臺灣博碩士論文加值系統

熱氧化的油、尤其是反覆使用的油炸油，所產生的醇類、醛類、酮類、酸類或碳氫化合物等極性化合物，與一些疾病是有相關性的，包括高血壓、脂肪肝、心血管疾病等。而先前對於荖葉的研究當中，已經證實了具有抑癌及抗突變、抗氧化、保護肝臟、降血脂的效用。而長期高油脂的飲食除了會增加身體和肝臟的重量，也極易造成脂肪肝，初期的脂肪肝病變並不會影響到肝功能指數的變化，但長期累積下來則會變成非酒精性脂肪性肝炎，非酒精性脂肪性肝炎是一個漸進的肝病，會導致肝細胞纖維化，最終進展為肝硬化。本研究的目的是探討荖葉對於使用油炸過的沙拉油餵食大鼠後對其血脂、肝功能以及腸道菌群的影響。將Sprague- Dawley雄性大鼠隨機分為三組，分別餵食以下飼料，為期十週：高油脂油炸沙拉油組(FSO)、高油脂油炸低劑量荖葉組(FSOL, FSO + Piper betle [50 mg/kg])、高油脂油炸高劑量荖葉組(FSOH, FSO + Piper betle [100 mg/kg])。十週後，犧牲大鼠，採集血液作血液生化值分析，並將其盲腸內容物進行細菌培養。實驗結果顯示荖葉具有降低血中ALT、TC指數，增加雙歧桿菌及乳酸桿菌的菌量，產生競爭性排除效應，進而改變腸道菌群生態。
關鍵詞：荖葉、高油脂飲食、油炸油、雙歧桿菌、乳酸桿菌

Thermal oxidative oil, especially repeatedly used frying oil, contains free fatty acids, small molecular alcohol, aldehyde, ketone, acid, lactone and hydrocarbon, diglyceride and monoglyceride, cyclic and epoxy compounds, trans isomers those are resulted from the chemical reactions of hydrolysis, oxidation, isomerization and polymerization during deep frying. Thermally oxidized oil generates reactive oxygen species that have been implicated in several pathological processes including hypertension, fatty liver, cardiovascular disease. Piper betel has been confirmed to have tumor suppressive, anti-mutagenic, antioxidant, hypolipidemic, and hepatoprotective effects. A long-term high-fat diet no only increases body and liver weight, and also leads to fatty liver. Fatty liver disease does not affect the liver function indices at first, but cumulative effects will lead to non-alcoholic steatohepatitis, a steatosis associated with inflammation, cell death, and fibrosis which can progress to cirrhosis. The aim of present study is to investigate the effects of Piper betel on serum lipids, liver function and gut microbiota in rats fed with thermally oxidized soybean oil/ high-cholesterol diets. Male Sprague-Dawley rats were divided into three groups and were fed with the following diets, respectively, for 10 weeks: soybean oil repeatedly used in frying food (FSO), FSO diet + Piper betle [50 mg/kg] (FSOL), and FSO diet + Piper betle [100 mg/kg] (FSOH). After 10 weeks, the rats were sacrificed, and blood samples were collected for blood biochemical parameters analysis and cecal contents for bacterial culture. Experimental results show that Piper betle reduced blood ALT, and TC data, increased the amount of Bifidobacterium and Lactobacillus, produced competitive exclusion effect, thereby changing the intestinal flora ecology.
Keywords：Piper betle、High-fat diet、thermally oxidized oil、Bifidobacterium、Lactobacilli

壹、中文摘要•••••••••••••••••••••••••••••••••••••••••••••••••••• 3
貳、英文摘要•••••••••••••••••••••••••••••••••••••••••••••••••••• 4
參、縮寫檢索表••••••••••••••••••••••••••••••••••••••••••••••••• 6
肆、緒論•••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 7
一、緒論•••••••••••••••••••••••••••••••••••••••••••••••••••• 7
二、荖葉(Piper betle)••••••••••••••••••••••••••••••••••• 9
三、油炸油••••••••••••••••••••••••••••••••••••••••••••••••• 11
四、腸道菌群(gut microbiota)•••••••••••••••••••••••••• 12
(一)、雙歧桿菌(Bifidobacterium)••••••••••••••••••••••••••• 14
(二)、乳酸桿菌(Lactobacilli) ••••••••••••••••••••••••••••••• 15
(三)、產氣莢膜梭菌(Clostridium perfringens) ••••••••••• 16
伍、實驗動機•••••••••••••••••••••••••••••••••••••••••••••••••••• 19
陸、研究架構•••••••••••••••••••••••••••••••••••••••••••••••••••• 21
柒、實驗器材與方法••••••••••••••••••••••••••••••••••••••••••• 23
一、實驗動物•••••••••••••••••••••••••••••••••••••••••••••• 23
二、實驗試劑藥品••••••••••••••••••••••••••••••••••••••• 23
三、實驗器材••••••••••••••••••••••••••••••••••••••••••••• 23
四、實驗設備•••••••••••••••••••••••••••••••••••••••••••••• 23
五、實驗方法•••••••••••••••••••••••••••••••••••••••••••••• 24
六、動物實驗•••••••••••••••••••••••••••••••••••••••••••••• 26
捌、實驗結果•••••••••••••••••••••••••••••••••••••••••••••••••••• 30
一、體重、體脂肪變化••••••••••••••••••••••••••••••••• 30
二、血液生化指數比較•••••••••••••••••••••••••••••••••• 30
三、三酸甘油脂與總膽固醇含量比較•••••••••••••••• 31
四、雙歧桿菌、乳酸桿菌菌量比較••••••••••••••••••• 31
五、產氣莢膜梭菌菌量比較•••••••••••••••••••••••••••• 31
玖、討論•••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 33
壹拾、參考文獻••••••••••••••••••••••••••••••••••••••••••••••••• 36
壹拾壹、圖表與說明••••••••••••••••••••••••••••••••••••••••••• 47
壹拾貳、附錄•••••••••••••••••••••••••••••••••••••••••••••••••••• 56

1.Tojo R, Suárez A, Clemente MG, de los Reyes-Gavilán CG, Margolles A, Gueimonde M, Ruas-Madiedo P Intestinal microbiota in health and disease: Role of bifidobacteria in gut homeostasis World J Gastroenterol. 2014 Nov 7;20(41):15163-76
2.Karczewski J, Poniedziałek B, Adamski Z, Rzymski P The effects of the microbiota on the host immune system Autoimmunity. 2014 Dec;47(8):494-504.
3.Chassard C, de Wouters T, Lacroix C Probiotics tailored to the infant: a window of opportunity Curr Opin Biotechnol. 2014 Apr;26:141-7
4.Casén C, Veb HC, Sekelja M, Hegge FT, Karlsson MK, Ciemniejewska E, Dzankovic S, Fryland C, Nestestog R, Engstrand L, Munkholm P, Nielsen OH, Rogler G, Simrén M, Öhman L7, Vatn MH, Rudi K Deviations in human gut microbiota: a novel diagnostic test for determining dysbiosis in patients with IBS or IBD Aliment Pharmacol Ther. 2015 Jul;42(1):71-83
5.Tojo R, Suárez A, Clemente MG, de los Reyes-Gavilán CG, Margolles A, Gueimonde M, Ruas-Madiedo P Intestinal microbiota in health and disease: Role of bifidobacteria in gut homeostasis World J Gastroenterol. 2014 Nov 7;20(41):15163-76
6.Sanders ME Probiotics: Definition, Sources, Selection, and Uses Clin Infect Dis. 2008 Feb 1;46 Suppl 2:S58-61
7.Gill HS, Guarner F Probiotics and human health: a clinical perspective Postgrad Med J. 2004 Sep;80(947):516-26
8.Ettinger G, MacDonald K, Reid G, Burton JP The influence of the human microbiome and probiotics on cardiovascular health Gut Microbes. 2014;5(6):719-28
9.Zhang Q, Saleh AS, Chen J, Shen Q Chemical alterations taken place during deep-fat frying based on certain reaction products: a review Chem Phys Lipids. 2012 Sep;165(6):662-81
10.Ng CY, Kamisah Y, Faizah O, Jaarin K The role of repeatedly heated soybean oil in the development of hypertension in rats: association with vascular inflammation Int J Exp Pathol. 2012 Oct;93(5):377-87
11.Kostecka M Eating habits of preschool children and the risk of obesity, insulin resistance and metabolic syndrome in adults Pak J Med Sci. 2014 Nov-Dec;30(6):1299-303
12.Gonzales JF, Barnard ND, Jenkins DJ, Lanou AJ, Davis B, Saxe G, Levin S Applying the precautionary principle to nutrition and cancer J Am Coll Nutr. 2014;33(3):239-46
13.Abilés J, Ramón AN, Moratalla G, Pérez-Abud R, Morón Jiménez J, Ayala A Effects of thermo-oxidised oils on lipid peroxidation in experimental animals Nutr Hosp. 2009 Jul-Aug;24(4):473-8
14.Venkadeswaran K, Muralidharan AR, Annadurai T, Ruban VV, Sundararajan M, Anandhi R, Thomas PA, Geraldine P Antihypercholesterolemic and Antioxidative Potential of an Extract of the Plant, Piper betle, and Its Active Constituent, Eugenol, in Triton WR-1339-Induced Hypercholesterolemia in Experimental Rats Evid Based Complement Alternat Med. 2014;2014:478973
15.Sazwi NN, Nalina T, Abdul Rahim ZH Antioxidant and cytoprotective activities of Piper betle, Areca catechu, Uncaria gambir and betel quid with and without calcium hydroxide BMC Complement Altern Med. 2013 Dec 11;13:351
16.Alam B, Akter F, Parvin N, Sharmin Pia R, Akter S, Chowdhury J, Sifath-E-Jahan K, Haque E Antioxidant, analgesic and anti-inflammatory activities of the methanolic extract of Piper betle leaves Avicenna J Phytomed. 2013 Spring;3(2):112-25
17.Konečná M, Urlacher SS Effect of chewing betel nut (Areca catechu) on salivary cortisol measurement Am J Phys Anthropol. 2015 Jun 8
18.衛生福利部國民健康署 檳榔E化網 檳榔的歷史及用途
19.Chang MC, Chen YJ, Chang HH, Chan CP, Yeh CY, Wang YL, Cheng RH, Hahn LJ, Jeng JH Areca Nut Components Affect COX-2, Cyclin B1/cdc25C and Keratin Expression, PGE2 Production in Keratinocyte Is Related to Reactive Oxygen Species, CYP1A1, Src, EGFR and Ras Signaling PLoS One. 2014 Jul 22;9(7):e101959
20.衛生福利部統計處 民國102年主要死因分析
21.Franke AA, Lai JF, Kawamoto CT, Pokhrel P, Herzog TA Areca (Betel) Nut Consumption: An Underappreciated Cause of Cancer Hawaii J Med Public Health. 2014 Dec;73(12):400-3
22.Lin MH, Wang CJ, Huang HP, Chou MY, Chou FP The tumorigenic characteristics of Lime-Piper betel quid-transformed JB6 cells Arch Toxicol. 2004 Mar;78(3):167-73
23.林易超 柯政全 謝天渝 檳榔及其添加物致突變性之研究 J Academy Formosan Stomatology 16: 273-295, 2000
24.Arawwawala LD, Arambewela LS, Ratnasooriya WD Gastroprotective effect of Piper betle Linn. leaves grown in Sri Lanka J Ayurveda Integr Med. 2014 Jan;5(1):38-42
25.D. Pradhan, Dr. K. A. Suri , Dr. D. K. Pradhan , P. Biswasroy Golden Heart of the Nature: Piper betle L. J Pharmacogn Phytochem Vol. 1 No. 6 2013
26.Amonkar AJ, Padma PR, Bhide SV Protection effect of hydroxychavicol, a phenolic component of betel leaf, against the tobacco-specific Mutat Res. 1989 Feb;210(2):249-53
27.Padma PR, Lalitha VS, Amonkar AJ, Bhide SV Anticarcinogenic effect of betel leaf extract against tobaccocarcinogens Cancer Lett. 1989 Jun;45(3):195-202
28.Young SC, Wang CJ, Hsu JD, Hsu JL, Chou FP Increased sensitivity of Hep G2 cells toward the cytotoxicity of cisplatin by the treatment of piper betel leaf extract Arch Toxicol. 2006 Jun;80(6):319-27
29.Milton Prabu S, Muthumani M, Shagirtha K Protective effect of Piper betle leaf extract against cadmium-induced oxidative stress and hepatic dysfunction in rats Saudi J Biol Sci. 2012 Apr;19(2):229-39
30.Young SC, Wang CJ, Lin JJ, Peng PL, Hsu JL, Chou FP Protection effect of piper betel leaf extract against carbon tetrachloride-induced liver fibrosis in rats Arch Toxicol. 2007 Jan;81(1):45-55
31.Kavousi P, Mirhosseini H, Ghazali H, Ariffin AA Formation and reduction of 5-hydroxymethylfurfural at frying temperature in model system as a function of amino acid and sugar composition Food Chem. 2015 Sep 1;182:164-70
32.Kamisah Y, Shamil S, Nabillah MJ, Kong SY, Hamizah NA, Qodriyah HM, Nur Azlina MF, Azman A, Jaarin K Deep-fried keropok lekors Increase Oxidative Instability in Cooking Oils Malays J Med Sci. 2012 Oct;19(4):57-62
33.Roberfroid M, Gibson GR, Hoyles L, McCartney AL, Rastall R, Rowland I, Wolvers D, Watzl B, Szajewska H, Stahl B, Guarner F, Respondek F, Whelan K, Coxam V, Davicco MJ, Léotoing L, Wittrant Y, Delzenne NM, Cani PD, Neyrinck AM, Meheust A Prebiotic effects: metabolic and health benefits Br J Nutr. 2010 Aug;104 Suppl 2:S1-63
34.楊宏志 腸道菌叢與人類的免疫系統 formosan J Med 2014;18:416-22
35.Verdu EF, Galipeau HJ, Jabri B Novel players in coeliac disease pathogenesis: role of the gut microbiota Nat Rev Gastroenterol Hepatol. 2015 Jun 9
36.Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, Gordon JI, Relman DA, Fraser-Liggett CM, Nelson KE. Metagenomic Analysis of the Human Distal Gut Microbiome Science. 2006 Jun 2;312(5778):1355-9
37.Deng P, Swanson KS Gut microbiota of humans, dogs and cats: current knowledge and future opportunities and challenges Br J Nutr. 2015 Jan;113 Suppl:S6-17
38.Guinane CM, Cotter PD Role of the gut microbiota in health and chronic gastrointestinal disease: understanding a hidden metabolic organ Therap Adv Gastroenterol. 2013 Jul;6(4):295-308
39.Roberfroid M, Gibson GR, Hoyles L, McCartney AL, Rastall R, Rowland I, Wolvers D, Watzl B, Szajewska H, Stahl B, Guarner F, Respondek F, Whelan K, Coxam V, Davicco MJ, Léotoing L, Wittrant Y, Delzenne NM, Cani PD, Neyrinck AM, Meheust A. Prebiotic effects: metabolic and health benefits Br J Nutr. 2010 Aug;104 Suppl 2:S1-63
40.Na JY, Park JM, Lee KS, Kang JO, Oh SH, Kim YJ Clinical Characteristics of Symptomatic Clostridium difficile Infection in Children: Conditions as Infection Risks and Whether Probiotics Is Effective Pediatr Gastroenterol Hepatol Nutr. 2014 Dec;17(4):232-8
41.Jiang Y, Kong Q, Roland KL, Wolf A, Curtiss R 3rd Multiple effects of Escherichia coli Nissle 1917 on growth, biofilm formation, and inflammation cytokines profile of Clostridium perfringens type A strain CP4 Pathog Dis. 2014 Apr;70(3):390-400
42.Schoster A, Kokotovic B, Permin A, Pedersen PD, Dal Bello F, Guardabassi L In vitro inhibition of Clostridium difficile and Clostridium perfringens by commercial probiotic strains Anaerobe. 2013 Apr;20:36-41
43.Picard C, Fioramonti J, Francois A, Robinson T, Neant F, Matuchansky C Bifidobacteria as probiotic agents – physiological effects and clinical benefits Aliment Pharmacol Ther. 2005 Sep 15;22(6):495-512
44.Goldstein EJ, Tyrrell KL, Citron DM Lactobacillus species: taxonomic complexity and controversial susceptibilities Clin Infect Dis. 2015 May 15;60 Suppl 2:S98-S107
45.Swain MR, Anandharaj M, Ray RC, Parveen Rani R Fermented Fruits and Vegetables of Asia: A Potential Source of Probiotics Biotechnol Res Int. 2014;2014:250424
46.Wyszyńska A, Kobierecka P, Bardowski J, Jagusztyn-Krynicka EK Lactic acid bacteria-20 years exploring their potential as live vectors for mucosal vaccination Appl Microbiol Biotechnol. 2015 Apr;99(7):2967-77
47.World Gastroenterology Organization WGO Practice Guideline - Probiotics and Prebiotics October 2011
48.Wu CC, Weng WL, Lai WL, Tsai HP, Liu WH, Lee MH, Tsai YC Effect of Lactobacillus plantarum Strain K21 on High-Fat Diet-Fed Obese Mice Evid Based Complement Alternat Med. 2015;2015:391767
49.Nagahama M, Ochi S, Oda M, Miyamoto K, Takehara M, Kobayashi K Recent Insights into Clostridium perfringens Beta-Toxin Toxins (Basel). 2015 Feb 3;7(2):396-406
50.Noel R.Kring,J.G.Holt, The Mycoplasmas,Bergry''s Manual of Systematic Bacteriology Vol2 p1179-1182 1984
51.Jiang Y, Kong Q, Roland KL, Wolf A, Curtiss R 3rd Multiple effects of Escherichia coli Nissle 1917 on growth, biofilm formation, and inflammation cytokines profile of Clostridium perfringens type A strain CP4 Pathog Dis. 2014 Apr;70(3):390-400
52.Novak-Weekley SM, Marlowe EM, Miller JM, Cumpio J, Nomura JH, Vance PH, Weissfeld A Clostridium difficile testing in the clinical laboratory by use of multiple testing algorithms J Clin Microbiol. 2010 Mar;48(3):889-93
53.Shindo Y, Dobashi Y, Sakai T, Monma C, Miyatani H, Yoshida Y Epidemiological and pathobiological profiles of Clostridium perfringens infections: review of consecutive series of 33 cases over a 13-year period Int J Clin Exp Pathol. 2015 Jan 1;8(1):569-77
54.Redondo LM, Carrasco JM, Redondo EA, Delgado F, Miyakawa ME Clostridium perfringens type E virulence traits involved in gut colonization PLoS One. 2015 Mar 23;10(3):e0121305
55.Allaart JG, van Asten AJ, Gröne A Predisposing factors and prevention of Clostridium perfringens-associated enteritis Comp Immunol Microbiol Infect Dis. 2013 Sep;36(5):449-64
56.Ma GC, Wu PF, Tseng HC, Chyau CC, Lu HC, Chou FP Inhibitory effect of Piper betel leaf extracts on copper-mediated LDL oxidation and oxLDL-induced lipid accumulation via inducing reverse cholesterol transport in macrophages Food Chem. 2013 Dec 15;141(4):3703-13
57.Dietrich CG, Kottmann T, Alavi M Commercially available probiotic drinks containing Lactobacillus casei DN-114001 reduce antibiotic-associated diarrhea World J Gastroenterol. 2014 Nov 14;20(42):15837-44
58.https://nei.nih.gov/news/pressreleases/050513
59.衛生福利部 健康食品之胃腸功能改善評估方法 920829衛署食字第0920401629號公告修正
60.Muñoa FJ, Pares R Selective medium for isolation and enumeration of Bifidobacterium spp. Appl Environ Microbiol. 1988 Jul;54(7):1715-8
61.Silvi S, Rumney CJ, Rowland IR An assessment of three selective media for bifidobacteria in faeces J Appl Bacteriol. 1996 Nov;81(5):561-4
62.Su P, Henriksson A, Mitchell H LAMVAB-a new selective medium for the isolation of lactobacilli from feces J Microbiol Methods. 29: 77-84 1997
63.May T, Mackie RI, Fahey GC Jr, Cremin JC, Garleb KA Effect of fiber source on short-chain fatty acid production and on the growth and toxin production by Clostridium difficile Scand J Gastroenterol. 1994 Oct;29(10):916-22
64.Cohen JC, Horton JD, Hobbs HH Human Fatty Liver Disease: Old Questions and New Insights Science. 2011 Jun 24;332(6037):1519-23
65.Dvorak K, Stritesky J, Petrtyl J, Vitek L, Sroubkova R, Lenicek M, Smid V, Haluzik M, Bruha R Use of non-invasive parameters of non-alcoholic steatohepatitis and liver fibrosis in daily practice--an exploratory case-control study PLoS One. 2014 Oct 28;9(10):e111551
66.Athyros VG, Tziomalos K, Katsiki N, Doumas M, Karagiannis A, Mikhailidis DP Cardiovascular risk across the histological spectrum and the clinical manifestations of non-alcoholic fatty liver disease: An update World J Gastroenterol. 2015 Jun 14;21(22):6820-6834
67.Milić S, Lulić D, Štimac D Non-alcoholic fatty liver disease and obesity: Biochemical, metabolic and clinical presentations World J Gastroenterol. 2014 Jul 28; 20(28): 9330–9337
68.Lecerf JM, de Lorgeril M Dietary cholesterol: from physiology to cardiovascular risk Br J Nutr. 2011 Jul;106(1):6-14
69.全民健康保險藥物給付項目及支付標準－第六編第八十三條「藥品給付規定」修正規定第2節心臟血管及腎臟藥物Cardiovascular-renal drugs