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研究生:曾健育
研究生(外文):Chien-Yu Tseng
論文名稱:探討紅豆及納紅豆之抗氧化性
論文名稱(外文):Study on the antioxidant activies of red bean and natto-red bean
指導教授:鍾雲琴
指導教授(外文):Yun-Chin Chung
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
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:2010
畢業學年度:98
語文別:中文
論文頁數:97
中文關鍵詞:納紅豆抗氧化
外文關鍵詞:natto-red beanantioxidant activies
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近年來研究發現,紅豆中的多酚化合物具有降低血壓及抗氧化之效果,且有研究指出,紅豆之50 %酒精萃取物,其清除α,α-diphenyl-β-picrylhydrazyl (DPPH)自由基的效果較其他種豆類 (綠豆、黑豆、黃豆及碗豆)佳。本研究旨在探討紅豆經Bacillus subtilis (枯草桿菌) 發酵後(稱之為納红豆)其水萃物的抗氧化及對人類肝癌細胞Hep G2細胞生長之影響。高雄五號紅豆以Bacillus subtilis於30℃發酵96小時後,評估其水萃物之DPPH自由基清除力、還原力、二價鐵離子螯合力等體外抗氧化活性;此外,本研究亦將納紅豆水萃物模擬人體腸胃道酵素作用後評估其抗氧化能力。納紅豆對體內抗氧化能力之影響,則是以Sprague-Dawley rats (SD) 雄鼠為動物模式,分為五組,分別為控制組 (去離子水)、紅豆低劑量 (0.5 g/kg BW) 及高劑量組 (1 g/kg BW) 與納紅豆低劑量 (0.5 g/kg BW) 及高劑量組 (1 g/kg BW),以管灌方式連續餵食28天後犧牲,進行體內酵素性抗氧化活性及非酵素性抗氧化物質濃度變化分析。最後以Hep G2細胞測試納紅豆水萃物對其細胞存活率之影響。本研究結果顯示,以B. subtilis 30℃發酵96小時之納紅豆水萃物對DPPH自由基清除能力與還原力較未發酵之紅豆高,但二價鐵離子螯合力則會降低;將納紅豆模擬人體腸胃道消化酵素作用後,其DPPH清除力及還原力之效果不受消化酵素影響,且經消化酵素作用後會提升二價鐵螯合力。體內抗氧化試驗結果顯示,餵食納紅豆具有增加肝臟CAT酵素活性之功能 ( p < 0.05);且經過納豆菌發酵之納紅豆,有較未發酵之紅豆在腦部維生素 C、維生素 E、GSH含量以及心臟之CAT 、GR酵素活性更高的趨勢,但在統計上無顯著差異 ( p > 0.05) ;而在心臟SOD、GR、GSH-Px酵素活性、肝臟GSH-Px酵素活性以及心臟、肝臟及腦部組織之GSSG,餵食納紅豆與紅豆組之趨勢相同,且在統計上二者無顯著差異 ( p >0.05)。此結果顯示納紅豆可提升動物體內之抗氧化能力,但其效果可能是經由攝食紅豆所提供,與納豆菌發酵並無關係。納紅豆水萃物與Hep G2細胞共培養30小時後,Hep G2細胞存活率會隨著納紅豆水萃物濃度的增加而增加,此結果顯示納紅豆水萃物具有促進Hep G2細胞生長之效果,但是否能夠促進正常肝細胞生長之效果則需進一步實驗測試。
The hypotension and antioxidant effects of polyphenol compounds extracted from red bean had been demonstrated. Previous studies also showed that 50% alcoholic extract of red bean obtained the greatest α,α-diphenyl-β-picrylhydrazyl (DPPH) scavenge effect compared to green bean, black bean, soy bean and garden pea. This study aimed to investigate the antioxidant activities and human hepatoma cells (Hep G2) proliferation effect of water extract from Bacillus subtilis fermented red bean (natto-red bean, NRB). The Kaohsiung NO. 5 red bean were fermentated by B. subtilis at 30℃ for 96hr, and then the antioxidant activities, including DPPH radical scavenging ability, ferrous iron chelating ability and reducing power, of water extract from NRB were determined in vitro. To simulate human gastrointestinal digestion, the antioxidant capacities of water extract from NRB were further assessed after enzymatic treatment. On the other hand, Sprague-Dawley rats (SD) male rats were used as animal model for antioxidant activity study in vivo. SD rats were divided into 5 groups, including the control (distilled water ), low dose (0.5 g/kg BW) of red bean, high dose (1 g/kg BW) of red bean, low dose (0.5 g/kg BW) of NRB, and high dose (1 g/kg BW) of NRB. The activities of antioxidant enzymes and the levels of antioxidant in SD rats were evaluated after oral administration of tested samples for 28 days. For the last part of this study, the effect of water extract from NRB on the growth of Hep G2 was estimated. The results of this study showed that NRB had greater DPPH scavenging effect and reducing power than unfermented red bean, however, ferrous ion chelating effect was lower in NRB than red bean. Gastrointestinal enzyme digestion raised NRB’s chelating ability on ferrous ion but did not change NRB’s DPPH scavenging effect nor reducing power. Animal study showed oral administration of NRB could increase significantly the vit C and E concentrations and catalase activity in liver ( p < 0.05). Compared to unfermented red bean, NRB groups had higher catalase activity and GR activity in hearts and Vit C, Vit E and glutathione concentrations in brains, but these differences were not significant ( p > 0.05). NRB and red bean groups had the same tendency to increase the SOD, GSH-Px and GR activities in hearts, GSH-Px activity in liver, and GSSG level in heart, liver and brain, but again, these changes had no statistic difference ( p > 0.05). In vivo study showed that B. subtillius fermentation did not raise the antioxidant effects of red beans. Growth of Hep G2 cells was stimulated by water extract of NRB after a 30 hr-incubation, and effect of water extract of NRB on the cell proliferation was in a dose-dependent matter. These results suggested the water extract of NRB had ability to stimulate the growth of Hep G2 cells, therefore, further study is needed to clarify whether the normal liver cells can be affected by the water extract of NRB as well.
目錄
頁次
目錄...........................................................................................................I
圖目錄.......................................................................................................V
表目錄....................................................................................................VII
中文摘要...............................................................................................VIII
英文摘要...................................................................................................X
I、 前言.................................................................................................1
II、 文獻回顧......................................................................................3
A. 氧化壓力之簡介.....................................................................3
a. 自由基的種類.................................................................4
b. 抗氧化物質.....................................................................8
1. 酵素性抗氧化物質........................................................9
2. 非酵素性抗氧化物質..................................................10
B. 納豆之保健功效...................................................................12
a. 納豆簡介.......................................................................12
1. 納豆菌 ( Bacillus natto ).............................................12
2. 納豆 (natto).................................................................12
b. 納豆之生理活性...........................................................13
1. 納豆激酶 (nattokinase).............................................13
2. 抗菌效果....................................................................13
3. 抗氧化效果................................................................13
C. 紅豆之保健功效..................................................................14
a. 紅豆簡介......................................................................14
b. 紅豆之生理活性..........................................................14
III、 研究目的....................................................................................16
IV、 材料與方法................................................................................17
A. 實驗架構...............................................................................17
B. 樣品來源與製備...................................................................17
a. 紅豆製備.......................................................................17
b. 固態發酵.......................................................................17
c. 納紅豆水萃液製備.......................................................17
C. 體外抗氧化試驗...................................................................19
a. 納紅豆水萃液之體外抗氧化能力...............................19
1. DPPH自由基清除能力................................................19
2. 二價鐵離子螯合能力...................................................20
3. 還原力...........................................................................22
b. 體內消化酵素作用後納紅豆水萃液之抗氧化能力...23
1. DPPH自由基清除能力............................................23
2. 二價鐵離子螯合能力...............................................23
3. 還原力.......................................................................23
D. 體內抗氧化試驗...................................................................25
a. 動物實驗設計...............................................................25
1. 老鼠品系....................................................................25
2. 實驗組別....................................................................25
3. 飼養場所與條件........................................................25
4. 試驗物質給予途徑....................................................26
5. 實驗劑量與給予其間................................................26
6. 動物犧牲方法…………………………..…………..26
7. 犧牲後臟器處理與保存………………..…………..26
b. 動物體內酵素性抗氧化表現……………….……..…26
1. 超氧歧化酶 (Superoxide dismutase, SOD) 活性測定……………………………………………………...26
2. 觸酶 ( Catalase, CAT) 活性測定………….…..…27
3. 麩胱甘肽過氧化酶 ( Glutathione peroxidase,
GSH-Px) 活性測定………………………...……28
4. 麩胱甘肽還原酶 (Glutathione reductase, GR) 活性
測定…………………………………………..….29
c. 體內非酵素性抗氧化表現……………………………30
1. 丙二醛 (Malondialdehyde, MDA) 濃度分 析…30
2. 還原型麩胱甘肽 (GSH) 與氧化型麩胱甘肽 (GSSG) 濃度分析…….………………...…….……31
3.維生素C濃度分析…………………………...……33
4.維生素E濃度分析……………………………...…34
5. 總蛋白濃度分析……………………………..……35
E. 納紅豆水萃液對人類肝癌細胞株 (Hep G2) 生長之影響………………………....…………………………………37
a. 人類肝癌細胞株 (Hep G2) 之細胞培養、繼代、保存及解凍條件………………………....……………………37
b. Hep G2細胞之生長曲線測定………………..………..38
c. 納紅豆對Hep G2細胞毒性測試…….…………….…38
F、 統計分析…………………….…………….…….……..…39
V、 結果與討論.………………………………………………………40
A、 納紅豆、紅豆水萃物之體外抗氧化能力比較………...……40
a. DPPH自由基清除能力…………………………….…...……40
b. 二價鐵離子螯合力……………………………………………40
c. 還原力…………………………………………………………41
d. 紅豆、納紅豆水萃物之體外抗氧化能力比較…………….…41
B、 消化酵素作用後納紅豆與紅豆之體外抗氧化能力之比較…46
a. 消化酵素作用後納紅豆、紅豆之DPPH自由基清除力…...46
b. 消化酵素作用後納紅豆、紅豆之二價鐵離子螯合力…........47
c. 消化酵素作用後納紅豆、紅豆還原力………………………47
d. 經消化酵素作用後之紅豆、納紅豆水萃物體外抗氧化能力比較………………………………………………………………47
C、 納紅豆、紅豆對於動物體內抗氧化活性之影響……………53
a. 納紅豆、紅豆對於動物體內酵素性抗氧化活性表現之影響..53
b. 納紅豆、紅豆對於動物體內非酵素性抗氧化活性表現之影響……………………………………………………………...54
c. 納紅豆、紅豆對於動物體內抗氧化系統之影響……....……55
D、 不同濃度納紅豆對Hep G2細胞毒性測試…….……………68
VI、結論…………………………………………………………..……71
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