Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51757
DC FieldValueLanguage
dc.contributor.advisor柯文慶zh_TW
dc.contributor.advisorMiao-Lin Huen_US
dc.contributor.advisor胡淼琳zh_TW
dc.contributor.author劉禧賢zh_TW
dc.contributor.authorHsien, Liu Hsien_US
dc.date2003zh_TW
dc.date.accessioned2014-06-06T08:54:51Z-
dc.date.available2014-06-06T08:54:51Z-
dc.identifier.urihttp://hdl.handle.net/11455/51757-
dc.description.abstract本研究以市售海參黃光參(Metriatyla scabra)為材料,分離與鑑定其內含機能性成分蛋白多醣(glycosaminoglycan, GAG),藉由腸道細胞(intestine 407)模式系統與添加致突變劑(4NQO與MNNG)探討其抗氧化與抗致突變之能力。此外,應用動物試驗分析,配合高膽固醇飲食與管餵GAG,評估海參體GAG對降膽固醇之功用與可能之作用機制。試驗結果如下: 1.乾海參之基本組成以重量百分率計,水分3%、蛋白質88%、碳水化合物 4%、脂質 2%、灰分 3%;復水後依次為 95%、4%、0.2%、0.1%、0.1%。依水、鹼、酸、熱處理之順序分畫海參體壁中之膠原蛋白,其含量分別為42%、6%、48%、2%,其中以水溶性(佔 42%)及酸溶性 (佔 48%)者居多,合佔總量之 90%。且以Type I與II collagen為主。 2.市售黃光參經由papain分解後所得之crude PG經離子交換管柱與膠體過濾層析,可分劃出2個peak (peak-1與peak-2),其分子量分別為200~500, 40~200 kDa,進一步分析內在成分發現,peak-1(GAG)含hexuronic acid、hexosamine為主,而peak-2(free glycan) 內含量以fucan為主。 3.單一細胞膠體電泳分析顯示,海參體蛋白多醣包括crude PG, GAG, free glycan等均可降低細胞DNA受傷指數;分析其抗氧化能力發現因具有清除DPPH自由基、超氧陰離子、清除過氧化氫以及螯合鐵與銅金屬離子的能力等抗氧化能力,因此可清除致突變劑所生成自由基與活性氧,進而保護細胞DNA。硝酸鹽還原測試,結果發現海參體蛋白多醣對亞硝基化合物不具有還原能力,可證明蛋白多醣對致突變劑4NQO與MNNG具有抑制與抗突變之效果。 4.動物試驗分析結果得知,海參體蛋白多醣GAG可降低大鼠血漿中總膽固醇、LDL-膽固醇濃度、肝臟與血漿過氧化程度及血清中抗氧化酵素活性SOD, GSH-Px活性。此影響與GAG之管餵量有關,在管餵量高於20mg/kg b.w.,隨著GAG濃度增加而有下降趨勢。 5.經由血清GSH與GSSG分析,餵食1%膽固醇之大鼠血清中GSH有顯著下降情況,但隨著餵食GAG含量增加而有上升趨勢,然而血清中GSSG其變化趨勢與GSH相同,由GSSG/GSH之比值可知管餵海參體GAG對於降低生物體內氧化壓力之途徑與GSH系統的相關性並不明顯。此外,餵食1%膽固醇與海參體GAG大鼠其糞便中膽酸排出並無顯著影響(p>0.05),因此推斷,GAG可能藉由其中影響脂質代謝或合成的途徑,達到降低血清與肝臟中脂質濃度。zh_TW
dc.description.abstractGlycosaminoglycan (GAG), recognized as a functional component with antioxidant, antimutagenic and hypolipidemic activities, was isolated and identified from body wall of sea cucumber Metriatyla scabra. Antioxidative and antimutagenic activities were investigated by using intestine 407 cell model system and additing of 4NQO (4-nitroquinoline-1-oxide) and MNNG (N-methyl-N-nitroso-guanidine)as mutagenic agents. Hypolipidemic effects and mechanism of the GAG were also evaluated through cholesterol supplemented diet with oral administration of GAG to rats. The results are: 1. Compositions of dried sea cucumber were: moisture 3%, protein 88%, carbohydrate 4%, lipid 2%, ash 3%; after rehydration they were 95%, 4%, 0.2%, 0.1%, 0.1%, respectively. Extraction in the order of water, alkali solution, acidic solution, and hot water indicates that major collagen contains in sea cucumber body wall were water-soluble (42%) and acid-soluble (48%), i.e., Type I and Type II collagens. 2. Two peaks (peak-1 and peak-2) were observed for the sea cucumber hydrolyzed with papain and followed by ion-exchange and gel-filtration chromatography. Peak-1 M.W. 200-500kDa, contained mainly GAG as hexuronic acid and hexosamine, while peak-2 M.W. 40-200kDa, contained mostly free glycan as fucose with little hexuronic acid or hexosamine. The peak-1 fraction was used to evaluate hypolipidemic effects. 3. Single cell gel electrophoresis (comet assay) shows that crude proteoglycan (crude PG), GAG (peak-1) and free glycan exhibited DNA damage, which may result from good antimutagenic effects, inducted by 4NQO and MNNG. GAG exhibited antioxidant capacity including scavenging of DPPH free radical, H2O2, superoxide anion and chelating Fe++ and Cu++. No ability for reduction of nitrate further verified the inhibition and antimutagenic activity. 4. Oral administration of GAG to male Wister rats shows that plasma levels of total cholesterol, LDL-cholesterol and atherogenic index, SOD, GSH-Px activity in plasma were significantly decreased, while HDL-cholesterol was significantly increased, and plasma and hepatric tissue MDA concentraction, although these effects of the GAG were only dose-dependent at dose higher than 20mg/kg b.w. Similarly, the GAG significantly prevented the increase (p<0.05) in hepatic contents of triglyceride, cholesterol and phospholipid. 5. GAG increased serum GSH levels. Howerever, the relationship between antioxidant activities and GSH levels was not obvious. Furthermore, the GAG did not significantly prevent the increase of bile acid in the fecal. Sea cucumber GAG probably was identified to attain the reduction of plasma and hepatic cholesterol.en_US
dc.description.tableofcontents中文摘要---------------------------------------------------------------------------------------------------- 1 英文摘要---------------------------------------------------------------------------------------------------- 3 研究動機與目的----------------------------------------------------------------------------------------- 5 研究背景---------------------------------------------------------------------------------------------------- 7 一.海參簡介----------------------------------------------------------------------------------------- 7 二.海參採集與乾燥品製造----------------------------------------------------------------------- 7 三.蛋白多醣簡介----------------------------------------------------------------------------------- 9 四.蛋白多醣之抗致突變與抗氧化性----------------------------------------------------------- 12 五.蛋白多醣與膽固醇及高脂血症-------------------------------------------------------------- 14 壹、 研究架構--------------------------------------------------------------------------------------- 18 貳、材料與方法 一.實驗材料--------------------------------------------------------------------------------------- 19 二.實驗試藥--------------------------------------------------------------------------------------- 19 三.實驗方法--------------------------------------------------------------------------------------- 21 (一)、海參復水與成分分析 1. 海參前處理與復水-------------------------------------------------------------------- 21 2. 彈性測定--------------------------------------------------------------------------------- 21 3. 重金屬含量分析------------------------------------------------------------------------ 21 4. 成分分析 4.1. 基本組成--------------------------------------------------------------------------- 22 4.2. 膠原蛋白--------------------------------------------------------------------------- 22 4.3. 胺基酸------------------------------------------------------------------------------ 22 4.4. Disk- Electrophoresis------------------------------------------------------------- 22 (二)、蛋白多醣分離與萃取 1. 酵素分解--------------------------------------------------------------------------------- 23 2. 管柱層析--------------------------------------------------------------------------------- 23 2.1. DEAE-cellulose chromatography----------------------------------------------- 23 2.2. Sephacyl S-400 chromatography------------------------------------------------ 23 3. 蛋白多醣成分分析 3.1. Hexuronic acid-------------------------------------------------------------------- 24 3.2. Hexosamine------------------------------------------------------------------------ 24 3.3. Fucose------------------------------------------------------------------------------ 24 3.4.胺基酸分析------------------------------------------------------------------------ 24 3.5.蛋白質分析------------------------------------------------------------------------ 24 3.6.電泳分析 a. Agarose Gel Electrophoresis------------------------------------------------ 24 b. 連續式膠體電泳(SDS-PAGE)------------------------------------------- 25 (三)、海參體蛋白多醣抗致突變與抗氧化分析 1. 細胞試驗 1.1. 細胞株來源----------------------------------------------------------------------- 25 1.2. 細胞株解凍與培養-------------------------------------------------------------- 26 1.3. 細胞存活分析(MTT cell viability assay)--------------------------------- 26 1.4. 單一細胞膠體電泳(Single cell electrophoresis;Comet assay)------- 26 2. 海參體蛋白多醣in vitro 抗氧化性 2.1. 清除1,1-diphanyl-2-picryl hydrazyl (DPPH)自由基能力測定----------- 27 2.2. 清除超氧陰離子能力(SOD-like)測定------------------------------------ 27 2.3. 清除過氧化氫(H2O2)能力測定------------------------------------------- 28 2.4. 螯合亞鐵離子能力測定-------------------------------------------------------- 28 2.5. 螯合銅離子能力測定----------------------------------------------------------- 28 3. 不飽和脂肪酸過氧化分析----------------------------------------------------------- 29 4. 細菌株來源 4.1. Salmonella typhimurium TA98, TA100活化與保存------------------------ 30 4.2. 毒性試驗-------------------------------------------------------------------------- 30 4.3. 安氏試驗法(Ames test)------------------------------------------------------- 31 5. 亞硝酸鹽還原能力(Nitrate reaction test)分析---------------------------------- 31 (四)、海參體蛋白多醣降血脂之分析 1. 動物試驗 1.1 動物來源---------------------------------------------------------------------------- 32 1.2 動物分組與飼養------------------------------------------------------------------- 32 1.3. 動物犧牲--------------------------------------------------------------------------- 34 2. 血液分析 2.1. 血漿分離--------------------------------------------------------------------------- 35 2.2. 血漿中脂質分析 a. 血漿總膽固醇含量測定----------------------------------------------------- 35 b. 血漿三酸甘油脂含量測定-------------------------------------------------- 35 c. 血漿磷脂質脂含量測定----------------------------------------------------- 35 d. 血漿高密度脂蛋白膽固醇(HDL-cholesterol)含量測定------------ 36 e. 血漿低密度脂蛋白膽固醇(LDL-cholesterol)含量測定------------- 36 f. 血漿過氧化TBARS分析---------------------------------------------------- 36 2.3. 血清分離 a. GOP指數分析----------------------------------------------------------------- 37 b. GPT指數分析------------------------------------------------------------------ 37 c. BUN含量分析----------------------------------------------------------------- 37 d. creatinine 含量分析---------------------------------------------------------- 38 2.4. 血清抗氧化酵素分析 a. GSH酵素活性分析----------------------------------------------------------- 38 b. GSSG酵素活性分------------------------------------------------------------ 38 3. 肝臟分析 3.1. 肝臟中脂質含量萃取------------------------------------------------------------ 39 3.2. 肝臟中總膽固醇含量分析------------------------------------------------------ 39 3.3. 肝臟中三酸甘油脂含量分析--------------------------------------------------- 39 3.4. 肝臟中磷脂質含量分析--------------------------------------------------------- 40 3.5. 肝臟中抗氧化酵素分析 a. 組織前處理-------------------------------------------------------------------- 40 b. SOD酵素活性分析----------------------------------------------------------- 40 c. GSH-PX酵素活性分析------------------------------------------------------- 41 d. Catalase酵素活性分析------------------------------------------------------- 41 3.6. 肝臟過氧化TBARS分析------------------------------------------------------- 41 4. 糞便分析 4.1. 糞便前處理------------------------------------------------------------------------ 41 4.2. 糞便中膽固醇萃取與分析------------------------------------------------------ 41 4.3. 糞便中總膽酸含量分析--------------------------------------------------------- 42 5. 組織切片--------------------------------------------------------------------------------- 42 6. 統計分析--------------------------------------------------------------------------------- 42 參、結果與討論 (一)、乾海參之復水成分分析 1. 乾海參之復水--------------------------------------------------------------------------- 43 2. 重金屬含量分析------------------------------------------------------------------------ 44 3. 海參之基本成分------------------------------------------------------------------------ 45 4. 海參膠原蛋白之分畫------------------------------------------------------------------ 46 5. 海參膠原蛋白之胺基酸組成與電泳分析------------------------------------------ 46 (二)、海參體蛋白多醣分離與分析 1. 蛋白多醣分離與成分分析------------------------------------------------------------ 50 2. 電泳分析--------------------------------------------------------------------------------- 55 (三)、海參體蛋白多醣抗致突變與抗氧化分析 1. 致突變劑劑量分析--------------------------------------------------------------------- 58 2. 細胞存活分析--------------------------------------------------------------------------- 60 3. 單一細胞膠體電泳分析--------------------------------------------------------------- 61 4. 抗氧化分析------------------------------------------------------------------------------ 66 5. 抗致突變分析(Ames test)----------------------------------------------------------- 68 6. 亞硝酸還原能力分析------------------------------------------------------------------ 50 (四)、海參體蛋白多醣降血脂之分析 1. 雄鼠體重變化與飲食狀況------------------------------------------------------------ 75 2. 血漿脂質濃度分析--------------------------------------------------------------------- 75 3. 血漿TBARS分析---------------------------------------------------------------------- 78 4. 血清中GOP, GPT, BUN 與 creatinine分析--------------------------------------- 78 5. 血清GSH與GSSG分析-------------------------------------------------------------- 81 6. 肝臟脂質含量分析--------------------------------------------------------------------- 83 7. 肝臟中抗氧化酵素SDO, GSH-Px與catalase之分析--------------------------- 83 8. 肝臟過氧化TBARS分析------------------------------------------------------------- 87 9. 糞便膽固醇與膽酸之分析------------------------------------------------------------ 88 10. 肝臟與小腸之組織切片觀察-------------------------------------------------------- 88 肆、結論---------------------------------------------------------------------------------------------- 93 伍、參考文獻--------------------------------------------------------------------------------------- 94zh_TW
dc.language.isoen_USzh_TW
dc.publisher食品科學系zh_TW
dc.subjectsea cucumberen_US
dc.subject海參zh_TW
dc.subjecthypercholesterolemiaen_US
dc.subjectantioxidanten_US
dc.subjectantimutagenicen_US
dc.subjectglycosaminoglycanen_US
dc.subjectproteoglycanen_US
dc.subject抗氧化zh_TW
dc.subject蛋白多醣zh_TW
dc.subject抗突變zh_TW
dc.subject高脂血症zh_TW
dc.titleIsolation of proteoglycan from sea cucumber and investigation on its antioxidant, antimutagenic and hypolipidemic activitiesen_US
dc.title海參體蛋白多醣之分離及其抗氧化、抗致突變性與降血脂之研究zh_TW
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
item.grantfulltextnone-
item.fulltextno fulltext-
item.cerifentitytypePublications-
Appears in Collections:食品暨應用生物科技學系
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