Immunstimulation und Induktion der Apoptose von Tumorzellen durch wirksame Verbindungen aus Ganoderma lucidum und Polygonum cuspidatum

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 80Ganoderma 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

Analysis of Vegetation Red Edge with Different Illuminated/Shaded Canopy Proportions and to Construct Normalized Difference Canopy Shadow Index

Shadows exist universally in sunlight-source remotely sensed images, and can interfere with the spectral morphological features of green vegetations, resulting in imprecise mathematical algorithms for vegetation monitoring and physiological diagnoses; therefore, research on shadows resulting from forest canopy internal composition is very important. Red edge is an ideal indicator for green vegetation’s photosynthesis and biomass because of its strong connection with physicochemical parameters. In this study, red edge parameters (curve slope and reflectance) and the normalized difference vegetation index (NDVI) of two species of coniferous trees in Inner Mongolia, China, were studied using an unmanned aerial vehicle’s hyperspectral visible-to-near-infrared images. Positive correlations between vegetation red edge slope and reflectance with different illuminated/shaded canopy proportions were obtained, with all R2s beyond 0.850 (p < 0.01). NDVI values performed steadily under changes of canopy shadow proportions. Therefore, we devised a new vegetation index named normalized difference canopy shadow index (NDCSI) using red edge’s reflectance and the NDVI. Positive correlations (R2 = 0.886, p < 0.01) between measured brightness values and NDCSI of validation samples indicated that NDCSI could differentiate illumination/shadow circumstances of a vegetation canopy quantitatively. Combined with the bare soil index (BSI), NDCSI was applied for linear spectral mixture analysis (LSMA) using Sentinel-2 multispectral imaging. Positive correlations (R2 = 0.827, p < 0.01) between measured brightness values and fractional illuminated vegetation cover (FIVC) demonstrate the capacity of NDCSI to accurately calculate the fractional cover of illuminated/shaded vegetation, which can be utilized to calculate and extract the illuminated vegetation canopy from satellite images

Antimicrobial, antioxidant, anti-inflammatory, and cytotoxic activities of Cordyceps militaris spent substrate.

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

Structural Characterization of a Polygonatum cyrtonema Hua Tuber Polysaccharide and Its Contribution to Moisture Retention and Moisture-Proofing of Porous Carbohydrate Material

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 × 103 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