臺灣博碩士論文加值系統

摘 要
茶是人類長期飲用的飲料，其中兒茶素(Catechins)佔茶粉抽提成分中20％以上，為主要的生理活性成分，主要成分為(-)-epigallocatechin gallate (EGCG)。兒茶素抗氧化之能力被認為是其抗細胞突變、防癌、預防心血管疾病的主因，但口服兒茶素之絕對生體可用率不到10%，因此未被吸收之兒茶素對腸道之效能亦應有所了解。基於預防醫學的觀點，體內兒茶素的抗氧化作用與其對腸道菌相之影響，是要長期觀察探討。本研究藉由長期餵予豬隻即溶綠茶粉，觀察豬隻體內血中自由基生物指標物質（MDA）與體內抗氧化生物指標物質（GSH）的增減，並檢測兒茶素的血中濃度相互評比，以明確瞭解兒茶素其抗氧化之功能。再藉由分析兒茶素成分，確認兒茶素之藥物動力學與其生體可用率，合併觀察未被吸收之兒茶素對腸內菌相的影響。
在兒茶素之抗氧化效能方面：豬隻連續口服Catechins即溶綠茶粉前後，在血漿中MDA濃度並無明顯增減趨勢，然而，血中GSH濃度則有明顯增加之趨勢。將口服投予即溶綠茶粉之後十九天內，生物指標物質的濃度與對照組（即投藥前三天之平均值）比較，發現血漿中MDA與GSH濃度並無明顯增減趨勢，且統計上無顯著差異存在；但於全血內GSH的濃度則有明顯增加之趨勢( P=0.013 )。由此得知在每日高劑量即溶綠茶粉口服投予下，未能影響血中MDA濃度，但血中GSH濃度有增加之趨勢，尤其以全血中還原型GSH濃度有較明顯的上升。
一簡單、精確且具有專一性的HPLC分析方法，已被發展用以同時偵測生物檢品中兒茶素成分（EGCG與ECG）的濃度。利用逆相層析管，配合280 nm之紫外光波長偵測，運用單一流動相合併使用不同的流速進行偵測。EGCG在血漿濃度5至200mg/ml的範圍內呈現良好線性關係（Y=-1138+765X，r2=0.9920），ECG在血漿濃度10至200mg/ml的範圍內呈現良好線性關係（Y=496+182X，r2=0.9970），同日內與異日內分析之CV值均在可接受範圍，顯示此為良好之分析方法。在8.64mg/kg劑量之靜脈投予六隻豬，分析其血中濃度，顯示EGCG與ECG符合二室性模式之藥物動力學性質。其排除半衰期分別為226.0±161.5、315.1±504.0分鐘，分布體積分別為34.4±21.0、43.8±65.0 ml/kg，清除率分別為0.11±0.35、0.10±0.40 ml/min/kg，而曲線下之面積（AUC）分別為12.0±3.4、17.0±10.1 mg×min/ml。
兒茶素對腸道之影響：豬隻連續口服Catechins即溶綠茶粉前後，腸道之總菌落數 (3.2±1.8 vs. 3.6±2.3)、厭氧性菌總菌落數 (3.8±1.9 vs. 3.5±1.8) 並無明顯之變化，有害菌大腸桿菌數 (6.3±3.1 vs. 4.8±1.9) 未見增減；然而，益生菌雙叉桿菌數 (1.6±1.2 vs. 6.7±3.0) 在投予即溶綠茶粉第八天後，具有顯著增加趨勢，其菌落數約為投予前之2-10倍。投予即溶綠茶粉第十二天後，乳酸桿菌菌落數 (4.2±2.4 vs. 8.9±1.9)顯著的微增，為未投予時之2倍。結果顯示益生菌雙叉桿菌及乳酸桿菌在腸菌叢中比例增高時，即相對的縮減有害菌的生存空間；雖然豬樣本少且腸道菌的差異性大，結果之標準偏差值較大，卻具有統計上顯著差異，因此口服連續投與高劑量即溶綠茶粉確實能改善腸道菌相，有利於腸道的正常生理機能。
本試驗連續口服即溶綠茶粉於豬隻體內，同時觀察其體內氧化壓力與腸道菌相之變化，發現長期服用即溶綠茶粉雖未能明顯降低體內脂質過氧化物（MDA）的產生，但卻能有效增加抗氧化物質（GSH）之含量；而且未被吸收之綠茶也能在腸道中明顯提高益生菌種（雙叉桿菌與乳酸桿菌）之數目，在有限的腸道空間內有害菌的生長即被抑制。因此口服連續投與高劑量兒茶素不僅能顯著提昇生理之抗氧化作用，也確實能改善腸道菌相有利於腸道的正常生理機能。

Abstract
More than 20% of catechins are in the extracted tea powder and (-)-epogallocatechin gallate (EGCG) is the major component of catechins. The antioxidative activity is thought as the main force to their antimutagenic, anticancer and protection of cardiovascular disease. While, The oral absolute bioavailability of catechins was less than 10%. Therefore, understanding the influence of unabsorbed catechins in GI tract became necessary. In the protecting medicine point, it is necessary to evaluate that the influence the antioxidative activity of catechins and catechins on the enterobacteria in pigs of a long term. The aim of this study is to evaluate the antioxidant activities of catechins and the influence of catechins on the enterobacteria in pigs.
Evaluation the antioxidative activity of catechins：After oral catechins 19 days, the plasma concentration of MDA did not changed while GSH concentration have an increasing tendency. Comparison of before and after oral catechins with t-test, plasma MDA concentration and plasma GSH concentration didn’t have statistical significant different. However, the GSH concentration in whole blood has statistical significant different（p=0.013）. Therefore, we infer that daily oral administration of high dose of catechins would increase the GSH concentration and enhance the ability of defense system. daily administration of high dose (520mg/kg, containing EGCG 60 mg/kg), but it did not change the MDA concentration, it infers that the damage of free radical causing from lipid peroxidation did not significant decreased or increased.
A simple, accuracy and specific analytical method based on HPLC was developed to detect the catechins in the biological sample. A C8 reversed phase column with UV detection at 280nm was used in chromatographic separation. Under the chromatographic conditions, the calibration curve of EGCG and ECG in plasma shows a good linearity within the concentration range of 5 to 200mg/ml（Y=-1138+765, r2=0.9920）and 10 to 200mg/ml（Y=496+182, r2=0.9970）. the validation of this analytical method are within the acceptable criteria. After intravenous administration（I.V.）of 8.64mg/kg green tea, the plasma concentration-time profiles of EGCG and ECG would be described by a bi-exponential equation. the elimination half-life are 226.0±161.5、315.1±503.9min, the distribution volume are 34.4±21.0、43.8±65.0 ml/kg，the clearance are 0.11±0.35、0.10±0.40 ml/min/kg，the area under the curves（AUC）are 12.0±3.4、17.0±10.1 mg×min/ml。
The influence of catechins on the enterobacteria：comparing with the colony before and after administration of catechins, total aerobic bacteria (3.2±1.8 to 3.6±2.3) and anaerobic bacteria (3.8±1.9 to 3.5±1.8) and E. coli (6.3±3.1 to 4.8±1.9) was not significantly changed. However, Bifidobacterium spp. and Lactobacillius spp. have an increasing tendency beginning at eighth day. Bifidobacterium spp. increased significantly and reached to 2 to 10 times of baseline data (1.6±1.2 to 6.7±3.0). Lactobacillius spp. increased slightly two times of control from the twelfth day (4.2±2.4 to 8.9±3.2). The ratio of colony of Bifidobacterium spp. to E. coli was increased accompanying to the times of administration. It indicated that catechins would be helpful to reconstruct or improve the normal enterobacteria in pigs by increase the Bifidobacterium spp..
In this study, after catechins administration daily, the blood GSH was also significantly increased and Bifidobacterium spp. and Lactobacillius spp. have an increasing tendency. Therefore, we inferred that oral administration of high dose of tea catechins daily would enhance the ability of defense system by increased whole blood GSH concentration and improve the GI flora by increase the Bifidobacterium spp. and Lactobacillius spp. to have the benefit to bowel normal conditions.

目 錄
頁次
目錄I
流程目錄IV
附表目錄V
附圖目錄VI
中文摘要IX
英文摘要XI
第一章 緒論1
一、茶的基本性質1
1、兒茶素(catechins)的結構分類2
2、兒茶素(catechins)的藥理作用4
3、兒茶素(catechins)的分析方法報告5
4、藥物動力學報告6
5、兒茶素的代謝7
二、體內的氧化壓力效能13
1、氧化壓力傷害13
1.1自由基簡介13
1.2評估體內自由基傷害13
1.3 MDA分析方法報告14
2、體內自由基的防禦機制19
2.1簡介19
2.2評估體內防禦機制的指標物19
2.3 GSH分析方法報告20
三、體內腸道菌相的改變與健康23
四、實驗目的24
第二章 研究方法及實驗26
一、實驗材料與儀器26
1、實驗試藥26
1.1氧化壓力效能26
1.2綠茶藥物動力學27
1.3腸道菌相實驗材料27
2、儀器28
3、試藥的配製29
3.1氧化壓力效能29
3.1.1 MDA試藥的配製29
3.1.2 GSH試藥的配製29
3.2綠茶含量分析29
二、實驗方法30
1、分析條件30
1.1氧化壓力效能30
1.1.1 MDA的分析條件30
1.1.2 GSH的分析條件30
1.2綠茶含量分析:生物檢品中綠茶各成分的分析條件30
1.3 腸道菌相34
2、血液檢品的處理34
2.1抗氧化壓力34
2.1.1 檢品中MDA的處理34
2.1.2 檢品中GSH的處理35
2.2綠茶含量分析35
3、標準檢量線的製作40
3.1 氧化壓力效能40
3.1.1 檢品中MDA的處理40
3.1.2 檢品中GSH的處理40
3.2 綠茶含量分析41
4、分析方法的確效41
4.1同次內分析的精確性與準確性41
4.2異次內分析的精確性與準確性41
5、動物實驗422
5.1實驗豬隻的準備42
5.2藥物投與溶液之組成42
5.3豬隻口服綠茶的方法43
5.4豬隻靜脈注射綠茶的方法43
第三章 結果與討論45
一、分析方法之結果與討論45
1、氧化應力效能45
1.1 MDA之分析方法45
1.2 GSH之分析方法48
2、綠茶含量分析51
二、確效試驗之結果與討論55
1、同次內分析的精確性與準確性55
2、異次內分析的精確性與準確性55
三、動物實驗之結果與討論60
1、氧化壓力效能60
1.1評估脂質過氧化產生之自由基傷害60
1.2評估生理抗氧化的防禦能力66
2、綠茶含量分析74
2.1靜脈注射綠茶之探討74
2.1.1綠茶成分之一EGCG74
2.1.2綠茶成分之二ECG75
2.2口服投與綠茶之探討81
2.2.1綠茶成分之一EGCG81
2.2.2綠茶成分之二ECG82
3、腸道菌相試驗88
第四章 結論93
第五章 參考資料95

第五章 參考資料
1. J.H. Weisburger, Tea and health: the underlying mechanisms., Proceeding of science experimental and biological medicine, 220, 271-275, 1999.
2. D.A. Balentine, S.A. Wiseman, and L.C.M. Bouwens, The chemistry of tea flavonoids., Critical Reviews in Food Science and Nutrition, 37, 693-704, 1997.
3. S.A. Wiseman, D.A. Balentine, and B. Frei, Antioxidants in tea., Critical Reviews in Food Science and Nutrition, 37, 693-704, 1997.
4. R.l. Prior, and G. Cao, Antioxidant capacity and polyphenolic components of teas: implications for altering in vivo antioxidant status., Proceeding of Social Experimental Biochemistry and Medicine., 220, 255-261, 1999.
5. 錢阜甯, Antibacterial and bactericidal properties of tea catechins.,食品工業月刊, 31, 16- 26, 1999.
6. Y. Hara, Influence of tea catechins on the digestive tract., Journal of Cellular Biochemistry Supplement, 27, 52-58, 1997.
7. K. Goto, S. Kanaya, T. Ishigami, and Y. Hara, The effects of tea catechins on fecal conditions of elderly residents in a long-term care facility., Journal of Nutrition Science and Vitaminology, 45, 135-141, 1999.
8. H.M. Merken, and G.R. Beecher, Measurement of food flavonoids by high-performance liquid chromatography: a review., Journal of Agricultural and Food Chemistry, 48, 577-599, 2000.
9. D. Treutter, Chemical reaction detection of catechins and proanthocyanidins with 4-dimethylaminocinnamaldehyde., Journal of Chromatography, 467, 185-193, 1989.
10. W.E. Bronner, and G.R. Beecher, Method for determining the content of catechins in tea infusions by high-performance liquid chromatography., Journal of Chromatography A, 805, 137-142, 1998.
11. J.J. Dalluge, B.C. Nelson, J.B. Thomas, and L.C. Sander, Selection of column and gradient elution system for the separation of catechins in green tea using high-performance liquid chromatography., Journal of Chromatography A., 793, 265-274, 1998.
12. G. Maiani, M. Serafini, M. Salucci, E. Azzini, and Ferro-Luzzi, Application of a new high-performance liquid chromatographic method for measuring selected polyphenols in human plasma., Journal of Chromatography B, 692, 311-317, 1997.
13. K. Nakagawa, and T. Miyazawa, Chemiluminescence-high-performance liquid chromatographic determination of tea catechins, (-)-epigallocatechin 3-gallate, at picomole levels in rat and human plasma., Analytical Biochemistry, 248, 41-49, 1997.
14. Y. Ho, Y-L Lee, and K-Y Hsu, Determination of (-)-catechin in plasma by high-performance liquid chromatography using fluorescence detection., Journal of Chromatography B, 665, 383-389, 1995.
15. P.C.H. Hollman, J.M.P. van Trijp, and M.N.C.P. Buysman, Fluorescence detection of flavonols in HPLC by postcolumn chelation with aluminum., Analytical Chemistry, 68, 3511-3515, 1996.
16. M-J Lee, Z-Y Wang, H. Li, L. Chen, Y. Sun, S. Gobbo, D.A. Balentine, and C.S. Yang, Analysis of plasma and urine tea catechins polyphenols in human subjects., Cancer Epideminology, Biomarkers and Prevention, 4, 393-399, 1995.
17. P.C.H. Hollman, L.B.M. Tijburg, and C.S. Yang, Bioavailability of flavonoids from tea., Critical Reviews in Food Science and Nutrition, 37, 719─738, 1997.
18. M. Zhu, Y. Chen, and R.C. Li, Oral absorption and bioavailability of tea catechins., Planta Medica, 66, 444-447, 2000.
19. L. Chen, M-J Lee, H. Li, and C.S. Yang, Absorption, distribution and elimination of tea polyphenols in rats., Drug Metabolism and Disposition, 25, 1045-1050, 1997.
20. K. Okushio, M. Suzuki, N. Matsumoto, F. Nanjo, and Y. Hara, Identification of (-)-epicatechin metabolites and their metabolic fate in the rat., Drug Metabolism and Disposition, 27, 309-316, 1998.
21. M.R. Meselhy, N.Nakamura, and M. Hattori, Biotransformation of (-)-epicatechin 3-o-gallate by human intestinal bacteria., Chemical Pharmacology Bull., 45, 888-893, 1997.
22. C. Li, M-J Lee, S. Sheng, X. Meng, S.Prabhu, B. Winnik, B. Huang, J.Y. Chung, S. Yan, C-T Ho, and C.S. Yang, Structural identification of two metabolites of catechins and their kinetics in human urine and blood after tea ingestion., Chemical Research and Toxicology, 13, 177-184, 2000.
23. B. Halliwell, and J.M.C. Gutridge, Free Radicals in Biology and Medicine.2nd ed., Clarendon press, Oxford, 1989.
24. L.L. de Zwart, J.H.N. Meerman, J.N.M. Commandeur, and N.P.E. Vermeulen, Biomarkers of free radical damage applications in experimental animals and in humans., Free Radical Biology and Medicine, 26, 202-226, 1999.
25. K. Moore, and L.J. Robberts II, Measurement of liquid peroxidation., Free Radical Research, 28, 659-671, 1998.
26. N. Volpi, and P. Tarugi, Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma., Journal of Chromatography B., 713, 433-437, 1998.
27. J. Chaudiere, and R. Ferrari-rliou, Intracellular antioxidants: frem chemical to biochemical mechanisms., Food and Chemical Toxicology, 37, 949-962, 1999.
28.F.J. Kelly, Glutathione: in defence of the lung., Food and Chemical Toxicology, 37, 963-966, 1999.
29. F. Michcelet, R. Gueguen, P. Leroy, M. Wellman, A. Nicolas, and G. Siest, Blood and plasma glutathione measured in healthy subjects by HPLC: relation to sex, aging, biological variables and life habits., Clinical Chemistry, 41, 1509-1517, 1995.
30. J. Chaudiere, and R. Ferrari-iliou, Intracellular antioxidants: from chemical to biochemical mechanisms., Food and Chemical Toxicology, 37, 949-962, 1999.
31. K. Shimada, and K. Mitamura, Derivatization of thiol-containing compounds., Journal of Chromatography B., 659, 227-241, 1994.
32. P. Leroy, A. Nocolas, M. Wellmann, F. Michelet, T. Oster, and G. Siet, Evaluation of o-phthalaldehyde as bifunctional fluorogenic post-column reagent for glutathione in LC., Chromatographia, 36, 130-134, 1993.
33. J-L Luo, F. Hammarqvist, I.A. Cotgreave, C. Lind, K. Andersson, and J. Wernerman, Determination of intracellular glutathione in human skeletal muscle by reversed-phase high-performance liquid chromatography., Journal of Chromatography B., 670, 29-36, 1995.
34. C-S Yang, S-T Chou, L. Liu, P-J Tsai, and J-S Kuo, Effect of aging on human plasma glutathione concentration as determined by high-performance liquid chromatography with fluorimetric detection., Journal of chromatography B., 674, 23-30, 1995
35. R. Paroni, E. Vecchi, G. Cighetti, C. Arcelloni, I. Fermo, A. Grossi, and P. Bonini, HPLC with o-phthalaldehyde precolunm derivatization to measure total, oxidized and protein-bound glutathione in blood, plasma and tissue., Clinical Chemistry, 41, 448-454, 1995.
36. T. Yoshida, Determination of reduced and oxidized glutathione in reythrocytes by high-performance liquid chromatography with ultraviolet absorbance detection., Journal of Chromatography B., 678, 157-164, 1996.
37. T. Oe, T. Ohyagi, and A. Naganuma, Determination of r-glutamylglutathione and other low-molecular-mass biological thiol compounds by isocratic high-performance liquid chromatography with fluorimetric detection., Journal of Chromatography B., 708, 258-289, 1998.
38. S. Arttamangkul, M.K. bhalgat, R.P. Haugland, Z. Diwu, J. Liu, and D.H. Klaubert, 5-(Pentafluorobenzoylamino)fluorescein: a selective substrate for the determination of glutathione concentration and glutathione transferase activity., Analytical Biochemistry, 269, 410-417, 1999.
39. K.J. Lenton, H.Therriault, and J.R. Wagner, Analysis of glutathione disulfide in whole cells and mitochondria by postcolumn derivatization high-performance liquid chromatography with ortho-phthalaldehyde., Analytical Biochemistry, 274, 125-130, 1999.
40. 王進琦編著，基礎微生物學，p.244-252，中華民國八十二年一月修訂版，藝軒圖書出版社.
41. 郭純育、莊靜芬著，腸內革命：乳酸菌，中華民國九十年一月初版，元氣齋出版社有限公司
42. T. Okubo, N. Ishihara, A. Oura, M. Serit, M. Kim, T. Yamamoto, and T. Mitsuoka, In vivo effects of tea polyphenol intake on human intestinal microflora and metabolism., Bioscince, Biotechology and Biochemistry, 56, 588-591, 1992.
43. Y. Hara, Influence of tea catechins on the digestive tract., Journal of Cellular Biochemistry Supplement, 27, 52-58, 1997.
44. K. Goto, S. Kanaya, T. Ishigami, and Y. Hara, The effect of tea catechins on fecal conditions of elderly residents in a long-term care facility., Journal of Nutrition Science and Vitaminology, 45, 135-141, 1999.
45. N. Volpi, and P. Tarugi, Improvement in high-performance liqiod chromatography malondialdehyde level determination in normal human plasma., Journal of Chromatography B., 713, 433-437, 1998.
46. C.S. Yang, L. Chen, M-J Lee, D. Balentine, M.C. Kuo, and S.P. Schantz, Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers., Cancer Epideminology, Biomaekers and Prevention, 7, 351-354, 1998.
47. W. Wang, and N. Ballatori, Endogenous glutathione conjugates: occurrence and biological functions., Pharmacological Reviews, 50, 335-355, 1998.
48. C.S. Tang, L. Chen, M-J Lee, and D. balentine, Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers., Cancer Epidermiology, Biomarkers and Prevention, 7, 351-354, 1998.
49. M.E. Anderson, Glutathione: an oerview of biosynthesis and modulation., Chemico-Biological Interactions, 111-112, 1-14, 1998.
50. D.O. Kennedy, M. Matsumoto, A. Kojima, and I. Matsui-Yuasa, Cellular thiols status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells., Chemico-Biological Interactions, 122, 59-71, 1999.
51.C.s. Yang, S. Kim, G-Y Yang, M-J Lee, J.Y. Chung, and C-T Ho, Inhibition of carcinogenesis by tea: Bioavailability of tea polyphenils and mechanisms of actions. Proceeding of Social Experimental Biochemistry and Medicine., 220, 231-217, 1999.