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研究生:林友梅
研究生(外文):You-Mei
論文名稱:以Balb/c小鼠為模式探討三種不同分子量之蒟蒻水解物對直結腸癌危險因子的影響
論文名稱(外文):Modulatory effects of three konjac hydrolysate fractions on risk factors of colon cancer in Balb/c mice
指導教授:陳曉鈴陳曉鈴引用關係
指導教授(外文):Hsiao-Ling Chen
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:營養學研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:91
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蒟蒻主要成分是葡甘聚醣(glucomannan),是一種可溶性纖維;由D-葡萄糖和D-甘露糖以ß-1,4醣甘鍵鍵結而成。本研究目的將比較未水解蒟蒻纖維、三種不同分子量之蒟蒻纖維部分水解物(平均聚合度為16,8,4)對大腸癌之預測生物指標(如糞便水液成分對人類大腸腺癌細胞株產生之毒性)及影響這些作用的因素(如糞便pH、短鏈脂肪酸濃度及致癌相關細菌酵素活性等)的調控作用。
將六週齡之Balb/c小鼠隨機分配,每組12隻,分別餵食不含纖維及含5%不同膳食纖維(Cellulose,C;Pectin,P;Konjac glucomannan,K;Konjac glucomannan hydrolysis fraction1、fraction 2、fraction 3,F1、F2、F3;Inulin,I;及Fructooligosaccharides,FO)實驗飲食21天。犧牲前三天(第18-20天)連續收集72小時糞便並馬上冷凍保存。隔夜禁食後第22天犧牲,採集血液及大腸,並秤取肝臟及盲腸內容物重冷凍儲存於-20℃冰箱。
結果顯示飼料攝取量以FO組較高,食物利用率方面則是以K組利用率最低,然而在體重增加方面各組間並無差異。相對器官重部份:相對肝臟重以FF組顯著高於其他各組,相對盲腸重部份則是以FO組最高,每日排便乾重部份,除FF組外攝取其他纖維皆可增加糞便乾重。糞便酵素方面ß-glucosidase以F1組活性最高;ß-galactosidase是以P組活性最高;ß-glucuronidase則是以F2組最高。在糞便pH值及短鏈脂肪酸含量部份,糞便pH值以P組最低與攝食其他膳食纖維的pH值相似,C組最高;在每日短鏈脂肪酸排出量方面,不論是乙酸、丙酸、正丁酸或異丁酸皆以F3組最高;乙酸、正丁酸或異丁酸FF組最低。
在糞便水細胞及DNA傷害測試部分,細胞毒性測試結果顯示;補充任何一種膳食纖維皆可以改善糞便水對細胞之毒性;DNA傷害測試方面,以FF組傷害程度最大,P及I組的DNA傷害程度最低;近一步以H2O2誘發DNA傷害發現,以FF組傷害程度最大,F3組的DNA傷害程度最低。
縱合上述結果顯示,蒟蒻水解物F3(DP≒4)在腸道發酵產生的短鏈脂肪酸最高,且對於改善糞便水對於細胞毒性及DNA傷害效果最顯著。


Konjac, a soluble dietary fiber, is rich in glucomannan polysaccharides which is composed of D-glucose and D-mannose by ß-1,4 glycosidic bond. The purpose of this study was to compare the effect of Konjac(K), Konjac hydrolysate fractions(F1, F2, F3; the degree of polymerization was 16, 8, 4 respectively), inulin(I), fructooligosaccharide(FO), pectin(P)and cellulose(C)on the concentrations of short chain fatty acids(SCFA)in the feces, and the fecal enzyme, fecal water pH, the cytotoxicity and DNA damage on Caco-2 cell.
The 6-wk-old Balb/c mice were randomly allocated to consume AIN-93 control fiber-free(FF), and 5% K, F1, F2, F3, I, FO, P or C diet for three weeks. The feces of mice were collected for three days before sacrifice. The mice were sacrificed on the 22nd day. After sacrificing, the main organs was collected and weighted to analyze.
There were no differences in body weight gain across groups. FO group has the highest daily food intake, and K group has the lowest food efficiency. FF group has the highest liver relative weight, and FO group has the highest cecum relative weight. All of the fiber used in this study can increase the daily feces dry weight.
On the fecal enzyme, F1 group has the highest ß-glucosidase activation; P group has the highest ß-galactosidase activation; F2 group has the highest ß-glucuronidase activation. Compare the fecal SCFA, F3 group has the highest concentrations of fecal acetate, propionate, and butyrate.
In the cytotoxicity on Caco-2 cell, all of the fiber used in this study can improve the fecal water induce cell toxicity. In the DNA damage on Caco-2 cell, P and I group has the lowest DNA damage. After using the H2O2 inducing DNA damage, F3 group has the best effect on prevent DNA from H2O2 damage.
In conclusion, the Konjac hydrolysate fractions 3(DP≒4)could increase the concentrations of fecal short chain fatty acids, and prevent the Caco-2 cell from fecal water damage.


中文摘要………………………………………………………………………………I
Abstract ………………………………………………………………………………II
第壹章、前言…………………………………………………………………………1
第貳章、文獻探討……………………………………………………………………2
第一節 膳食纖維 ……………………………………………………………………2
一、膳食纖維之定義…………………………………………………………………2
二、膳食纖維之分類…………………………………………………………………2
三、本研究所探討之膳食纖維………………………………………………………3
第二節 寡醣 …………………………………………………………………………7
一、寡醣之定義………………………………………………………………………7
二、寡醣種類…………………………………………………………………………7
第三節 難消化性碳水化合物之生理功能 …………………………………………8
一、產生短鏈脂肪酸…………………………………………………………………8
二、調節腸道菌相……………………………………………………………………8
三、影響胃排空的時間………………………………………………………………9
四、增加糞便體積……………………………………………………………………9
五、增加糞便的水分…………………………………………………………………10
六、促進黏膜細胞分化………………………………………………………………10
七、促進腸道蠕動……………………………………………………………………10
八、輔助治療或預防發炎性腸疾病…………………………………………………11
九、預防直結腸癌……………………………………………………………………11
第四節 難消化性碳水化合物聚合度對生理功效影響……………………………12
一、果寡醣與菊醣之生理功能比較…………………………………………………12
二、膳食纖維與其部分水解物功效之比較…………………………………………12
第五節 難消化性碳水化合物降低直結腸癌之可能機……………………………13
一、直結腸癌發生之步驟……………………………………………………………13
二、難消化性碳水化合物與直結腸癌………………………………………………14
(一)促進腸道蠕動及排便…………………………………………………………14
(二)產生短鏈脂肪酸………………………………………………………………15
(三)提高腸道益生菌………………………………………………………………17
(四)降低與致癌相關之細菌酵素…………………………………………………17
(五)降低糞便二級膽酸之比例或含量……………………………………………18
(六)抗氧化作用……………………………………………………………………18
(七)調節免疫力……………………………………………………………………19
(八)改善發炎性腸疾病……………………………………………………………19
(九)改變腸道細菌代謝途徑………………………………………………………19
第六節 飲食對糞便水液毒性之影響………………………………………………20
一、直結腸癌的危險指標……………………………………………………………20
二、彗星影像分析……………………………………………………………………20
第參章、研究目的……………………………………………………………………21
第肆章、材料與方法…………………………………………………………………22
第一節 實驗動物及飼料配方………………………………………………………22
一、實驗動物 ………………………………………………………………………22
二、實驗飼料 ………………………………………………………………………22
(一)動物飼料配方 ………………………………………………………………22
(二)蒟蒻水解產物製備方法 ……………………………………………………22
(三)飼料成分來源 ………………………………………………………………23
第二節 實驗設計……………………………………………………………………26
第三節 分析測定方法………………………………………………………………26
一、不同膳食纖維醣類聚合度之測定 ……………………………………………26
(一)總醣含量測定 ………………………………………………………………26
(二)還原醣含量測定 ……………………………………………………………27
(三)聚合度計算 …………………………………………………………………28
(四)所需試劑 ……………………………………………………………………28
二、糞便收集與冷凍乾燥 …………………………………………………………29
(一)糞便收集 ……………………………………………………………………29
(二)含水率分析 …………………………………………………………………29
三、糞便酵素活性之測試 …………………………………………………………29
(一)糞便酵素ß-glucosidase、ß-galactosidase、ß-glucuronidase酵素活性測定方法 ……………………………………………………………………………………29
(二)所需試劑………………………………………………………………………30
四、糞便短鏈脂肪酸的萃取及分析…………………………………………………30
(一)糞便水pH值測定……………………………………………………………30
(二)糞便短鏈脂肪酸的萃取方法…………………………………………………31
(三)測定方法………………………………………………………………………31
(四)所需試劑………………………………………………………………………32
五、糞便水製備………………………………………………………………………32
六、糞便水細胞DNA毒性試驗……………………………………………………32
(一)細胞實驗………………………………………………………………………32
(二)細胞存活率……………………………………………………………………34
(三)DNA傷害分析 ………………………………………………………………34
(四)所需試劑與儀器………………………………………………………………35
第四節 常用儀器及藥品……………………………………………………………36
一、常用儀器…………………………………………………………………………36
二、常用藥品…………………………………………………………………………37
第伍章、統計方法……………………………………………………………………38
第陸章、結果…………………………………………………………………………39
第一節 不同聚合度之蒟蒻水解產物………………………………………………39
第二節 不同膳食纖維對小鼠平均增重、攝取量及食物利用率的影響…………40
第三節 不同膳食纖維對小鼠相對器官重的影響…………………………………40
第四節 不同膳食纖維對小鼠每日排便量及含水率之影響………………………46
第五節 不同膳食纖維對小鼠的糞便酵素影響……………………………………48
第六節 不同膳食纖維對小鼠糞便水pH值之影響 ………………………………51
第七節 不同膳食纖維對小鼠的糞便短鏈脂肪酸影響……………………………54
第八節 餵食不同膳食纖維對小鼠糞便水之細胞毒性……………………………60
第九節 餵食不同膳食纖維小鼠糞便水對DNA傷害 ……………………………67
第柒章、討論…………………………………………………………………………74
第一節 不同聚合度之蒟蒻水解物製備方法………………………………………74
第二節 不同膳食纖維對小鼠平均增重、攝取量、食物利用率及器官探討……75
第三節 不同膳食纖維對小鼠每日排便量、含水率及糞便細菌酵素活性探討…76
第四節 不同膳食纖維對小鼠糞便水pH值及短鏈脂肪酸探討 …………………78
第五節 餵食不同膳食纖維對小鼠糞便水之細胞及DNA傷害探討 ……………79
第捌章、結論 ………………………………………………………………………82
第玖章、參考文獻 …………………………………………………………………83
附錄一、餵食不同膳食纖維對小鼠糞便水DNA傷害情形之彗星影像攝影……89
附錄二、以H2O2誘發DNA傷害觀察餵食不同膳食纖維對Caco-2 cell DNA傷害情形之彗星影像攝影 ……………………………………………………………90
附錄三、以Trolox與細胞共同培養觀察Fiber-free糞便水對Caco-2 cell DNA傷害情形之彗星影像攝影 ……………………………………………………………91


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