臺灣博碩士論文加值系統

甲殼素是由幾丁質去乙醯基所製成的，在自然界中幾丁質存在於甲殼類動物、昆蟲的外骨骼或真菌類的細胞壁中。雖然在之前的研究中發現甲殼素具有一些生理上的活性，例如能使傷口癒合加快、能降低血脂和抗凝血作用等，但是對於其抗氧化的效用卻知道的很少。
在本篇實驗中，我們致力於探討甲殼素的抗氧化功用和其作用的機制。我們使用從蝦殼或蟹殼萃取而來的水溶性甲殼素，以TBA method檢測其抗脂質過氧化的程度，以1,10-phenanthroline當作指示劑檢測鐵離子的濃度。發現甲殼素具有很強的抗脂質過氧化能力，能抵抗由鐵離子引起的脂質過氧化；而且在脂質過氧化的過程中的initiation和propagation stage皆有作用。Shrimp CM-chitosan、crab CM-chitosan和trolox抑制脂質過氧化的IC50濃度分別是54.09、58.88和64.09μg/ml。而經由實驗證明它們的抗脂質過氧化能力是藉由螯合鐵離子所產生的，對於鐵離子的濃度和氧化還原狀態皆有影響。接下來測試甲殼素是否具有清除超氧自由基 (superoxide anion) 、過氧化氫 (hydrogen peroxide) 和2,2′-azobis (2-amidino-propane) dihydrochloride (ABAP)、tert-butyl hydroperoxide所產生的自由基的能力，發現並沒有這樣的效果。
在紅血球細胞實驗中，我們使用鐵離子引起細胞膜脂質過氧化，發現甲殼素有抑制細胞膜脂質過氧化的效果。另外在HL-60細胞實驗中，我們使用鐵離子引起細胞產生活性氧 (reactive oxygen species)、使細胞膜破損和粒腺體膜電位下降，發現甲殼素有很好的禦防效果。總和以上結果，甲殼素具抗脂質過氧化和螯合過度金屬離子的能力，可當作一個有潛力的抗氧化劑。

Chitosan is the deacetylated product of chitin, an ubiquitous biopolymer found in the exoskeleton of insects and marine invertebrates or the cell wall of fungi. Althought chitosan has been found to have some physiological and biological activities such as hypocholesterolemic activity, wound dressing and blood anticoagulant, little is known about the utility of chitosan in anti-lipid peroxidation effect.
In this study, we examined the effects of water-soluble carboxylmethyl chitosan (CM-chitosan) on antioxidant activities and its possible mechanism of action. We used water-soluble CM-chitosan derived from shrimp or crab as material to make following experiments. Percent inhibition of the lipid peroxidation was used as an index of the antioxidant effect. The products of lipid peroxidation were measured as malondialdehyde-thiobarbituric acid (MDA-TBA). Ferrous ion content was analyzed by the indicator 1,10-phenanthroline. We found that CM-chitosan could significantly reduce iron-induced lipid peroxidation at the initial and propagation stages in rat liver microsomes. The IC50 for shrimp CM-chitosan, crab CM-chitosan and trolox were 54.09, 58.88 and 64.09 μg/ml, respectively. And its antioxidant activities were mediated by chelating iron. We also test whether CM-chitosan has antioxidant activities on scavenging superoxide anion, hydrogen peroxide or free radicals produced by 2,2′-azobis (2-amidino-propane) dihydrochloride (ABAP) or tert-butyl hydroperoxide, CM-chitosan has no effect on them. We then used rat red blood cells to evaluate the effects of CM-chitosan on cell membrane lipid peroxidation induced by ferrous ion. It was found that CM-chitosan could prevent cell membrane from lipid peroxidation induced by ferrous ion. In HL-60 promyelocytic leukemia cell line experimemts, we used ferrous ion to cause cell membrane damage, induce cell to produce reactive oxygen species (ROS), and make mitochondrial membrane potential decreasing. CM-chitosan could protect those cells from oxidative stress. Taken together these observations, CM-chitosan may have potential use as an antioxidant for the decrease of lipid peroxidation by chelating iron.

Abstract in Chinese…………………………………..…...……..………....1
Abstract in English………………………………..……...…………….…..2
Introduction…………………..………………………...…………………..4
(I).Lipid peroxidation is induced by ROS and by transition metal ions………………………………...………………………………………...5
(II).Chitosan……………………………………..…………………………..9
Instruments and Materials…………………………………………….....10
Methods……………………………….………….………………………..14
Preparation of water-soluble carboxylmethyl chitosan…………………….15
Preparation of rat liver microsomes………………..………………………16
Lipid peroxidation system……………………………..…………………...16
Measurement of TBA-RS………………………………………..…………17
Wavelength spectra measurement………………………………….………17
Measurement of ferrous ions (Fe2+) concentration…………………………18
Ferrous iron (Fe2+)—stimulated oxidation of linoleic acid.……………..…..18
Measurement of superoxide anions production by phenazine methosulphate-NADH system……………………………………………………………...18
Measurement of superoxide anions production by xanthine-xanthine oxidase system……………………………………………………………………....19
Measurement of hydrogen peroxide degradation……………………..……19
Lipid peroxidation induced by ABAP or tert-butyl hydroperoxide………..20
Rat RBC…………………...………………………………………………..21
Measurement of lipid peroxidation by flow cytometry…………………….21
HL-60 cell line……………………..……………………………………….22
Cell culture…………………………..……………………………………..22
Cell membrane integrity determination by trypan blue dye exclusion…….22
Cell integrity determination by propidium iodide………………….……....23
Measurement of ROS by flow cytometry…………………………………..23
Measurment of mitochondrial membrane potential………………….…….24
Results………………………………….…………….…………………….26
Antioxidative effect on ascorbic acid or NADPH—induced lipid peroxidation in microsomes…………………………………..……………………………….…..27
Effect on terminination of radical chain reaction……….…………….……28
Effect of iron level on the lipid peroxidation…………………...………….28
Effect on superoxide anions scavenging capacities………………………..29
Effect on H2O2 scavenging capacities……………………………………...30
Effect on cleaning free radical produced by ABAP or tert-butyl hydroperoxide...30
Effect on lipid peroxidation induced by iron in rat RBC…………………..30
Effect on membrane damage induced by ferrous ion in HL-60 cells………31
Effect on ROS production induced by ferrous ion in HL-60 cells…………32
Effect on mitochondrial membrane potential in HL-60 cells………………32
Discussion………………….………………………………………………34
References………………………………...….……………………………40
Figures and Tables……………………..……………………….…………48

Aubrey E., Harvey JR., John AS., Edward SA. (1955) Simultaneous spectrophotometric determination of iron (II) and total iron with 1,10-phenanthroline. Analytical Chemistry. 27 (1) : 26-29.
Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72 : 248-254.
Cacciuttolo MA., Trinh L., Lumpkin JA., Rao G. (1993) Hyperoxia induces DNA damage in mammalian cells. Free Radical Biology & Medicine. 14 (3) : p267-267.
Cai H., Harrison DG. (2000) Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circulation Research. 87 (10) : 840-844.
Casalino E., Sblano C., Landriscina C. (1996) A possible mechanism for initiation of lipid peroxidation by ascorbate in rat liver microsomes. International Journal of Biochemistry & Cell Biology. 28 (2) : 137-149.
Dahle LK., Hill EG., Holman RT. (1962) The thiobarbituric acid reaction and the autoxidations of polyunsaturated fatty acid methyl esters. Archives of Biochemistry and Biophysics. 98 : 253-261.
Davies KJ. (1987) Protein damage and degradation by oxygen radicals. I. general aspects. Journal of Biological Chemistry. 262 (20) : 9895-9901.
Duniec Z., Robak J., Gryglewski R. (1983) Antioxidant properties of some chemicals vs their influence on cyclooxygenase and lipoxidase activities. Biochemical Pharmacology. 32 : 2283-2286.
Gallagher R., Collins S., Trujillo J., McCredie K., Ahearn M., Tsai S., Metzgar R., Aulakh G., Ting R., Ruscetti F., Gallo R. (1979) Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood. 54 : 713-733.
Galley HF., Davies MJ., Webster NR. (1996) Ascorbyl radical formation in patients with sepsis: effect of ascorbate loading. Free Radical Biology ＆ Medicine. 20 (1) : 139-143.
Gao D., Sakurai K., Chen J., Ogiso T. (1995) Protection by baicalein against ascorbic acid-induced lipid peroxidation of rat liver microsomes. Research Communications in Molecular Pathology and Pharmacology. 90 (1) : 103-114.
Garle MJ., Knight A., Downing AT., Jassi KL., Clothier RH., Fry JR. (2000) Stimulation of dichlorofluorescin oxidation by capsaicin and analogues in RAW 264 monocyte/macrophages: lack of involvement of the vanilloid receptor. Biochemical Pharmacology. 59 (5) : 563-572.
Hanlon MC., Seybert DW. (1997) The pH dependent of lipid peroxidation using water-soluble azo initiators. Free radical Biology & Medicine. 23 (5) : 712-719.
Hirano S. (1996) Chitin biotechnology applications. Biotechnology Annual Review. 2 : 237-258.
Illum L. (1998) Chitosan and its use as a pharmaceutical excipient. Pharmaceutical Research. 15 (9) : 1326-1331
Ishii M., Shimizu S., Nawata S., Kiuchi Y., Yamamoto T. (2000) Involvement of reactive oxygen species and nitric oxide in gastric ischemia-reperfusion injury in rats: protective effect of tetrahydrobiopterin. Digestive Diseases & Sciences. 45 (1) : 93-98.
Kelley EE., Wagner BA., Buettner GR., Burns CP. (1999) Nitric oxide inhibits iron-induced lipid peroxidation in HL-60 cells. Archives of Biochemistry and Biophysics. 370 : 97-104.
Klokkevold PR., Vandemark L., Kenney EB., Bernard GW. (1996) Osteogenesis enhanced by chitosan (poly-N-acetyl glucosaminoglycan) in vitro. Journal of Periodontology. 67 (11) : 1170-1175.
Lahiri-Chatterjee M., Katiyar SK., Mohan RR., Agarwal R. (1999) A flavonoid antioxidant, silymarin, affords exceptionally high protection against tumor promotion in the SENCAR mouse skin tumorigenesis model. Cancer Research. 59 (3) : 622-632.
Lavelli V., Hippeli S., Peri C., Elstner EF. (1999) Evaluation of radical scavenging activity of fresh and air-dried tomatoes by three model reactions. Journal of Agricultural & Food Chemistry. 47 (9) : 3826-3831.
Leal AM., Begona Ruiz-Larrea M., Martinez R., Lacort M. (1998) Cytoprotective actions of estrogens against tert-butyl hydroperoxide-induced toxicity in hepatocytes. Biochemical Pharmacology. 56 : 1463-1469.
Leprat P., Ratinaud MH., Maftah A., Petit JM., Julien1 R. (1990) Use of nonyl acridine orange and rhodamine 123 to follow biosynthesis and functional assembly of mitochondrial membrane during L1210 cell cycle. Experimental Cell Research. 186 : 130-137.
Li W., Li Z., Liao W., Feng XD. (1993) Chemical modification of biopolymers--mechanism of model graft copolymerization of chitosan. Journal of Biomaterials Science, Polymer Edition. 4 (5) : 557-566.
Ma Y., Ogino T., Kawabata T., Li J., Eguchi K., Okada S. (1999) Cupric nitrilotriacetate-induced apoptosis in HL-60 cells association with lipid peroxidation, release of cytochrome c from mitochondria, and activation of caspase-3. Free Radical Biology ＆ Medicine. 27 (1-2) : 227-233.
Machlin LJ., Bendich A. (1987) Free radical tissue damage: protective role of antioxidant nutrients. FASEB Journal. 1 (6) : p441-445.
Mattiasson B., Kumar A., Galaev IY. (1998) Affinity precipitation of proteins: design criteria for an efficient polymer. Journal of Molecular Recognition. 11 (1-6) : 11-16.
Maulik G., Kassis AI., Savvides P., Makrigiorgos GM. (1998) Fluoresceinated phosphoethanolamine for flow-cytometric measurement of lipid peroxidation. Free Radical Biology & Medicine. 25 (6) : 645-653.
McCord JM. (1987) Oxygen-derived radicals: a link between reperfusion injury and inflammation. Federation Proceedings. 46 : 2402-2412.
Miles PR., Wright JR., Bowman L., Colby HD. (1980) Inhibition of hepatic microsomal lipid peroxidation by drug substrates without drug metabolism. Biochemical Pharmacology. 29 : 565-570.
Minotti G. (1993) Sources and role of iron in lipid peroxidation. Chemical Research in Toxicology. 6 : 134-146.
Minotti G., Aust SD. (1992) Redox cycling of iron and lipid peroxidation. Lipids. 27 (3) : 219-226.
Multhaup G., Ruppert T., Schlicksupp A., Hesse L., Beher D., Masters CL. Beyreuther K. (1997) Reactive oxygen species and Alzheimer's disease. Biochemical Pharmacology. 54 (5) : 533-539.
Noro T., Oda Y., Miyase T., Ueno A., Fukushima S. (1983) Inhibitors of xanthine oxidase from the flowers and buds of Daphne genkwa. Chemical & Pharmaceutical Bulletin. 31 (11) : 3984-3987.
Okada S. (1996) Iron-induced tissue damage and cancer: The role of reactive oxygen species-free radicals. Pathology International. 46 : 311-332.
Onishi H., Machida Y. (1999) Biodegradation and distribution of water-soluble chitosan in mice. Biomaterials. 20 : 175-182.
Ormrod DJ. ,Holmes CC., Miller TE. (1998) Dietary chitosan inhibits hypercholesterolaemia and atherogenesis in the apolipoprotein E-defient mouse model of atherosclerosis. Atherosclerosis. 138 : 329-334.
Pick E., Keisari Y. (1980) A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. Journal of Immunological Methods. 38 : 161-170.
Reddy AC., Lokesh BR. (1992) Studies on spice principles as antioxidants in the inhibition of lipid peroxidation of rat liver microsomes. Molecular & Cellular Biochemistry. 111 (1-2) : 117-124.
Rezazadeh H., Athar M. (1998) Effect of iron overload on the benzoyl peroxide-mediated tumor promotion in mouse skin. Cancer Letters. 126 (2) : 135-142.
Richardson DR. (1997) Potential of iron chelators as effective antiproliferative agents. Can. J. Physiol. Pharmacol. 75 : 1164-1180.
Richardson SC., Kolbe HV., Duncan R. (1999) Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA. International Journal of Pharmaceutics. 178 : 231-243.
Robak J., Gryglewski RJ. (1987) Flavonoids are scavengers of superoxide anions. Biochemical Pharmacology. 37 (5) : 837-841.
Roller S., Covill N. (1999) The antifungal properties of chitosan in laboratory media and apple juice. International Journal of Food Microbiology. 47 (1-2) : 67-77.
Sato T., Kawamoto A., Tamura A., Tatsumi Y., Fujii T. (1992) Mechanism of antioxidant action of pueraria glycoside (PG)-1 (an isoflavonoid) and mangiferin (a xanthonoid). Chemical & Pharmaceutical Bulletin. 40 (3) : 721-724.
Schaich KM. (1992) Metals and lipid oxidation. contemporary issues. Lipid. 27 (2) : 209-218.
Shimojoh M., Fukushima K., Kurita K. (1998) Low-molecular chitosans derived from β-chitin: preparation, molecular characteristics and aggregation activity. Carbohydrate Polymers. 35 : 223-231.
Sugano M., Watanabe S., Kishi A., Izume M., Ohtakara A. (1988) Hypocholesterolemic action of chitosans with different viscosity in rats. Lipids. 23 (3) : 187-191.
Tanaka M., Anzai S., Takeno K., Nakagawa M. (1994) Antioxidant action of thiopalmitic acid on microsomal lipid peroxidation. Chemical & Pharmaceutical Bulletin. 17 (9) : 1151-1154.
Thierry G., Arthur IC. (1999) NADPH-dependent microsomal electron transfer increases dedradation of CYP2E1 by the proteasome complex: role of reactive oxygen species. Archives of Biochemistry and Biophysics. 370 (2) : 258-270.
Vahouny GV., Satchithanandam S., Cassidy MM., Lightfoot FB., Furda I. (1983) Comparative effects of chitosan and cholestyramine on lymphatic asorption pf lipids in the rat. The American Journal of Clinical Nutrition. 38 : 278-284.
Wagner BA., Buettner GR., Oberley LW., Burns CP. (1998) Sensitivity of K562 and HL-60 cells to edelfosine, an ether lipid drug, correlates with production of reactive oxygen species. Cancer Research. 58 : 2809-2816.
Willmore LJ. and Willian JT. (1991) Iron-induced lipid peroxidation and brain injury responses. Integrative Physiological & Behavioral Science 9 (2) : 175-180.