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To investigate the influence of molecular weight (Mw) 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 × 106 g/mol) solution to obtain two degraded fractions with molecular weight of 6.53 × 105 g/mol (GLPC) and 3.49 × 104 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 Mw 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 Mw of β-D-glucan influenced its anti-inflammatory activity and decreasing of Mw would improve the activity, which provided evidence for the potential use of β-D-glucan from G. lucidum as anti-colitis ingredients.

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 (p0.05), but it was not significantly related to the ability of the mushrooms to scavenge reactive oxygen species (p0.05). 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.

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 HClO4 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 μL. The results showed that all the calibration curves had excellent linearity (R2>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 μg/mL, and the quantification (LOQ) was between 0.10 and 19.53 μg/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.

Title page and contents 1\. Introduction 1 2\. Materials and Methods 9 3\. Results 22 4\. Discussion 58 5\. Summary 66 6\. References 68 Publications, Acknowledgements 80, Ganoderma lucidum, a traditional Chinese medicine, harbors a well-known capacity to modulate immunoactivity and inhibit tumour cell growth. GLIS is a proteoglycan fraction purified from Ganoderma lucidum through different chromatographic steps. This fraction has been proven to induce the proliferation and differentiation of lymphocytes. Its effect on B cells and macrophages was further investigated. GLIS was also found to be the active fraction of crude extract Ganoderma lucidum in the activation of macrophages. After being stimulated with GLIS, the cells were more spread out and elongated than those of controls. It can activate macrophages to increase the secretion of IL-1.... , TNF-á and reactive nitrogen intermediate (RNI) production. The percentage of phagocytosis was significantly enhanced in the presence of GLIS, and it triggered macrophage activation for tumour cytotoxicity. The macrophages from tumour-bearing mice demonstrated more sensitivity to the stimulus of GLIS. GLIS selectively increased the proliferation of B cells, and enhanced the population of CD25 and CD71 positive cells of B cells, but not that of non-B cells, so the cell-type specificity of GLIS was B cells. After being stimulated by GLIS, B cells were activated into antibody producing plasma cells, and secreted significant amounts of IgM. With the respect to the response to MSLs, the relative amount of IgM obtained from B cells induced by GLIS was lower in the absence of accessory cells. When 0.5% macrophages was added to B cells, B cell survival and IgM secretion increased significantly after stimulation with GLIS. The interaction of B cells and macrophages was not only due to the substances secreted by macrophages, but also to the direct effect. The B cells from tumour-bearing mice could secrete 3 - 4 times IgM than that of TMSLs. In vivo experiments also found a significant increase of IgM secretion after injection with GLIS in comparison to the control. Characterisation of the active fraction GLIS was performed. Polymyxin B, a selective inhibitor of LPS, abolished LPS-induced NO production of macrophages, whereas it did not inhibit the action of GLIS on macrophages. Through HPLC, the active compound of GLIS was determined with the molecular weight ca. 2000 k. The activity of GLIS did not change after treatment with pronase E, but the activity was significantly reduced after treatment with NaIO4. . When GLIS was digested with â -1,3-glucanase, the activation rate of macrophage was reduced significantly. FITC-labeled GLIS can bind to macrophage surfaces as confirmed using FACS analysis. Incubation of macrophages in the presence of either laminarin or mannose, which are soluble carbohydrate antagonists of macrophage â -glucan and mannose receptors, significantly reduced macrophage NO production following treatment with GLIS. Treatment of macrophages with anti-CD14 Ab significantly blocked GLIS-induced nitrite production. According to their traditional use in China, eight traditional Chinese medicines were chosen to be screened for their inhibition activity of tumour cells. Polygonum cuspidatum demonstrated strong tumour growth inhibition. The active compounds of the crude extractwere sought through bioassay-directed fractionation. HZ-3-1-b was shown to be the active compound in the crude extract of Polygonum cuspidatum that inhibited the growth of tumour cells. HZ-3-1-b could inhibit the proliferation of various kinds of tumour cells in a dose-dependent manner. HZ-3-1-b was confirmed to induce SW 620 cells apoptosis by stained with annexin V-FITC conjugate and analysis of DNA fragments. In addition, it was found that SW620 cells were arrested in S phase. HZ-3-1-b was identified as resveratrol using HPLC and TLC. Through resveratrol affinity column, a protein with a molecular weight of ca. 35-40 k was found. It was identified as GAPDH through Maldi-TOF analysis. Reveratrol could inhibit the activity of GAPDH. Vmax of the enzyme was down to 21% by 100 µm reveratrol, whereas Km remained unchanged., Ganoderma lucidum-Extrakt, eine traditionelle chinesische Medizin, ist für seine immunmodulatorische Wirkung bekannt. GLIS ist ein durch verschiedene chromatographische Schritte aus Ganoderma lucidum isoliertes Proteoglykan. Es wurde gezeigt, dass diese Fraktion Makrophagen zu erhöhter Sekretion von IL- 1b, TNF-a und RNI stimuliert. In Anwesenheit von GLIS stieg der Anteil phagozytierender Zellen erheblich an und durch Makrophagen vermittelte Tumor- Zytolyse wurde angeregt. Makrophagen von Mäusen, die Tumoren trugen, wiesen eine erhöhte Sensitivität für die Stimulation mit GLIS auf. GLIS begünstigte selektiv die Proliferation von B-Zellen. Es erhöhte den Anteil aktivierter, CD25- und CD71-positiver Zellen unter den B-Zellen, nicht jedoch unter den Nicht-B-Lymphocyten. Nach Stimulation mit GLIS entwickelten sich B-Zellen zu Plasmazellen, die hohe Mengen an IgM produzierten. Die Überlebensrate von B-Zellen und die IgM-Produktion in Gegenwart von Makrophagen erhöhten sich nach Stimulation mit GLIS wesentlich. Die Interaktion von B-Zellen und Makrophagen ist nicht nur auf die von den Makrophagen sekretierten Substanzen, sondern auch auf den direkten Zellkontakt zurückzuführen. Experimente in vivo zeigten ebenfalls eine deutlich gesteigerte IgM-Produktion nach i.p.-Gabe von GLIS. HPLC Analyse und Bioassay ergaben, daß die Aktivität von GLIS auf dessen Polysaccharidwirkstoffe zurückzuführen ist, welche ein relatives Molekulargewicht von etwa 2000 k aufweisen. Der Rohextrakt von Polygonum cuspidatum (HZ) hat auf Tumoren (-zellen) eine stark wachstumshemmende Wirkung. Als wirksamer Bestandteil des Rohextraktes wurde durch eine Bioassay- gelenkte Fraktionierung HZ-3-1-b angereichert. HZ-3-1-b hemmte das Wachstum von Tumorzellen dosisabhängig. Immunfluoreszenzfärbung von Annexin-V und der Nachweis von DNA Fragmentation bestätigten, daß HZ-3-1-b bei SW 620 Zellen Apoptose auslöste. Ferner wurden SW 620 Zellen in der S-Phase angehalten. HZ-3-1-b wurde durch HPLC und TLC als Resveratrol identifiziert. Ein Resveratrol-bindendes Protein mit einem Molekulargewicht von etwa 35-40 k wurde durch Affinitätschromatographie isoliert und mit Hilfe von MALDI-TOF- Massenspektrometrie als GAPDH identifiziert. Durch den Nachweis einer nichtkompetitiven Hemmung, die Maximalgeschwindigkeit der Enzymreaktion wurde durch 100 µM Resveratrol um 79 % reduzierten, während Km unverändert blieb, konnte GADPH als Zielmolekül von HZ-3-1-b/Resveratrol bestätigt werden.