosis (neutral red uptake) was significantly enhanced by LEP1/-2. Levels of NO, TNF-α and IL-6 were higher in LEP1/-2-treated groups than in cLEP-treated group. qRT-PCR analysis showed that LEP1/-2 had greater enhancing effect on mRNA transcription of iNOS, TNF-α, and IL-6 genes. Western blotting analysis revealed that LEP1/-2 strongly promoted phosphorylation of kinases ERK and JNK, and suggested that they exert immunoenhancing effects via MAPK signaling pathway.

Alpha-terpineol affects synthesis and antitumor activity of triterpenoids from Antrodia cinnamomea mycelia in solid-state culture.

To enhance production of Antrodia cinnamomea triterpenoids (ACTs) from mycelia in solid-state culture, α-terpineol was added to the medium as an elicitor at an optimal concentration of 0.05 mL L-1. Multi-stage solvent extraction and HPLC analysis were performed, and the compositions of ACTs-E (from culture with elicitor) and ACTs-NE (from culture without elicitor) were found to be quite different. In assays of in vitro antitumor activity, ACTs-E, in comparison with ACTs-NE, produced stronger viability reduction in several tumor cell lines and stronger apoptosis induction in HeLa in a dose-dependent manner. Several related proteins involved in the mitochondrial pathway of apoptosis (p53, Bax, caspase-3) did not show expression upregulation by ACTs-E, suggesting that apoptosis induction occurred through a p53-independent process. Further analysis revealed that ACTs-E strongly inhibited synthesis of topoisomerase I (TOP1) and tyrosyl-DNA phosphodiesterase I (TDP1), which are involved in DNA repair, at both transcriptional and protein levels. Our findings suggest that ACTs-E have potential for applications in the pharmaceutical, clinical, and functional food industries, as a novel antitumor agent and a dual TOP1/TDP1 inhibitor.

A polysaccharide from Antrodia cinnamomea mycelia exerts antitumor activity through blocking of TOP1/TDP1-mediated DNA repair pathway.

A polysaccharide (termed ACPS-1) from mycelia of Antrodia cinnamomea under submerged culture was purified by hot water extraction and successive DEAE-52 cellulose and Sephadex G-100 column chromatography, and structurally characterized by FTIR, NMR, periodate oxidation, Smith degradation, and GC-MS. ACPS-1 (MW 2.296 × 104 Da) was composed primarily of Man, Xyl, Ara, Fuc and Rha with a molar ratio of 31.27:1.77:1.44:1.34:1.00, and its backbone consisted of repeating α-(1 → 3), α-(1 → 6), α-(1 → 2), and α-(1 → 4) glycosidic linkages. ACPS-1 displayed strong in vitro growth-inhibitory effects on several human and mouse cancer cell lines (HeLa, A431, H22 and S180), and were not cytotoxic to normal mouse spleen cells. Studies of the inhibitory mechanism revealed that ACPS-1 induced apoptosis and cell cycle arrest (cells remained in G2/M phase) through blocking of topoisomerase I/tyrosyl-DNA phosphodiesterase I (TOP1/TDP1)-mediated DNA repair pathway. Our findings suggest that ACPS-1 has strong potential applications in pharmaceutical and food industries, and as a novel anticancer agent based on its dual TOP1/TDP1 inhibitory effect.

Low molecular weight chitosan is an effective antifungal agent against Botryosphaeria sp. and preservative agent for pear (Pyrus) fruits.

Antifungal activity and preservative effect of a low molecular weight chitosan (LMWC) sample, derived from chitosan by enzymatic hydrolysis, were investigated in vitro and in vivo. A pathogenic fungal strain was isolated from decayed pear (Pyrus bretschneideri cv. "Huangguan") fruit and identified as Botryosphaeria sp. W-01. LMWC was shown to strongly inhibit W-01 growth based on studies of minimum inhibitory concentration (MIC) and effects on mycelial biomass and radial growth of the fungus. LMWC treatment of W-01 cells reduced ergosterol synthesis and mitochondrial membrane potential (ΔY), early events of apoptosis. Transmission electron microscopy and confocal laser scanning microscopy studies revealed that LMWC penetrated inside W-01 hyphae, thereby inducing ultrastructural damage. LMWC coating had a significant preservative effect on wounded and nonwounded pear fruits, by inhibiting postharvest decay and browning processes. LMWC activated several defense-related enzymes (polyphenol oxidase, peroxidase, chitinase), maintained nutritional value, and slowed down weight loss. Our findings indicate the strong potential of LMWC as a natural preservative agent for fruits and vegetables.

A novel polysaccharide from mycelia of cultured Phellinus linteus displays antitumor activity through apoptosis.

Two novel polysaccharides termed PLPS-1 and PLPS-2 were isolated from mycelia of cultured Phellinus linteus by hot water extraction, purified by DEAE-52 cellulose and Sephadex G-100 column chromatography, and structurally characterized by FTIR and NMR spectroscopy, GC-MS, periodate oxidation/Smith degradation, and methylation analysis. The monosaccharide compositions of PLPS-1 (MW 2.5×10(5)Da) and PLPS-2 (MW 2.8×10(4)Da) were respectively Glc, Ara, Fuc, Gal, and Xyl in molar ratio 21.964:1.336:1.182:1:1, and Glc, Gal, Man, Ara, Fuc, Xyl in molar ratio 14.368:2.594:1.956:1.552:1.466:1; i.e., both were heteropolysaccharides. The backbone of PLPS-1 consisted primarily of repeating α-d-Glc(1→4)-α-d-Glc(1→6) units, while that of PLPS-2 consisted of α-(1→3)-d-Glc and α-(1→6)-d-Glc. The side branches were also different in their carbohydrate components. In in vitro antitumor assays, PLPS-1 displayed strong anti-proliferative effect against S-180 sarcoma cells through apoptosis, whereas PLPS-2 had no such effect. The difference in antitumor activity between the two PLPS evidently results from their structural differences. PLPS-1 has potential as a novel anticancer agent.

Isolation, characterization, and antitumor activity of a novel heteroglycan from cultured mycelia of Cordyceps sinensis.

A novel heteroglycan, Cordyceps sinensis polysaccharide 1 (molecular weight 1 17 × 10(5) Da), was isolated and purified from mycelia of the fungus C. sinensis obtained by solid-state culture. Structural characterization by chemical analysis, GC-MS, FTIR, and NMR spectroscopy showed that C. sinensis polysaccharide 1 was mainly composed of (1 → 6)-linked α-D-Glc and α-D-Gal, with minor ß-(1 → 4)-D-Xyl and ß-(1 → 4)-D-Man residues probably located in the side chains with a trace amount of α-(1 → 3)-L-Rha residue. In biological assays, C. sinensis polysaccharide 1 significantly inhibited proliferation of sarcoma 180 cells and induced apoptosis in a dose-dependent manner. Further studies will elucidate the antitumor mechanism of C. sinensis polysaccharide 1 and promote its utilization for the development of novel, effective anticancer drugs.

Protective effect of chitooligosaccharides against cyclophosphamide-induced immunosuppression in mice.

Chitooligosaccharides (COS) display a variety of important biological activities, including antimicrobial, antitumor, anti-inflammatory, and immunoenhancing. In the present study, a COS sample (degree of polymerization 4-11, analyzed by FTIR and MALDI-TOF-MS) prepared by hydrolysis with a recombinant chitosanase was orally administered to mice for evaluation of its effect on cyclophosphamide (Cy)-induced immunosuppression. Thymus and spleen indices, delayed-type hypersensitivity (DTH) reaction, macrophage phagocytosis, and certain enzyme activities were significantly higher in mice treated with COS+Cy than in mice treated with Cy alone. ELISA experiments showed that COS treatment enhanced production of the cytokines IL-2, IL-12, and IFN-γ but decreased production of IL-10 in sera of Cy-treated mice. The well-defined COS product studied displayed strong immunoenhancing activity and a protective effect against Cy-induced immunosuppression. COS-derived products should be further investigated for possible immunostimulatory applications in the food and pharmaceutical industries.