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研究生:温淨錞
研究生(外文):WEN, CHING-CHUN
論文名稱:探討納豆菌及乳酸菌共同發酵紅豆 ( Phaseolus angularis ) 萃取物之抗氧化活性及其對人類肝癌細胞生長之影響
論文名稱(外文):Studies on the Antioxidant Activities and the Effects of Bacillus subtilis and Lactobacillus delbrueckii Co-cultured Red Bean ( Phaseolus angularis ) Extract on Cellular Growth of Human Hepatoma Cells
指導教授:周淑姿周淑姿引用關係
指導教授(外文):CHOU, SU-TZE
口試委員:鍾雲琴許成光
口試委員(外文):CHUNG, YUN-CHINHSU, CHENG-KUANG
口試日期:2020-07-22
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養學系
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:2020
畢業學年度:108
語文別:中文
論文頁數:92
中文關鍵詞:納豆菌乳酸菌紅豆人類肝癌細胞株 ( Hep G2 cells )
外文關鍵詞:Bacillus subtilisLactobacillus delbrueckiired beanHep G2 cells
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肝癌為十大癌症死因之一,因此對於肝癌的預防及治療是重要的。紅豆 ( Phaseolus angularis ) 為豆科豇豆屬植物,為台灣一重要全穀物,研究指出各種食用豆類中以紅豆最具良好體外抗氧化活性,故選擇紅豆為發酵基質。發酵菌株選用枯草桿菌 (Bacillus subtilis subsp. subtilis) 與保加利亞乳桿菌 (Lactobacillus delbrueckii subsp. bulgaricus) 共同發酵,研究指出經納豆菌與乳酸菌共同發酵紅豆萃取物 (Bacillus subtilis and Lactobacillus delbrueckii co-culture fermented red bean, BLR) 具有良好體外抗氧化活性與抗發炎效力,但對於人類肝癌細胞株 (Hep G2 cells) 之作用並不清楚,故本研究將進一步探討BLR對Hep G2細胞抑制生長之影響。實驗將納豆菌及乳酸菌活化後共同接種於紅豆基質中於37 °C下靜置培養,每24小時翻攪一次,發酵120小時,之後以熱水、50%或95%乙醇萃取,其萃取物分別為BLR-W、BLR-E50及BLR-E95。實驗先測其體外抗氧化活性,再以細胞模式探討BLR對於Hep G2生長之影響,以及利用高效液相層析法 (high-performance liquid chromatography, HPLC) 分析其組成份。結果顯示BLR-W、BLR-E50及BLR-E95皆具還原力、清除DPPH自由基、螯合亞鐵離子、抑制脂質過氧化之能力以及降低Hep G2細胞的存活率並改變其細胞型態,其中以BLR-E95效果為最佳,因此後續細胞實驗皆以BLR-E95進行。經DAPI染色及彗星試驗得知BLR-E95會造成DNA氧化損傷的發生及促使細胞內ROS及Ca2+的釋放,並降低粒線體膜電位,誘導凋亡相關因子caspase-3、8、9的活性增加,使catalase與glutathione調適性上升,並使MDA含量下降。經HPLC結果分析catechin、rutin、quecertin為BLR主要之組成份。綜合上述,經納豆菌與乳酸菌共同發酵紅豆萃取物具有良好之體外抗氧化活性與抑制Hep G2細胞生長與誘導凋亡的作用。
Hepatic carcinoma is one of the ten leading causes of cancer death, so how to prevent or delay the hepatic carcinoma development is important. The red bean (Phaseolus angularis) is a leguminous seed and is mainly used as an important whole grain in Taiwan. Previously studies have demonstrated that red bean showed the highest antioxidative potential among several edible beans, so choose red beans as fermentation substrate. Previously studies have shown that Bacillus subtilis and Lactobacillus delbrueckii co-cultured red bean (BLR) extract possessed anti-oxidant and anti-inflammation activities. However, the effect of BLR on human liver cancer cell lines (Hep G2) is still unclear. The objectives of this study were to evaluate the effects of BLR extract on the cellular growth of Hep G2 and analysis phenolic compounds with high-performance liquid chromatography (HPLC). The fermentation of red beans by a co-culture of B. subtilis and L. bulgaricus incubated at 37 °C for 120 hours and stirred every 24 hours. Then the fermented red bean extracted by using different solvents (H2O, 50% ethanol, 95% ethanol) to obtained different solvent extracts, including BLR-W, BLR-E50 and BLR-E95. First, the in vitro antioxidant activities of different solvents BLR extracts were evaluated; then, each extract also was subjected to cell viability by using HepG2 cells. The results of in vitro study had shown that BLR possessed anti-oxidative characteristic including reducing power, DPPH radical-scavenging activity, Fe2+-chelation activity and lipid peroxidation inhibitory activity. Also, the BLR can reduce Hep G2 cell viability and change cell morphology. It was obvious that BLR-E95 was more effective ability. DAPI stain and comet assay were conducted and they indicated that BLR-E95 induced Hep G2 cell DNA damage, change of ROS, MMP, Ca2+ releasing, and increased the activity of caspase-3, caspase-8 and caspase-9. The catechin, rutin and quercetin were the most abundant phytochemicals in BLR-E95. In conclusion, this study indicates that BLR have antioxidant activities, inhibit Hep G2 cell growth and induce apoptosis.
目錄 Ⅰ
表目錄 Ⅲ
圖目錄 Ⅳ
中文摘要 Ⅴ
英文摘要 Ⅵ
第一章、前言 1
第二章、文獻回顧 3
第一節 肝癌 3
一、肝癌的簡介 3
二、肝癌的成因 3
三、臨床治療肝癌的方法 3
四、天然植物對肝癌細胞之影響 4
第二節 氧化壓力與抗氧化防禦系統 4
一、自由基與活性氧物質之介紹與形成 4
二、自由基與活性氧物質對生物體之影響 5
三、抗氧化作用機制 5
四、抗氧化防禦系統 5
第三節 紅豆介紹 10
一、紅豆簡介 10
二、紅豆的營養成分及加工應用 11
三、紅豆的生理活性 13
第四節 枯草桿菌介紹 14
一、枯草桿菌簡介 14
二、枯草桿菌發酵產品其生理功能 14
第五節 乳酸菌介紹 15
一、乳酸菌簡介 15
二、乳酸菌發酵產品其生理功能 15
第六節 經納豆菌與乳酸菌共同發酵紅豆產品之研究 16
第七節 細胞凋亡 ( cell apoptosis) 16
一、定義 16
二、細胞凋亡相關因子 17
三、細胞凋亡途徑 18
第三章、研究目的 20
第四章、實驗流程 21
第五章、材料與方法 23
第一節 儀器設備與器材 23
第二節 藥品名稱 25
第三節 實驗方法 27
一、納豆菌與乳酸菌共同發酵紅豆萃取物之製備 27
二、體外抗氧化試驗 28
三、細胞試驗 33
四、高效液相層析 44
第四節 統計方法 46
第六章、結果與討論 47
一、BLR其不同溶劑萃取物之體外抗氧化活性 47
二、BLR萃取物對人類肝癌細胞株 ( Hep G2 cells )生長之影響 57
三、BLR-95對Hep G2細胞DNA損傷之影響 58
四、利用流式細胞儀檢測BLR-95對Hep G2細胞凋亡相關因子之分析 64
五、BLR-95對Hep G2細胞抗氧化系統恆定與過氧化物含量之影響 74
六、利用HPLC分析BLR-E95之組成份 82
第七章、結論 84
第八章、參考文獻 86

行政院衛生福利部統計資料https://dep.mohw.gov.tw/DOS/lp-4927-113.html
Cayuela MM. Oxygen free radicals and human disease. Biochimie. 1995; 77:147-61.
Chou ST, Chao WW, Chung YC. Antioxidative activity and safety of 50% ethanolic red bean extract (Phaseolus radiatus L. var. Aurea). Int J Food Sci Technol. 2008;43:1371-8.
Sumi H, Hamada H, Nakanishi K, Hiratani H. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinases. Acta Haematol. 1990;84:139-43.
Juan MY, Chou CC. Enhancement of antioxidant activity, total phenolic and flavonoid content of black soybeans by solid state fermentation with Bacillus subtilis BCRC 14715. Food Microbiol. 2010;27:586-91.
Hose H, Sozzi T. Biotechnology group meeting probiotics-fact or fiction. J Chem Technol Biotechnol. 1991;51:540-4.
Hirayama K, Rafter J. The role of probiotic bacteria in cancer prevention. Microbes Infect. 2000;2:681-6.
Qiu GH, Xie X, Xu F, Shi X, Wang Y, Deng L. Distinctive pharmacological differences between liver cancer cell lines HepG2 and Hep3B. Cytotechnology. 2015;67:1-12.
Singh AK, Kumar R, Pandey AK. Hepatocellular carcinoma:causes, mechanism of progression and biomarkers. Curr Chem Genom Transl Med. 2018;12:9-26.
Yang JD, Roberts LR. Hepatocellular carcinoma: a global view. Nat Rev Gastroenterol Hepatol. 2010;7:448-58.
Peers F, Bosch X, Kaldor J, Linsell A, Pluijmen M. A flatoxin exposure, hepatitis B virus infection and liver cancer in Swaziland. Int J Cancer. 1987;39:545-53.
何信治癌中心醫院 http://www.kfsyscc.org
潘日鴻。大型樟芝發酵液對肝癌細胞Hep G2生長抑制之探討。臺灣大學食品科技研究所碩士論文。2009。
盧泯任。五爪金英誘導人類肝癌細胞Hep G2之凋亡作用。台東大學生命科學系碩士論文。2011。
劉于禎。金錢薄荷萃取物誘導人類肝癌細胞株 (Hep G2 cells) 凋亡之作用及其機制探討。靜宜大學食品營養學系碩士論文。2017。
Vishal Tandon MD, Gupta BM, Tandon R. Free radicals/reactive oxygen species. JK Pract. 2005;12:143-8.
Machlin LJ, Bendich A. Free radical tissue damage: protective role of antioxidant nutrients. FASEB J. 1987;1:441-5.
Valko M, Leibfritz D, Moncol J, Cronin Mark TD, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. IJBCB. 2007;39:44-84.
謝明哲。營養生化學。華藤文化股份有限公司。2004。
Scandalios JG. Oxygen stress and superoxide dismutase. Plant Physiol. 1993;101:7-12.
Carmagnol F, Sinet PM, Jerome H. Selenium-dependent and non-selenium-dependent glutathione peroxidases in human tissue extracts. Biochim Biophys Acta Gen Subj. 1983;759:49-57.
Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, Chen S, Corpe C, Dutta A, Dutta SK, Levine M. Vitamin C as an antioxidant: evaluation of its role in disease prevention. J Am Coll Ntur. 2003;22:18-35.
Sies H, Stahl W. Vitamins E and C, beta-carotene, and other carotenoids as antioxidants. Am J Clin Nutr. 1995;62:1315-21.
Frei B, Kim MC, Ames BN. Ubiquinol-10 is an effective lipid-soluble antioxidant at physiological concentrations. Proc Natl Acad Sci USA. 1990;87:4879-83.
行政院農業委員會https://kmweb.coa.gov.tw
陳玉如。紅豆新品種高雄9號之育成。高雄區農業改良場研究彙報。2015。
高雄區農業改良場技術專刊。2019。
食品營養成分資料庫(新版)。2015。
施明智。食物學原理。藝軒圖書出版社。2009。
石依萍。紅豆50%乙醇萃取物抗氧化及抗發炎活性之探討。靜宜大學食品營養學系碩士論文。2010。
Mukai Y, Sato S. Polyphenol-containing azuki bean (Vigna angularis) seed coats attenuate vascular oxidative stress and inflammation in spontaneously hypertensive rats. JNB. 2011;22:16-21.
Sato S, Mukai Y, Yamate J, Kato J, Kurasaki M, Hatai A, Sagai M. Effect of polyphenol-containing azuki bean (Vigna angularis) extract on blood pressure elevation and macrophage infiltration in the heart and kidney of spontaneously hypertensive rats. CEPP. 2009;35:43-9.
Sato S, Yamate J, Hori Y, Hatai A, Nozawa M, Sagai M. Protective effect of polyphenol-containing azuki bean (Vigna angularis) seed coats on the renal cortex in streptozotocin-induced diabetic rats. JNB. 2005;16:547-53.
Sato S, Hori Y, Yamate J, Saito T, Kurasaki M, Hatai A. Protective effect of dietary azuki bean (Vigna angularis) seed coats against renal interstitial fibrosis of rats induced by cisplatin. Nutrition. 2005;21:504-11.
Han KH, Fukushima M, Shimizu K, Kojima M, Ohba K, Tanaka A, Shimada KI, Sekikawa M, Nakano M. Resistant starches of beans reduce the serum cholesterol concentration in rats. J Nutr Sci Vitaminol. 2003;49:281-6.
Han KH, Fukushima M, Kato T, Kojima M, Ohba K, Shimada KI, Sekikawa M, Nakano M. Enzyme-resistant fractions of beans lowered serum cholesterol and increased sterol excretions and hepatic mRNA levels in rats. Lipids. 2003;38:919-24.
Harwood CR. Introduction to the biotechnology of Bacillus. Biotechnology hand book:1-4.
Omura K, Hitosugi M, Zhu X, Lkeda M, Maeda H. A newly derived protein from Bacillus subtilis natto with both antithrombotic and fibrinolytic effects. J Pharmacol Sci. 2005;99:247-51.
吳佳璇。發酵溫度影響bacillus subtilis BCRC 14715發酵黑豆中機能性 成分之改變。國立台灣大學生物資源暨農學院食品科技研究所碩士論 文。2008。
Caplice E, Fitzgerald GF. Food fermentations: role of microorganisms in food production and preservation. Int J Food Microbiol. 1999;50:131-49.
謝宗翰。以乳酸菌發酵苦瓜萃取液之抗氧化及抗菌能力探討。國立宜蘭大學食品科學系碩士論文。2018。
Yamamoto N, Akino A, Takano T. Antihypertensive effects of different kinds of fermented milk in spontaneously hypertensive rats. Biosci Biotechnol Biochem. 1994;58:776-8.
Wan Y, Xin Y, Zhang C, Wu D, Ding D, Tang L, Owusu L, Bai J, Li W. Fermentation supernatants of Lactobacillus delbrueckii inhibit growth of human colon cancer cells and induce apoptosis through a caspase 3‑dependent pathway. Oncol Lett. 2014;7:1738-42.
Jhan JK, Chang WF, Wang PM, Chou ST, Chung YC. Production of fermented red beans with multiple bioactivities using co-cultures of Bacillus subtilis and Lactobacillus delbrueckii subsp. bulgaricus. LWT-Food Sci Technol. 2015;63:1281-87.
Ghobrial IM, Witzig TE, Adjei AA. Targeting apoptosis pathway in cancer therapy. CA Cancer J Clin. 2005;55:178-94.
Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998;281:1322-6.
Nicholson DW, Thornberry NA. Caspases: killer proteases. Trends Biochem Sci. 1997;22:299-306.
Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495-516.
Parrino J, Hotchkiss RS, Bray M. Prevention of immune cell apoptosis as potential therapeutic strategy for severe infections. Emerg Infect Dis. 2007;13:191-8.
Biologicals N. https://www.novusbio.com/apoptosis-pathway.
Willians WB, Cuvelier ME, Berset C. Use of free radical method to evaluate antioxidant activity. Technol. 1995;28:25-30.
Oyaizu M. Studies on products of browning reaction antioxidative activities of products of browning reaction prepared from glucosamine. Jpn J nutri. 1986;44:307-15.
Yusof NM, Hasan MH, Armayni UA, Ahmad MS, Mohsin HF, Wahab IA. The ferrous ion chelating assay of pandanus extracts. ICNP. 2013;4:155.
Taga MS, Miller E, Pratt D. Chia seeds as a source of natural lipid antioxidants. J Am Oil Chem Soc. 1984;61:928-931.
Julkunen TR. Phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics. J Agric Food Chem. 1985; 33: 213-7.
Sultana B, Anwar F, Ashraf M. Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules. 2009; 14:2167-80.
Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2001.
Royall JA, Ischiropoulos H. Evaluation of 2',7'-dichlorodihydrofluorescein and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys. 1993;194:389-97.
Kruman II, Mattson MP. Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis. J Neurochem. 1999;72:529-40.
Petit PX, Oconnor JE, Grunwald D, Brown SC. Analysis of the membrane potential of rat-and mouse-liver mitochondrial by flow cytometry and possible applications. Eur J Biochem. 1990;194:389-97.
Kondo T, Kitano T, Iwai K, Watanabe M, Taguchi Y, Yabu T, Umehara H, Domae N, Uchiyama T, Okazaki T. Control of ceramide-induced apoptosis by IGF-1: involvement of PI-3 kinase, caspase-3 and catalase. Cell Death Differ. 2002;9:682-92.
Schneider SR, Mawrin C, Motsch C, Boltze C, Peters B, Hartig R, Buhtz P, Giers A, Rohrbeck A, Freigang B. Retention of the arginine allele in codon 72 of the p53 gene correlates with poor apoptosis in head and neck cancer. AJP. 2004;164:1233-41.
Kassie F, Parzefall W, Knasmüller S. Single cell gel electrophoresis assay: a new technique for human biomonitoring studies. Mutat Res Rev Mutat Res. 2000;463:13-31.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchomic acid. Anal Biochem. 1985;15:76-85.
Marklund S and Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. FEBS J. 1974;47:469-74.
Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967;70:158-69.
Aebi H. Catalase in: methods of enzymatic analysis. Verlag Chemie. 1983;3:273-86.
Tatum VL, Changchit C, Chow CK. Measurement of malondialdehyde by high performance liquid chromatography with fluorescence detection. Lipids. 1990;25:226-9.
Chen SJ, Chung JG, Chung YC , Chou ST. In vitro antioxidative and antiproliferative activity of the stem extracts from Graptopetalum paraguayense. Am J Chinese Med. 2008;36:369-83.
陳怡樺。以動物模式評估經納豆菌及乳酸菌共同發酵紅豆 (Phaseolus angularis) 萃取物之安全性及抗氧化活性。靜宜大學食品營養學系碩士論文。2010。
Tan TW, Tsai HR, Lu HF, Lin HL, Tsou MF, Lin YT, Tsai HY, Chen YF, Chung JG. Curcumin-induced cell cycle arrest and apoptosis in human acute promyelocytic leukemia HL-60 Cells via MMP changes and caspase-3 activation. Anticancer Res. 2006;26:4361-72.
Mondal A, Bennett LL. Resveratrol enhances the efficacy of sorafenib mediated apoptosis in human breast cancer MCF7 cells through ROS, cell cycle inhibition, caspase 3 and PARP cleavage. Biomed Pharmacother. 2016;84:1906-14.
Gao X, Wang B, Wei X, Men K, Zheng F, Zhou Y, Zheng Y. Anticancer effect and mechanism of polymer micelle-encapsulated quercetin on ovarian cancer. Nanoscale. 2012;4:7021-30.
Yu Y, Deng Y, Lu BM, Liu YX, Li J, Bao JK. Green tea catechins: a fresh flavor to anticancer therapy. Apoptosis. 2014;19:1-18.
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