臺灣博碩士論文加值系統

海巴戟天(Morinda citrifolia)，屬於茜草科（Rubiaceae），其俗名為Noni，主產於夏威夷群島、大溪地與熱帶亞洲國家，依據民間傳統療法的資料顯示，海巴戟天的樹皮、莖、根、葉、果皆可調製，相傳有糖尿病、高血壓與癌症的預防及治療等醫療保健?效C本論文首要目的是針對不同的海巴戟天之葉、莖及果實粗萃取物進行抗氧化活性分析；並藉抗氧化活性分析的結果確立最佳材料來源與萃取條件。萃取條件使用 80℃ 熱水、50 % 與 99.5% 乙醇、乙酸乙酯及超臨界二氧化碳等萃取方法，抗氧化活性分析採用 Trolox 當量的抗氧化能力、還原力、清除 DPPH 自由基能力、清除活性氧的能力（超氧陰離子、氫氧自由基及過氧化氫等）以及螯合鐵能力。結果顯示海巴戟天的葉在不同溶劑萃取下，存在各式抗氧化活性的能力，顯示海巴戟天的葉中存在極性與非極性的抗氧化成分；高螯合鐵能力成分傾向存在於葉中極性部分，還原力、清除O2-•與 H2O2 的能力傾向存在於葉中非極性部分，清除氫氧自由基能力則多存在於葉中醇溶性部分。海巴戟天之褐莖萃取物，以超臨界二氧化碳萃取的方式所得之抗氧化活性較高，顯示褐莖中抗氧化物質多屬於非極性。不論是何種萃取條件，海巴戟天果實及青莖萃取物的抗氧化活性普遍較差。綜合來說，就材料而言，海巴戟天的葉中萃取物能偵測出較多樣的抗氧化特性與較高的活性，值得作為進一步純化抗氧化物質的材料來源；就萃取條件而言，以乙醇萃取的方法能得到較高的總抗氧化活性，其中又以清除氫氧自由基能力為主。
本論文第二個目的是評估海巴戟天粗萃取物的抗氧化活性與其化學成份的相關性，結果顯示了海巴戟天葉之乙醇萃取物含有較高的總酚類化合物及類黃酮並且與其抗氧化活性有關，但海巴戟天超臨界二氧化碳萃取物的總酚類化合物含量普遍較低。第三個目的是評估海巴戟天萃取物在保護生物細胞膜及 DNA免於氧化性傷害之能力，結果顯示海巴戟天葉之 99.5% 乙醇萃取物對LYD三品系肉豬肝臟微粒體脂質過氧化具有顯著性的抑制能力，此結果可能與其清除氫氧自由基的能力為主有關，並與多酚類物質相關。另一方面海巴戟天葉之熱水及 50% 乙醇萃取物在保護氫氧自由基誘導無細胞膜之人類淋巴球 DNA 氧化傷害上卻出現了較佳的能力，這可能是因其具有強的金屬離子螯合能力及有力的清除氫氧自由基能力。相較之下，海巴戟天葉之99.5% 酒精及乙酸乙酯萃取物在較高劑量（10-100 μg/mL）時反而呈現了促氧化之現象，推測會造成此現象的原因可能是海巴戟天葉之 99.5% 乙醇及乙酸乙酯萃取物具有較高的還原力，會將金屬離子還原而造成促氧化作用。

Morinda citrifolia（Rubiaceae）, commonly known as Noni, is a plant typically found in the Hawaiian, Tahitian and tropical Asia. The bark, stem, roots, leaves, and fruits have been used traditionally as a folk remedy for many diseases including diabetes, hypertension, and cancer. The first purpose of this study was focused on the antioxidative properties of various crude extracts from leaves, stems and fruits of Noni. With the results of antioxidative analysis, the optimal material of Noni plant and extract condition would be defined. Hot water (80℃), 50% aqueous or absolute ethanol, ethyl acetate and supercritical fluid carbon dioxide (SF-CO2) were used as solvents to extract. The antioxidative activity of crude extracts were measured by Trolox equivalent antioxidant capacity (TEAC) assay, the reducing power assay, the bleaching of 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical, the scavenging ability of reactive oxygen species (superoxide ion, hydroxyl radical and hydrogen peroxides) and the chelating activity on ferrous ions. The results suggested that antioxidative activity of Noni’s extracts might be due to both polar and non-polar compounds. Hot water and 50 % aqueous ethanol extracts from leaves and stems exhibited higher chelating activity on ferrous ions. Ethyl acetate extracts from leaves showed the highest scavenging ability of superoxide ion, and SF-CO2（1500 psi）extracts from leaves displayed significantly the scavenging ability of hydrogen peroxides. On the other hand, the absolute ethanol extracts of Noni’s leaves and green stems, and the SF-CO2 （3500 psi） extracts of brown stems had higher scavenging ability to hydroxyl free radical. However, fruits extracts were showed very poor as antioxidants. To sum up, leaves of Noni exhibited higher and variety of antioxidative activity, which is comparable to that of both stems and fruits. Noni leaf is worth further research in purification and identification of active compounds. As for extraction condition, ethanol extracts had higher total antioxidative activity, especially in scavenging of hydroxyl radical.
The second purpose was to study the relationship between the antioxidative activities and chemical constituents in the crude extracts of Noni. The data showed that ethanolic extracts of Noni’s leaves had higher total polyphenolic and flavonoid contents and revealed that these compounds played a part in antioxidative activities. However, SF-CO2 extraction of Noni generally had lower total polyphenolic contents. The third study was to prove the Noni’s extracts in the protection ability of biological cell membrane and DNA which were attacted by oxidative damage. The results indicated that the absolute ethanol extracts of Noni’s leaves display significantly the inhibition ability of lipid peroxidation of microsomes from LYD pig liver. The scavenging mechanism of the absolute ethanol extracts of Noni’s leaves might have relation with hydroxyl radical. The antioxidant components might be attributed to higher content of polyphenolic compounds. On the other hand, the hot water and 50% ethanolic extracts of Noni’s leaves display the better protection ability from hydroxyl radical-induced, DNA damage in human lymphocyte without cell membrane. The antioxidant mechanism may be attributed to a strong metal-chelating ability and their effectiveness as good scavengers of hydroxyl radicals. By contrast, the absolute ethanol and ethyl acetate extracts of Noni’s leaves showed the pro-oxidative activity in the higher dosage extracts (10-100 μg/mL). The higher reducing power, which reduced the ferric ion to ferrous ion, appeared to be responsible for the pro-oxidative activity of the absolute ethanol and ethyl acetate extracts of Noni’s leaves.

目 錄

中文摘要…………………………………………………………………….I

英文摘要…………………………………………………………………….III

謝誌………………………………………………………………………….VII

目錄………………………………………………………………………….IX

表次………………………………………………………………………….XIII

圖次………………………………………………………………………….XIV

縮寫對照表………………………………………………………….……....XVII

壹、前言…………………………………………………………………….1
貳、文獻回顧……………………………………………………………….4
一、自由基相關文獻回顧……………………………………………….4
(一) 自由基之定義及反應型式………….………………………….4
(二) 自由基的種類…………….………….……………………...….5
(三) 自由基對生物體分子的傷害.................................................….12
(四) 抗氧化劑作用原理及機制..........................................................13
二、海巴戟天相關文獻回顧.....................................................................15
(一) 海巴戟天的民間傳統用法……………………………………...16
(二) 海巴戟天之主要成分………………………………….................18
(三) 海巴戟天之生物活性.....................................................................18
參、材料與方法................................................................................................23
一、實驗架構……………………………………………………………....23
二、實驗材料................................................................................................26
(一)原料...................................................................................................26
(二)藥品...................................................................................................27
(三)溶劑...................................................................................................29
(四)豬肝...................................................................................................29
(五)人體血液….......................................................................................29
三、儀器設備................................................................................................29
四、實驗方法................................................................................................30
(一)樣品的製備.......................................................................................30
1.超臨界二氧化碳萃取.......................................................................30
2.乙酸乙酯萃取...................................................................................31
3. 99.5%乙醇萃取................................................................................31
4.50%乙醇萃取....................................................................................32
5.純水萃取...........................................................................................32
(二).抗氧化活性之測定..........................................................................32
1.總抗氧化能力(TEAC)之測定..........................................................32
2.還原力測定.......................................................................................33
3.清除自由基及活性氧能力之測定...................................................34
(1).清除DPPH自由基能力之測定..................................................34
(2).清除超氧陰離子能力之測定......................................................35
(3).清除氫氧自由基能力之測定......................................................37
(4).清除過氧化氫能力之測定..........................................................38
4.螯合鐵能力之測定...........................................................................39
(三)抗氧化活性成分分析……………………………………………...40
1.總酚類化合物含量測定...................................................................40
2.類黃酮含量測定...............................................................................40
3.海巴戟天葉之99.5%及50%乙醇萃取物有效成分之初步分離...41
(四).對生物體脂質及DNA氧化傷害之保護作用測定.......................42
1.對豬肝微粒體脂質過氧化之保護作用測定..................................42
2.對人類淋巴球DNA氧化傷害之保護作用測定............................43
五、統計分析................................................................................................45
肆、 結果與討論..............................................................................................46
一、不同萃取方法對海巴戟天葉、莖、果實之粗萃取率的影響............46
二、海巴戟天葉、莖、果實之粗萃取物的抗氧化活性分析....................46
(一) 總抗氧化力(TEAC)........................................................................46
(二) 還原力.............................................................................................48
(三) 清除自由基及活性氧能力.............................................................49
　 1.清除DPPH自由基之能力...............................................................49
　 2.清除超氧陰離子之能力...................................................................51
　 3.清除氫氧自由基之能力...................................................................53
　 4.清除過氧化氫之能力.......................................................................54
(四)螯合亞鐵離子之能力.......................................................................55
(五)討論...................................................................................................56
三、海巴戟天葉、莖、果實之粗萃取物的抗氧化成分分析....................57
(一)總酚類化合物含量...........................................................................57
(二)類黃酮含量.......................................................................................58
(三)海巴戟天葉之99.5%及50%乙醇萃取物有效成分之初步分離...59
1.海巴戟天葉99.5%乙醇萃取物初步分離的結果.........................59
2.海巴戟天葉50%乙醇萃取物初步分離的結果............................60
(四)討論.................................................................................................61
四、海巴戟天葉溶劑萃取物對生物體脂質及DNA氧化傷害之保護作用
(一)對豬肝微粒體脂質過氧化之保護作用.........................................61
(二).對人類淋巴球DNA氧化傷害之保護作用.................................62
(三)討論.................................................................................................65
伍、 結論........................................................................................................100
陸、參考文獻..................................................................................................102

1. Halliwell B. and Gutteridce J. M. C., Free radical in biologv and medicine, 2nd ed., Clarendon Press, Oxford, 1989.
2. Halliwell B., Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans, Free Radical Research., 25: 55-74, 1996.
3. Gutteridge J. M. C. and Halliwell B., Antioxidants in nutrition health and disease, 1th edition, Oxford: Oxford University Press, 1994.
4. Hirazumi A., Furusawa E., Chou SC. and Hokama Y., “Anticancer activity of Morinda citrifolia (noni) on intraperitoneally implanted Lewis lung carcinoma in syngeneic mice.”Proceedings of the Western Pharmacology Society, 37: 145-6, 1994.
5. Hirazumi A., Furusawa E., Chou S. C. and Hokama Y.,“Immuno -modulation contributes to the anticancer activity of Morinda citrifolia (noni) fruit juice.” Proceedings of the Western Pharmacology Society, 39: 7-9, 1996.
6. Liu G., Bode A., Ma WY., Sang S., Ho CT. and Dong Z., “Two novel glycosides from the fruits of Morinda citrifolia (noni) inhibit AP-1 transactivation and cell transformation in the mouse epidermal JB6 cell line.”Cancer Research, 61(15): 5749-56, 2001.
7. Sang S., He K., Liu G., Zhu N., Cheng X., Wang M., Zheng Q., Dong Z., Ghai G., Rosen RT. and Ho CT.,“A new unusual iridoid with inhibition of activator protein-1 (AP-1) from the leaves of Morinda citrifolia L.”Organic Letters, 3(9): 1307-9, 2001.
8. Hirazumi A. and Furusawa E.,“An immunomodulatory polysaccharide-rich substance from the fruit juice of Morinda citrifolia (noni) with antitumour activity.”Phytotherapy Research, 13(5): 380-7, 1999.
9. Sang S., Cheng X., Zhu N., Stark RE., Badmaev V., Ghai G., Rosen RT. and Ho CT.,“Flavonol glycosides and novel iridoid glycoside from the leaves of Morinda citrifolia. ”J.Agric.& Food Chem., 49(9): 4478-81, 2001.
10. Zin ZM., Abdul-Hamid A., and Osman A.,“Antioxidative activity of extracts from Mengkudu (Morinda citrifolia L.) root,fruit and leaf.” Food Chemistry, 78: 227 –231, 2002.
11. Cacciuttolo MA., Trinh L., Lumpkin JA., Rao G.,“Hyperoxia induces DNA damage in mammalian cells. ”Free Rad. Biol. Med., 14: 267-276, 1993.
12. Halliwell B., Murcia MA., Chirico S., Aruoma OI.,“ Free radicals and antioxidants in food and in vivo: What they do and how to work.”Crit Rev. Food Sci. Nutr., 35: 7-20, 1995.
13. Kehrer J. P.,“Mechanisms and effect of lipid peroxidation.”Crit. Rev. Toxicol., 23: 21-48, 1993.
14. Lander H. M.,“An essential role for free radicals and derived species in signal transduction. ”FASEB J., 11: 118-124,1997.
15. 郭靜娟，“薏苡籽實之抗氧化成分及其抑制自由基傷害之研究”。國立台灣大學食品科技研究所博士論文，2001。
16. Young T. A., Cunningham C. C., Bailey S. M., ”Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol. ”Archives of Biochem. Biophy., 405: 65-72, 2002.
17. Anthony W. S., Arie A.,“The biochemical basis of the NADPH oxidase of phagocytes”. TIBS., 18: 43-47, 1993.
18. James LG., Sareen SG.,“Advanced nutrition and human metabolism ”. 3rd ed., Wadsworth Press, USA, 2000.
19. Guerciolini R., Szumlanski C., Weinshiboum R. M.,“Human liver xanthineoxidase: nature and extent of individual variation”. Clin. Pharmzcol. Ther., 50:663-672, 1991.
20. Ünlü A., Sucu N., Tamer L., Coskun B., Yücebilgiç G., Ercan B., Gül A., Dikmengil M., Atik U.,“Effects of Daflon on oxidative stress induced by hindlimb ischemia/reperfusion”. Pharmacological Res., 48:11–15, 1998.
21. Diplock A. T., Charleux J. L., Crozier-Willi G, Jok F. J., Rice-Evans C., Roberfroid M., Stahl W., Vina-Ribes J.,“Functional food science and defense against reactive oxidative species”. British J. Nutr., 80:S77-S112, 1998.
22. Przedborski S., Jackson-Lewis V., Yokoyama R., Shibata T., Dawson V. L., Dawson T. M.,“Role of neuronal nitric oxide in 1-methyl-4 -phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced dopaminergic neurotoxicity”. Proc. Natl. Acad. Sci.,93:4546-4571,2000.
23. Kristal B. S., Park B. K., Yu B. P.,“4-hydroxylhexenal is potent inducer of the mitochondrial permeability transit”. J. Biol. Chem., 271:6033-6038, 1996.
24. Marnett L. J., Burcham P. C.,“Endogeneous DNA adducts: potential and paradox”. Chem. Res. Toxicol., 6:771-785, 1993.
25. Gutteridge J., Halliwell B.,“The measurement and mechanism of lipid peroxidation in biological systems”. Trends Biochem. Sci., 15:129-135, 1990.
26. Berliner J. A., Navab M., Fogelman A. M., Frank J. S., Demer L. I., Edwares P. A., Watson A. D., Lusis A. J.,“Atherosclerosis: basic mechanisms, oxidation, inflammation, and genetics”. Circulation., 91:2488-2496, 1995.
27. Branen A. L.,“Toxicology and biochemistry of butylated hydroxy anisole and butylated hydroxytoluene”. J. Am. Oil Chem. Soc., 52: 59-63 ,1975.
28. Ito N., Fukushima S., and Tsuda H.“Carcinogenicity andmodification of the carcinogenic response by BHA, BHT and other antioxidants”. CRC Crit. Rev. Toxicol., 15: 109-150 , 1985.
29. Dziezak J. D.,“ Preservatives: antioxidant”. Food Technol. 40:94-102 , 1986.
30. 拱玉郎。“天然抗氧化劑發展近況”。食品工業，29（3）：29-37，1997。
31. 蕭培根。“中國本草圖錄”。商務印書館有限公司。香港。1988-1997。
32. Mian-Y., et al., “Morinda citrifolia(Noni):A literature review and recent advances in Noni research”. Acta. Pharmacologica Sinica., 23(12):1127-1141, 2002.
33. 張慧敏。“神奇的諾麗”。生智出版社。台灣。2002。
34. Levand O., Larson H. O., “Some chemical cons tituents of Morinda citrifolia”. Planta Med., 36: 186-7, 1979.
35. Heinicke R.,“The pharmacologically active ingredient of Noni”.Bulletin of the National Tropical Botanical Garden, 1985
36. Atkinson N.,“Antibacterial substances from flowering plants. ”. Australian J. Exper. Biol., 34: 17-26, 1956.
37. Bushnell O. A., Fukuda M, Makinodian T.,“The antibacterial properties of some plants found in Hawaii”. Pacific Science., 4: 167-83, 1950.
38. Leach A. J., Leach D. N., Leach G. J.,“Antibacterial activity ofsome medicinal plants of Papua New Guinea”. Sci. New Guinea., 14: 1-7, 1988.
39. Duncan S. H., Flint H. J., Stewart C. S.,“Inhibitory activity of gut bacteria against Es cherichia coli 0157 mediated by dietary plant metabolites”. FEMS Microbiol Lett.,164: 283-58, 1998.
40. Umezawa K.,“Isolation of 1-methoxy-2-foremyl-3-hydroxyl- anthraquinone from Morinda citrifolia and neoplasm inhibitors containing the same”. Japan Kokai Tokyo Koho J. P., 736: 94-87, 1992.
41. Author unlis ted.,“Noni plant may help TB”. AIDS patient care STDS., 15: 175, 2001.
42. Hiramatsu T., Imoto M., Koyano T., Umezawa K., “Induction of normal p henotypes in ras -trans fo rmed cells bydamnacanthal from Morinda citrifolia”. Cancer Lett., 73:161-6, 1993.
43. Hiwasa T., Arase Y., Chen Z., Kita K., Umezawa K., Ito H., et.al. “Stimulation of ultraviolet-induced apoptos is of humanfibroblast UVr-1 cells by tyrosine kinas e inhibitors”. FEBS Lett.,444: 173-6, 1999.
44. Raj R. K.,“Screening of indigenous plants for anthelmintic action against humanAscaris Lumbricoides: Part-II”. Indian J. Physiol. Pharmacol., 19: 47-9, 1975.
45. Joseph B.,“Noni: Polynesia’s natural pharmacy”. Pride Publishing , p 13. 1997.
46. Younos C, Rolland A, Fleurentin J, Lanhers MC, Miss lin R,Mortier F. Analgesic and behavioural effects of Morinda citrifolia”. Planta. Med., 56: 430-4, 1990.
47. Youngken H. W.,“A study of the root of Morinda citrifolia Linn I”. J. Am. Pharm. Assoc., 47: 162-5, 1958.
48. Youngken H. W., Jenkins H. J., Butler C. L.. “Studies on Morinda citrifolia L. II”. J. Am. Pharm. Assoc., 49: 271-3, 1960.
49. Moorthy N. K., Reddy G. S.,“Preliminary phytochemical and pharmacological study of Morinda citrifolia Linn”. Antiseptic., 67: 167-71, 1970.
50. 潘懷宗、劉晉魁、周良穎、謝秉甫、李沐勳 ，“利用超臨界二氧化碳萃取肉桂中精油成分：並與水蒸氣蒸鎦法進行比較”。 J. Chin. Med., 5(3):199-207, 1994.
51. 葉東柏、郭建民，“市售罐裝茶飲料中兒茶素類及咖啡因含量之分析”。 藥物與食品分析， 6:447-454， 1998。
52. Yu T. W., and Ong C. N., “Lag-Time Measurement of antioxidant capacity using myoglobin and 2,2’-azino-bis (3-ethylbenzthiazoline-6- sulfoni cacid): rationale, application, and limitation”. Anal. Biochem., 275:217-223, 1999.
53. Oyaizu M., “Antioxidative activity of browning products of glucosamine fractionated by organic solvent and thin-layer chromatography”. Nippon Shokuhin Kogyo Gakkaishi., 35:771-775, 1986.
54. Shimada K., Fujikawa K., Yahara K. and Nakamura T., “Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion” . J. Agric. Food Chem., 40: 945-948, 1992.
55. Brand-Williams W., Cuvelier M. E. and Berset C.“Use of a free radical method to evaluate antioxidant activity”. Lebensm-Wiss. U.Technol., 28: 25-30, 1995.
56. Ghiselli A., Nardini M., Baldi A. and Scaccini C., “Antioxidant activity of different phenolic fractions separated from an Italian red wine” . J. Agric. Food Chem., 46: 361-367, 1998.
57. Halliwell B., et al., “The deoxyribose method: a simple ''test tube'' assay for determination of rate constants for reaction of hydroxyl radicals” . Anal. Biochem., 165:215, 1987.
58. Rinkus, S., et al., “Analysis of hydrogen peroxide in freshly prepared co.ees”. Food and Chemical Toxicology, 28, 323–331, 1990.
59. Aruoma O. I. and Haillwell B., “The iron-binding and hydroxyl radical scavenging action of anti-inflammatory drugs” . Xenobiotica., 18:459-70, 1988.
60. Germano, M. P., et al., “Evaluation of Extracts and Isolated Fraction from Capparis spinosa L. Buds as an Antioxidant Source ”. J. Agric. Food Chem.,50(5); 1168-1171, 2002.
61. Jia Z. S., Tang M. C., Wu J. M.,“The determination of flavonoid contents in mulberry and their scavenging effects on radicals”. Food Chem., 64:555-559. 1999.
62. 日本健康營養食品協會。健康食品規則基公示。平成7年6月1 日
63. Anderson D., et al., “Flavonoids modulate Comet assay responses to food mutagens in human lymphocytes and sperm”. Mutation Research ,269-277, 1998
64. McConkey D. J., et al.,“Calcium-activated DNA fragmentation kills immature thymocytes”. FASEB J., 3:1843-1849, 1989
65. 吳明娟，梁桂容，王麗淑，郭玫君。“芫荽(Coriandrum sativum)抽出液對DNA氧化傷害之保護”。嘉南學報，26:192-199，2000。
66. Pitotti A.,Elizalde B., and Anese M.,“Effectof charamelization and Maillard reaction products on peroxidase activity”. J. Food Biochem., 18:445-457, 1995.
67. Kim S. J., Han D. and Rhee J. S.,“Measurement of superoxide dismutase-like activity of natural antioxidants. Biosci”. Biotech. Biochem., 59:5.822-826, 1995.
68. 謝秋蘭，“杜仲水萃取物抗氧化性機能之研究”。國立中興大學食品科學研究所博士論文，2000。
69. Gordon M. H.,“The mechanism of antioxidant action in vitro”., Elsevier Applied Science, London and New York, 1990.
70. Aruoma O. I., Halliwell B. and Williamson G.,“In vitro methods for characteracizing potential prooxidant and antioxidant actions of nonnutrive substances in plant foods”. AOCS press , Champaign, Illinois, 1998.