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Title: 不同分子量不同去已醯度幾丁聚醣的抗氧化性質
Antioxidative Properties of Different Molecular Weights, Different Degrees of Deacetylation Chitosan
Authors: 陳榮輝
Contributors: NTOU:Department of Food Science
Keywords: 幾丁聚醣;去已醯度;分子量;抗氧化;結構與活性關係;體外抗氧化方法;纖維母細胞;角質細胞;活性氧物質;細胞增生;細胞活性;細胞膜完整性;脂質過氧化
Chitosan;degree of deacetylation;molecular weight;antioxidative properties;qualitative structure-activity relationship;in vitro antioxidation methods;fibroblasts;keratinocytes;reactive oxygen species;cell proliferation;viability;membrane integrity;lipid peroxidation
Date: 2008-08
Issue Date: 2011-06-28T05:52:08Z
Publisher: 行政院國家科學委員會
Abstract: 摘要:本研究計畫的目地是探討幾丁聚醣的去已醯度或/與分子量影響它的抗氧化性質與 應用。幾丁聚醣因資源豐富,具再生性、有生物分解性不會污染環境、有生物相容性(毒 性低、不會產生抗體等)、有許多功能性質如成膜性、成膠性、在酸性溶液帶正電(抗 菌、吸附、止血)等等,再加上有許多生理活性如降膽固醇、降血脂,降血壓,增加免 疫功能。故在食品、生物技術、醫藥、保健食品都有應用潛力。 最近有文獻報告幾丁聚醣有抗氧化能力而低分子量的幾丁聚醣的抗氧化能力比較 好。以高溫強鹼方法製備幾丁聚醣時,低分子量的幾丁聚醣其去已醯度必然高於高分子 量者。以結構與活性關係的觀點看,帶有正電荷的胺基比沒有帶電荷的胺基較能與電子 結合。與電子結合的反應是還原反應。帶有正電荷的胺基是還原劑,故以結構與活性關 係的觀點推論,去已醯度對氧化反應的影響必然高於分子量的影響。所以本研究擬以三 年的時間來探討幾丁聚醣的去已醯度或/與分子量影響它的抗氧化性質與應用。 第一年的計畫擬以三種不同分子量、兩種不同去已醯度的幾丁聚醣以體外抗氧化方 法來釐清幾丁聚醣的去已醯度或/與分子量對抗氧化性質的影響。三種分子量擬設定在5 x 105、3 x 103、與1 x 103 道爾敦以下,以利依序在乳化食品或化妝品、保健食品、醫 藥上的應用。兩種不同去已醯度則選65%與95%。以體外抗氧化方法如清除α , α- diphenyl-β-picryhydrazyl (DPPH)自由基能力、清除超氧陰離子能力、清除過氧化氫能 力、螯合亞鐵離子能力、清除氫氧離子能力等來判定是否具有抗氧化並可依具不同能力 來設計應用的系統。 第二年的計畫擬以上述之三種不同分子量、兩種不同去已醯度的幾丁聚醣以本研究 室較純熟的纖維母細胞、角質細胞兩種細胞株探討在有活性氧物質(ROS)的壓力下的 保護能力例如以[3H]-thymidine 法測細胞株的增生(proliferation)能力、以MTT 法測細 胞株的活性(viability)保護由ascobate 引發的活性氧物質的細胞毒殺性質、細胞株的 合成fibronectin 的能力。 第三年的計畫擬以上述之三種不同分子量、兩種不同去已醯度的幾丁聚醣以本研究 室較純熟的纖維母細胞、角質細胞兩種細胞株探討在有或沒有紫外線照射下纖維母細 胞、角質細胞兩種細胞株的細胞膜破壞、脂質過氧化(peroxidation)、分泌elastin 與 matrixmetalloproteinase 1(MMP-1)之差異,以瞭解不同分子量、不同去已醯度的幾丁 聚醣對紫外線照射傷害的保護能力。細胞膜破壞以測定乳酸去氫酶的釋放量的多寡來表 示細胞膜破壞程度。以量測methylene blue 衍生物的生成量來表示脂質過氧化的程度, 以量測MMP-1 與elastin 的活性與量來表示真皮層的受紫外線照射傷害的程度。 本計畫的完成將有助於釐清幾丁聚醣的去已醯度或/與分子量影響它的抗氧化性質 以及何種去已醯度或/與分子量的幾丁聚醣在什麼條件下有最好的抗氧化能力。進一步 設計適當去已醯度或/與分子量的幾丁聚醣以應用在食品、生物技術、醫藥、保健食品 等工業上。
Abstract:The objectives of this 3 fiscal years project are to explore the effect of degree of deacetylation and/or molecular weight of chitosan used on its antioxidative properties and applications. Chitosan is a deacetylated derivates of chitn, the 2nd most abundant biopolymer next to cellulose. it is biodegradable and regenerative resource so there is no stress to environment, biocompatible (low toxicity and low antigenicity), have many functional properties such as film or gel forming ability, a polyelectrolytes with positive charge thus facilitate its bactericide, absorption, and hemostat properties, have many biological activities such as lowering the hypocholesterollemia, lowering blood pressure and enhancing immunity. Therefore, chitin chitosan are potential biomaterials could be applied in many foods, biotech, biomedical, nutraceutical foods etc areas. Recent several literatures revealed antioxidative properties of chitosan. They reported low molecular weight (MW) chitosans show better antioxidative properties than the higher MW ones. But most commercial chitosan products are produced with high temperature & high concentration alkali deacetylation process. Lower MW chitosan usually have a high degree of deacetylation (DD). From qualitative structure-activity relationship (QSTA) point of view, a protonated amine interacts with an electron is a reduction reaction. Therefore a molecule with a protonated amine group in solution is itself as a reductant or an antioxidant. Therefore, form QSTA point of view, the effect of DD on antioxidantive property should be a dominant factor than the effect of MW. Therefore, we try to elucidate the effect of degree of deacetylation and/or molecular weight of chitosans used on their antioxidative properties and application. In the first fiscal year, three different MWs, Two different DDs chitosan will be tested their antioxditive properties with in vitro antioxidation methods to reveal the effect of the mentioned two parameters. Three different MWs will be controlled around 5 x 105, 3 x 103, and 1 x 103 Da. The chosen is considered for easier adapted in future applications in emulsifying foods product, cosmetics; immunoadjuvant functional foods, and biomedical carrier respectively. Whereas, two DDs value will be 95% and 65%. The in vitro antioxidantive methods chosen will be scavenging of α , α- diphenyl-β-picryhydrazyl (DPPH) free radical, superoxide radical, or hydrogen peroxide, hydroxyl radical eliminating, metal chelating activity to evaluate the antioxidative properties to suit for the future applications. In the 2nd fiscal year the above mentioned chitosans will be used to protect the fibroblast and keratinocyte under the stress of reactive oxygen species (ROS) generated by Na ascorbate. In the culture medium the concentration of Na ascorbate higher than one mM will show cytotoxicity to to cultured fibroblast or keratinocytes. The antioxdative property of these different chitosans will be tested by proliferation enhancement, cell viability, protection against ascobate-induced cytotoxicity and fibronectin biosynthesis ability. In the 3rd fiscal year, chitosans will be used to protect against membrane damage, lipid peroxidation and express of elastin and MMP-1 of the fibroblasts and kerationocytes exposed to UV radiation. The data will be analysis the protection effect of different DDs and/or different MWs on cell exposured to UV radiation. Membrane integrity will be determined by measuring lactate dehydrogenase release from cell via the LDH assay kit. Lipid peroxidation will be measured by the reaction of lipid hydroperoxides with a methylene blue derivative in the present of hemoglobin to yield methylene blue that can be detected spectrophotometrically. The elastin and matrixmetalloproteinase 1 (MMP-1) protein level will be determined by indirected ELISA. The completion of this three years project will enable us to elucidate the effect of degree of deacetylation and/or molecular weight of chitosan used on its antioxidative properties and which DDs and/or MWs chitosan under what conditions have the optimal antioxidavie property. Furthermore, it will help us to design the appropriate DDs and/or MWs chitosan to apply in foods, biotech, biomedical, nutraceutical foods etc areas.
Relation: NSC97-2313-B019-009
URI: http://ntour.ntou.edu.tw/ir/handle/987654321/8667
Appears in Collections:[食品科學系] 研究計畫

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