香菇是一種重要的藥用蘑菇。數千年來，香菇一直被人們作為食物和藥物來使用。許多研究表明，香菇的提取物具有抗氧化活性，而且他們的抗氧化活性與他們的酚類化合物的含量相關。然而，到目前為止該研究大多集中在對香菇子實體的研究，對香菇菌絲體分泌物的研究就少見報導。 / 在本課題研究中，使用不同的體外抗氧化測定方法和酚類化合物含量測定方法來研究兩種香菇菌絲體分泌物（1358DE 和L5458DE）。實驗結果表明， 在不同的體外抗氧化實驗中1358DE 和L5458DE均具有明顯不同的抗氧化活性。在清除DPPH自由基，清除氫氧根離子，清除超氧陽離子，清除過氧化氫離子，螯合亞鐵離子，還原能力，抑制老鼠紅細胞溶血和抑制脂質過氧化的實驗中，1358DE 和L5458DE的IC50 分別為3.3和132.6; 44.5和 > 1000; 26.9和53.7; 153.6和 >175.0; 176.0和521.0; 26.7和746.4; 47.8和736.9; 3.1和 > 1000 μg/ml。他們的多酚化合物的含量分別為237.33 and 24.08 mg (GAE)/g of DE。實驗資料表明，1358DE的抗氧化活性高於L5458DE，其原因可能是1358DE的酚類化合物含量較高。 / 由於1358DE具有較好的抗氧化活性，採用有機溶劑萃取的方法將其分成水溶性部位和乙酸乙酯部位。體外抗氧化實驗表明，水溶性部位的抗氧化活性與1358DE相近，而乙酸乙酯部位則沒有表現出抗氧化活性。因此，使用聚醯胺柱色譜（可以將多酚類化合物從其他成分中分離出來）對水溶性部位進行進一步的分離，可以得到兩個聚醯胺洗脫部位（P-1和P-2）。與原來的水溶性部位比較，P-1的糖的含量明顯增加，而多酚化合物含量明顯減少，抗氧化活性也明顯降低；相反，P-2的糖的含量明顯減少，而多酚化合物含量明顯增加，抗氧化活性也明顯增加。該實驗結果表明，糖對抗氧化活性的貢獻遠不及多酚化合物。因此，多酚化合物是1358DE的抗氧化活性成分。基質輔助鐳射解吸電離飛行時間質譜和三氯化鐵試劑測定結果表明，P-2是一類水溶性多酚低聚物（WSP），它的分子量在600~1200Da之間。 / 水溶性多酚低聚物（WSP）是1358DE的主要抗氧化活性成分。採用過氧化氫引導細胞毒性的細胞(V79-4)模型來進一步研究WSP的抗氧化活性。在細胞毒性試驗中，在所有測試濃度，WSP在濃度6.25~50 μg/ml均能明顯抑制過氧化氫引致的細胞毒性。此外，WSP還能明顯抑制由過氧化氫引起的丙二醛（MDA）增加和抗氧化酶（SOD，CAT，GSH-Px）的減少。 / 許多抗氧化劑被報導具有抗血管增生活性，該活性與其抗氧化活性相關。由於WSP具有非常好的抗氧化活性，因此，採用斑馬魚模型來研究WSP的抗血管增生活性。在內源性鹼性磷酸酶測定實驗結果表明，WSP在濃度50，100，150，200，250 μg/ml，斑馬魚（野生型）的血管生成明顯分別減低為87.2, 85.6, 74.8, 69.4, and 62.8%（與空白對照組相比）。此外，在螢光顯微鏡下可觀察到WSP在濃度為250μg/ml能明顯抑制螢光斑馬魚（fli1a:EGFP）的節間血管形成。 / 本研究表明，水溶性多酚低聚物（WSP）是香菇菌絲體分泌物的抗氧化成分，WSP不僅具有抗氧化活性，同時還具有抗血管增生活性。此外，本研究結果表明香菇菌絲體分泌物是很好的天然抗氧化劑的來源。 / Shiitake mushroom (Lentinus edodes), known in China as Xiang-gu, is one of the most valuable medicinal mushrooms, and has been used for thousands of years both as food and medicine. Shiitake mushroom extracts have also been found to have antioxidant properties and their antioxidant ability is positively correlated with their phenolic content. However, thus far, investigation of the antioxidant ability of shiitake mushroom has mainly focused on the fruiting body, and the antioxidant properties of its mycelial exudates are rarely reported. / In this study, exudates (DE) secreted from two shiitake mushroom mycelia (strains 1358 and L5458) were evaluated for their antioxidative properties and phenolic content. 1358DE and L5458DE showed distinct antioxidant activity in different in vitro assays, including scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radical, superoxide anions and hydrogen peroxide; the ability to chelate ferrous ions; reducing power; hemolysis inhibition activity in rat erythrocyte; and lipid peroxidation inhibition (IC₅₀ values of 1358DE and L5458DE were 3.3 and 132.6; 44.5 and > 1000; 26.9 and 53.7; 153.6 and >175.0; 176.0 and 521.0; 26.7 and 746.4; 47.8 and 736.9; and 3.1 and > 1000 μg/mL, respectively). Their total phenolic content was 237.33 and 24.08 mg gallic acid equivalent (GAE)/g of dry DE, respectively. Overall, these results show that 1358DE generally possesses better antioxidant properties than L5458DE, possibly due to its larger total phenolic content. / 1358DE were selected to further investigate for its better antioxidant effect. 1358DE was fractionated using water-solvent partition and two fractions [water soluble fraction (WF) and ethyl acetate fraction (EF)] were obtained. The antioxidant effects of WF were similar to those of the original 1358DE, while EF did not possess any antioxidant activities. The WF was further isolated with polyamide column, which can separated the polyphenols from other components, and two sub-fraction (P-1 and P-2) were obtained. After the WF passing through the polyamide column, carbohydrate content in the sub-fraction 1 (P-1) was significantly increased, while its total phenolic content reduced dramatically, and its antioxidant activity decreased. However, the sub-fraction 2 (P-2) was the opposite. Carbohydrate content in P-2 was significantly reduced, while its total phenolic content increased dramatically, and its antioxidant activity increased. Apparently, carbohydrate contributed little to the antioxidant effect than that of the phenolic compounds as shown from this investigation. These results suggest that the antioxidant effect in 1358DE was contributed by the presence of polyphenols. Besides, results from Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ferric trichloride reaction suggested that P-2 was oligomers of water soluble polyphenols (WSP) with the molecular weight of about 600~1200 Da. / The water soluble polyphones (WSP) were the potent antioxidant components in 1358DE and further study its protective effect against the hydrogen peroxide which induced cytotoxicity in V79-4 cells. In the cell viability experiments, pretreatment of WSP at the concentrations of 6.25~50 μg/ml increased the cell viability significantly more than at the presence of H₂O₂ only. Besides, the pretreatment of cells with WSP significantly inhibited the increase of Malondialdehyde (MDA, which is a by-product of lipid peroxidation) production and the decrease of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) activities induced by H₂O₂. / Quite a few antioxidant compounds have been reported that a causative relationship may exist between the anti-angiogenic activity and antioxidant effect. Therefore, a zaebrafish model was using to investigate the anti-angiogenic activity of the WSP because of its excellent antioxidant activity. In quantitative of endogenous alkaline phosphatase (EAP) assay, after the embryos treated with WSP at final concentrations of 50, 100, 150, 200, 250 μg/ml, and the vessel formation were significantly (p < 0.05) reduced to 87.2, 85.6, 74.8, 69.4, and 62.8% of the control value, respectively. Moreover, from the microscope, compare to the control, WSP at the concentration of 250 μg/ml also showed potent inhibition on the intersegmental blood vessels (ISVs) formation in Tg(fli1a:EGFP)y1 zebrafish embryos. Thus, the finding indicated that WSP could inhibit vessel formation in zeabrafish. / Overall, this study revealed that water soluble polyphenols (WSP) was the active components of 1358DE. Besides of the antioxidant effect, the WSP could inhibit vessel formation significantly in zebrafish. The findings indicate that exudates of shiitake mushroom mycelia have good potential as a source of natural antioxidants. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Huang, Weihuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 90-103). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.v / List of Abbreviations --- p.vii / List of Figures --- p.ix / List of Tables --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Oxidation and antioxidant --- p.1 / Chapter 1.1.1 --- Reactive oxygen species (ROS) --- p.1 / Chapter 1.1.2 --- Sources of ROS --- p.1 / Chapter 1.1.3 --- The role of ROS in normal physiology --- p.2 / Chapter 1.1.4 --- Oxidative damage to DNA, lipids and proteins --- p.2 / Chapter 1.1.5 --- Antioxidant defense systems in vivo --- p.4 / Chapter 1.1.6 --- Sources of antioxidants --- p.6 / Chapter 1.2 --- Assessment of antioxidant capacity in vitro and in vivo (Antioxidant methodology) --- p.9 / Chapter 1.2.1 --- Assessment of Free Radical Scavenging Capacity in vitro --- p.9 / Chapter 1.2.2 --- Antioxidant capacity in cultured Cells --- p.10 / Chapter 1.2.3 --- Antioxidant capacity in vivo --- p.11 / Chapter 1.3 --- Mushrooms --- p.12 / Chapter 1.3.1 --- Mushroom life cycle --- p.12 / Chapter 1.3.2 --- Nutritional and medicinal values of mushroom --- p.14 / Chapter 1.4 --- Shiitake mushroom (Lentinus edodes) --- p.15 / Chapter 1.5 --- Objectives --- p.17 / Chapter Chapter 2 --- Antioxidant activity and total phenolic content in Shiitake mycelial exudates --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.2 --- Materials and methods --- p.19 / Chapter 2.2.1 --- Materials --- p.19 / Chapter 2.2.2 --- Sample preparation --- p.20 / Chapter 2.2.3 --- In vitro antioxidant activity assays --- p.20 / Chapter 2.2.4 --- Determination of total phenolic content --- p.25 / Chapter 2.2.5 --- Statistical analysis --- p.25 / Chapter 2.3 --- Results and discussion --- p.25 / Chapter 2.3.1 --- Antioxidant activity --- p.25 / Chapter 2.3.2 --- Total phenolic content --- p.37 / Chapter 2.3.3 --- Antioxidant activity (IC₅₀ values) and phenolic content --- p.37 / Chapter 2.4 --- Conclusion --- p.39 / Chapter Chapter 3 --- The antioxidant components of Shiitake mycelial exudates (1358DE) --- p.42 / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Materials and methods --- p.42 / Chapter 3.2.1 --- Materials --- p.42 / Chapter 3.2.2 --- Sample preparation --- p.43 / Chapter 3.2.3 --- HPLC analytical condition --- p.43 / Chapter 3.2.4 --- Sample fractionated using solvent-water partition and polyamide column chromatographic method guided by in vitro antioxidant assays --- p.43 / Chapter 3.2.5 --- Determination of total phenolic content --- p.44 / Chapter 3.2.6 --- Determination of the contents of carbohydrate --- p.45 / Chapter 3.2.7 --- MALDI-TOF MS analysis --- p.45 / Chapter 3.2.8 --- Statistical analysis --- p.46 / Chapter 3.3 --- Results and discussions --- p.46 / Chapter 3.3.1 --- HPLC analytical results --- p.46 / Chapter 3.3.2 --- Samples fractionation using solvent-water partition --- p.47 / Chapter 3.3.3 --- Water soluble fraction (WF) was further isolated using polyamide column chromatographic --- p.50 / Chapter 3.3.4 --- Molecular weight determination of P-2 --- p.54 / Chapter 3.4 --- Conclusion --- p.56 / Chapter Chapter 4 --- Antioxidative effect of water soluble polyphenols (WSP) in Shiitake mycelial exudates (1358DE) against H2O2-induced cytotoxicity in V79-4 cells --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Sample preparation, materials and methods --- p.58 / Chapter 4.2.1 --- Preparation of the water soluble polyphenols (WSP) --- p.58 / Chapter 4.2.2 --- Materials --- p.58 / Chapter 4.2.3 --- Cell culture and treatment --- p.59 / Chapter 4.2.4 --- MTT assay --- p.60 / Chapter 4.2.5 --- Lactate dehydrogenase (LDH) release assay --- p.60 / Chapter 4.2.6 --- Assay for lipid peroxidation measuring the malondialdehyde (MDA) --- p.61 / Chapter 4.2.7 --- Assay for antioxidant enzymes --- p.62 / Chapter 4.2.8 --- Protein determination --- p.62 / Chapter 4.2.9 --- Statistical analysis --- p.63 / Chapter 4.3 --- Results and discussion --- p.63 / Chapter 4.3.1 --- Cytotoxicty of WSP in V79-4 cells --- p.63 / Chapter 4.3.2 --- Determined the time of WSP pretreatment in V79-4 cell against H₂O₂-induced cytotoxicity --- p.63 / Chapter 4.3.3 --- Protective effect of WSP treated cell against H₂O₂-induced cytotoxicity --- p.64 / Chapter 4.3.3 --- Inhibition of WSP on lipid peroxidation --- p.66 / Chapter 4.3.4 --- Effects of WSP on antioxidant enzyme activities --- p.66 / Chapter 4.4 --- Conclusion --- p.72 / Chapter Chapter 5 --- Anti-angiogenic property of water soluble polyphenols (WSP) in Shiitake mycelial exudates (1358DE) --- p.73 / Chapter 5.1 --- Introduction --- p.73 / Chapter 5.1.1 --- Angiogenesis --- p.73 / Chapter 5.1.2 --- Angiogenesis as a therapeutic target --- p.73 / Chapter 5.1.3 --- Tumors angiogenesis --- p.75 / Chapter 5.1.4 --- Reactive oxygen species (ROS) and tumor angiogenesis --- p.75 / Chapter 5.1.5 --- Anti-angiogenic effects of polyphenols --- p.76 / Chapter 5.1.6 --- Experimental model for studying anti-angiogenic agents --- p.76 / Chapter 5.2 --- Sample preparation, materials and methods --- p.78 / Chapter 5.2.1 --- Preparation of the water soluble polyphenols (WSP) --- p.78 / Chapter 5.2.2 --- Materials --- p.78 / Chapter 5.2.3 --- Methods --- p.79 / Chapter 5.2.4 --- Statistical analysis --- p.80 / Chapter 5.3 --- Results --- p.81 / Chapter 5.3.1 --- Anti-angiogenic effect of WSP on zebrafish model --- p.81 / Chapter 5.3.2 --- Microscopic imaging --- p.82 / Chapter 5.4 --- Discussion and conclusion --- p.82 / Chapter Chapter6 --- Conclusions --- p.84 / Chapter 6.1 --- Conclusion --- p.86 / Chapter 6.2 --- Future works --- p.88 / References --- p.89 / Chapter Appendix 1 --- Publication --- p.104
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328274 |
| Date | January 2012 |
| Contributors | Huang, Weihuan., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xiv, 104 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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