臺灣博碩士論文加值系統

本實驗針對蜂膠成份之主要活性物-CAPE能抑制結腸癌細胞、白血病細胞、多種被病毒感染的細胞、皮膚癌細胞的生長。以化學療法的觀點，發展並研究出蜂膠的有效成份，以供臨床上抗發炎，抗過敏，抗突變及抗病毒之應用。首先設計出 (1) 結構具有兩個芳香環，(2)中間有脂類連接和含有羥基的9種化合物，命名為 3-(3,4-dihydroxyphenyl)acrylic acid phenethyl ester (CAPE), 3-(3,4-dimethoxyphenyl)acrylic acid phenethyl ester (PEDMC), 3-(3,4-dihydroxyphenyl)acrylic methyl ester (MC), 3-(3,4-dihydroxyphenyl) acrylic acid ethyl ester (EC), 3-(4-bromophenyl)acrylic acid phenethyl ester (BrCAPE), 3-(3,4-dimethoxyphenyl)acrylic acid ethyl ester, 3-(4-methoxyphenyl)acrylic acid ethyl ester, 2,3-dibromo-3-(3,4-dimethoxyphenyl)propoinic acid ethyl ester, and 2,3-dibromo-3-(4-methoxyphenyl)propoinic acid ethyl ester。並以acyl chloride的中間產物進行酯化反應，得到高產率的合成方法及縮短反應時間。本實驗以CAPE、PEDMC和MC作用於抑制HIV複製上，利用三株不同的HIV-1 isolates M-tropic (JRCSF), T-tropic (NL-4-3) and Dual tropic (89.6) ，結果顯示CAPE和MC對於HIV之複製有明顯抑制作用，於濃度10 M至100 M之間尤其明顯，PEDMC的抑制作用沒有CAPE和MC明顯。在口腔癌方面，我們選用四株人類口腔正常細胞 (BF)和癌細胞(OSF, GNM, TSCCa)，以MTT assay分析上述9種不同化合物(analogues)對於正常及癌細胞的細胞毒性，結果顯示以CAPE、MC和EC對OSF(癌前細胞)、GNM（齒齦癌細胞）和TSCCa(舌癌細胞) 有明顯細胞毒性，其毒性隨濃度增加而增加，但MC對於正常BF細胞有少許細胞毒性；含Br-基的化合物，除BrCAPE對GNM（齒齦癌細胞）有明顯細胞毒性外，2,3-dibromo-3-(3,4-dimethoxyphenyl)-propionic acid ethyl ester和2,3-dibromo-3-(4-methoxyphenyl)-propionic acid ethyl ester並無法有效抑制癌細胞生長，故可推知caffiec acid系列的結構具有兩個芳香環或中間有脂類連接和含有羥基，在治療口腔癌部分有明顯效果，因此可深入探討作用機制，預期成為一治療口腔癌的新藥。

Propolis, a honey bee hive product, exhibits a broad spectrum of medical application including antibiotic, antiviral, anti-inflammatory and tumor growth inhibition; some of the observed biological effects may be due to caffeic acid (cinnamic acid) esters that are present in propolis. In the present work we synthesized five caffeic acid esters, namely 3-(3,4-dihydroxyphenyl)acrylic acid phenethyl ester (CAPE), 3-(3,4-dimethoxyphenyl)acrylic acid phenethyl ester (PEDMC), 3-(3,4-dihydroxyphenyl)acrylic methyl ester (MC), 3-(3,4-dihydroxyphenyl) acrylic acid ethyl ester (EC), 3-(4-bromophenyl)acrylic acid phenethyl ester (BrCAPE), and four structure-like compounds, namely 3-(3,4-dimethoxyphenyl)acrylic acid ethyl ester, 3-(4-methoxyphenyl)acrylic acid ethyl ester, 2,3-dibromo-3-(3,4-dimethoxyphenyl)propoinic acid ethyl ester, 2,3-dibromo-3-(3,4-dimethoxyphenyl)propoinic acid ethyl ester and tested them against the PBMC cell infected by M-tropic (JRCSF), T-tropic (NL-4-3) and Dual tropic (89.6) of HIV-1 isolates. The CAPE and MC show significant inhibition on the growth of HIV-replication in PBMC cell, but the PEDMC shows less inhibition effect than the CAPE and MC. In addition, the effect of these agents on the growth of normal human buccal mucosal fibroblast (BF), oral submucosus fibroblast (OSF), neck metastasis of Gingiva carcinoma (GNM), tongue squamous cell carcinoma (TSCCa) was studied by the MTT assay. Treatment with 25-200 M CAPE and 50-400 M EC shown significant cytotoxicity on OSF, GNM, TSCCa cells, but not on BF cell. The results suggests that CAPE-like compounds be acting as good chemotherapy agents against HIV infection and oral cancer.

目 錄
頁碼
中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………2
緒論………………………………………………………………………3
研究目的…………………………………………………………………13
實驗材料及方法…………………………………………………………14
結果………………………………………………………………………32
討論………………………………………………………………………41
結論………………………………………………………………………45
參考文獻…………………………………………………………………47
表…………………………………………………………………………55
圖…………………………………………………………………………66
附件一、NMR光譜圖……………………………………………………78
附件二、已發表之文章…………………………………………………93
附表(圖)

1. Rous, P. A sarcoma of the fowl transmissble by an agent separable from the tumor cell. J. Exp., 13: 397-411, 1911.
2. Burkitt, D., O’Connor, G. Malignant lymphoma in African children. I. Clinical syndrome. Cancer, 14: 258-269, 1961.
3. Epstein, M., Achong, B., Barr, Y. Virus partical in cultured lymphoblasts from Burkitt’s lymphoma. Lancet., 1: 702-702, 1964.
4. Gallo, R., Kalyanaraman, V., Sarngadharan, M. Association of the human type C retrovirus with a subset of adult T-cell cancer. Cancer Res., 43: 3892-4899, 1983.
5. Hinuma, Y., Nagata, K, Hanaoka, M. Adult T-cell leukemia antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc. Natl. Acad. Sci. USA, 78: 6476-6480, 1982.
6. Bishop, J. The molecular genetics of cancer. Leukemia, 2: 199-208, 1988.
7. Beasley, R. Hepatitis B virus-the major etiology of hepatocellular carcinoma. Cancer, 61: 1942-1956, 1988.
8. London, T., Blumberg, B. A cellular model for the role of hepatitis B virus in the pathogenesis of hepatocellular carcinoma. Hepatology, 2: 10-14, 1982.
9. Zur, H. Papillomavirvus in human cancer. Cancer, 59: 1692-1696, 1987.
10. Lasky, L., Groopman, J., Fennine, C. Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. Science, 33: 209-212, 1986.
11. Kensler, T. W., and Taffe, B. G. Free radicals in tumor promotion. Adv. Free Radical Biol. Med., 2: 347-387, 1986.
12. Harris, M., Patenaude, P., Cooperberg, P., Fillipenko, D., Thorne, A., Raboud, J., Rae, S., Dailey, P., Chernoff, D., Todd, J., Conway, B., Julio, S., Montaner, G., and the INCAS Study Group. Correlation of virus load in plasma and lymph node tissue in hamn immunodeficiency virus infection. J. I. D., 176: 1388-1392, 1997.
13. Mitsya,. H., Border, S.: Strategies for antiviral therapy in AIDS. Nature, 325: 773-778, 1987.
14. Yarchoan, R., Broder, S. Anti-retroviral therapy of AIDS and related disorders: General principles and specific development of dideoxynucleosides. Pharmaco. Ther., 40: 329-348, 1989.
15. Roxenbaum, W., Dormnt, D. Antimonitunsstate (HPA 23) treatment of three patient with AIDS and one with prodrome. Lancet i., 450-451, 1985.
16. Mitsuya, H., Popvic, M. Suramin protection of T cell in vitro against infectivity and cytopathic effect of HTLV-III. Science, 266: 172-174, 1984.
17. Richman, D., Fischl, M. The toxicity of azidothymidine (AZT) in the treatment of patient with AIDS and AIDS-related complex. N. Eng. J. Med., 317: 192-197, 1987.
18. Mitsuya, D., Jarrett, R. Long-term ingibition of human T-lymphotropic virus type III/ lymphadenopathy-associated virus (human immundeficiency virus) DNA synthesis and RNA expression in T cells protected by 2’, 3’-dideoxynucleosides in vitro. Proc. Nalt. Acad. Sci. USA., 84: 2033-2037, 1987.
19. Lader, B., Darby, G. HIV wuth reduced senitivity to Zidovudine (AZT) isolated during prolong therapy. Science, 231: 1731-1734, 1989.
20. 蔡崇弘，邱清華：臺灣地區口腔癌發生率研究．中華牙誌　9: 104-115, 1990.
21. Chen CH：An epidemiological study of oral squamous cell carcinoma in southern Taiwan. J. Formosan Dent. Assoc., 10: 268-274, 1987.
22. Kwan HW：A statistical study on oral carcinomas in Taiwan with emphasis on the relationship with betel nut chewing：A preliminary report. J. Formosan Med. Assoc., 75: 497-505, 1976.
23. 關學婉, 蕭裕源: 台灣口腔癌之流行病學研究. 科學發展月刊. 4: 25-29, 1979.
24. 黃穰基, 張哲壽, 周明哲, 藍中孚: 國人頰部口腔癌與嚼檳榔關係. 中華民國口腔顎面外科學會雜誌. 3: 23, 1991.
25. 黃湧澧, 林立民, 葛應欽: 檳榔塊與口腔癌. 公共衛生雜誌. 19: 371-383, 1993.
26. Cancer registry annual report in Taiwan area, 1982-1986.
27. 賴德榮, 陳鴻榮: 細胞學在口腔癌之診斷價值..高雄醫學科學雜誌. 2: 122-130, 1986.
28. 邱顯斌, 謝地: 用甲苯氨藍染口腔的病變. 中華民國耳鼻喉科醫學會雜誌. 20: 168-173, 1985.
29. Liu, B.U., Chiang, C. P., Yao, Y. T. How SW: Histological observation on oral cancer lesions after adjuvant chemotherapy. J. Formosan Med. Assoc., 87: 164-171, 1988.
30. 謝地: 頭頸部癌之Bleomycin治療效果臨床及病理組織學研究. 中華民國耳鼻喉科醫學會雜誌. 5: 87-107, 1970.
31. 林宗洲: 頭頸部癌症之全身性合用化學藥物治療法.中華民國耳鼻喉科醫學會雜誌. 11: 29-33, 1976.
32. Jeddar, A., Khasany, A., Ramsaroop, V. G., Bhamjei, A., Haffejce, I. E., and Moosa, A. The antibacterial action of honey: an in vitro study. S. Afr. Med. J., 67: 257-258, 1985.
33. Hladon, B., Bylka, W., Wojtaszek, M. E., Skyzypczale, L., Szafarele, P., Chodera, A., and Kowalewski, Z. In vitro studies on the cytostatic activity of propolis extracts. Arzneim.-Forsh. Drug Res., 30: 1847-1848, 1980.
34. Koshihara, Y., Neichi, T., Murota, S. L., Lao, A., Fujimoto, Y., and Tarsuno, T. Caffeic acid is selective inhibitor for leukotriene biosynthesis. Biochim. Biophys. Acta, 792: 92-97, 1984.
35. Gribel, N, V., and Pashinskii, V. G. Antitumor properties of honey. Vopr. Onkol., 36: 704-709, 1990.
36. Greenaway, W., Scaysbrook, T., and Whatley, F. R.. The analysis of bud exudate of Populus×euramericana and of propolis by gas chromatography-mass spectrometry. Proc. R. Soc. Lnd. B. Biol. Sci., 232: 249-272, 1987.
37. Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E., Efors, L, Caldwell, M., Estevez, V., and Nakanishi, K. Perferential cytoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia, 44: 230-232, 1988.
38. Wattenberg, L. W., Coccia, J. B., and Lans, L. K. Inhibitory effects of phenolic compounds on benzo(a)pyrene-induced neoplasia. Cancer Res., 40: 2820-2823, 1980.
39. Su, Z-z., Grunberger, D., and Fisher, P. Suppression of adenovirus type 5 E1A-mediated transformation and expression of the transformed phenotype by caffeic acid phenethyl ester (CAPE). Mol. Carcinog., 4: 231-242, 1991.
40. Guarini, L., Su, Z-z., Zucker, S., Lin, J., Grunberger, D., and Fisher, P. Growth inhibition and modulation of antigenic phenotype in human melanoma and glioblastoma multiforme cells by caffeic acid phenethyl ester (CAPE). Cell. Mol. Biol., 38: 513-527, 1992.
41. Rao, C., Desai, D., Kaul, B., Amin, S., and Reddy, R. Effect of caffeic acid esters on carcinogen-induced mutagenicity and human colon adenocarcinoma cell growth. Chem. Biol. Interact., 84: 277-290, 1992.
42. Rao, C. V., Desai, D., Simi, B., Kulkarni, N., Amin, S., and Reddy, B. S. Inhibitory effects of caffeic acid esters on azoxymethane-induced biochemical changes and aberrant crypt foci formation in rat colon. Cancer Res., 53: 4182-4188, 1993.
43. Frankel, K., Wei, H., Bhimani, R., Zadunaisky, J. A., Ferraro, T., Huang, M. T., Conney, A. H., and Grunberger, D. inhibition of tumor promoter-mediated processes in mouse skin and bovine lens by caffeic acid phenethyl ester. Cancer Res., 53: 1255-1261, 1993.
44. Chen, J. H., Shao, Y., Huang, M. T., Chin, C. K., and Ho, C. T. Inhibitory effects of caffeic acid phenethyl ester on human leukemia HL-60 cells. Cancer Lett., 108: 211-214, 1996.
45. Sud’ina, G., Mirzoeva, O., Puskareva, M., Korshunova, G., Sumbatyan, N., and Varfolomeev, S. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett., 329: 21-24, 1993.
46. Chiao, C., Carothers, A., Gruberger, D., Solomon, G., Preston, G., and Barrett, J. Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells. Cacer Res., 55: 3576-3583, 1995.
47. Natarajan, K., Singh, S., Burke, T., Grunberger, D., and Aggarwal, B. Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor, NF-B. Proc. Nttl. Acad. Sci. USA, 93: 9090-9095, 1996.
48. Zheng, Z. S., Xue, G. Z., Grunberger, D., and Prystowsky, J. H. Caffeic acid phenethyl ester inhibits proliferation of human keratinocytes and interferes with the EGF regulation of ornithine decarboxylase. Oncology Res., 7(9): 445-452, 1995.
49. Courtois, S. J., Segaert, S., Degreef, H., Bouillon, R., and Garmyn, M. Ultraviolet B suppresses vitamin D receptor gene expression in keratinocytes. Biochem. Biophy. Res. Comm., 246(1): 64-69, 1998.
50. Fesen, M. R., Kohn, K. W., Leteurtre, F., and Pommier, Y. Inhibitors of human immunodeficiency virus integrase. Pro. Natl. Acad. Sci. USA. 90: 2399-2403, 1993.
51. Fesen, M. R., Pommier, Y., Leteurtre, F., Hiroguchi, S., Yung, J., and Kohn, K. W. Inhibition of HIV-1 integrase by flavones, caffeic acid phenethyl ester (CAPE) and related compounds. Biochem. Pharm., 48(3): 595-608, 1994.
52. Burke, T. R., Fesen, M. R., Mazumder, A., Wang, J., Carothers, A. M., Grunberger, D., Driscoll, J., Kohn, K., and Pommier, Y. Hydroxylated aromatic inhibitors of HIV-1 integrase. J. Med. Chem., 38(21): 4171-4178, 1995.
53. Mazumder, A., Wang, S., Neamati, N., Nicklaus, M., Sunder, S., Chen, J., Miline, G. W., Rice, W. G., Burke, T. R., and Pommier, Y. Antiretroviral agents as inhibitors of both human immundeficiency virus type 1 integrase and protease. J. Med. Chem., 39(13): 2472-2481, 1996.
54. Nicklaus, M. C., Neamati, N., Hong, H., Mazumder, A., Sunder, S., Chen, J., Milne, G. W., and Pommier, Y. HIV-1 integrase pharmacophore: discovery of inhibitors through three-dimensional database searching. J. Med. Chem., 40(6): 920-929, 1997.
55. Bohm, K. The flavonoids. Editio Cantor, Aulendar Wurtt. 1968.
56. Gabor, M. The anti-inflammatory action of flavonoids. Budapest: Akadcmiai Kiado. 1972.
57. Gabor, M. Anti-inflammatory substances of plant origin. In Vane JR, Ferreira SH(eds): “Handbook of experimental pharmacology: Anti-inflammatory drugs.” New York: Springer Verlag 1979, 698-739.
58. Middieton, E., Drzewiccki, G. Effects of flavonoids and transtional metal cations on antigen induced histamine release from human basophils. Biochem Pharmaco, 31: 1449-1453, 1982.
59. Pamukcu, A., Yalciner, S., Hatcher, J., Bryan, G. Quercetin, a rat intestinal bladder carcinogen present in bracken fern. Cancer Res., 40: 3468-3472, 1980.
60. Hardigree, A., Epler, J. Comparative mutagenesis of plant flavonoids in microbial system. Mutation Res., 58: 231-239, 1978.
61. Macgregor, J., Jurd, L. Mutagenicity of plant flavonoids: Structural requiremets for mutagenic activity in Salmonella typhimurium. Mutation Res., 54: 297-309, 1978.
62. Pusztai, R., Beladi, I., Bakai, M., Musi, I., Kukan, E. Study on the effect of flavooids and related substances. I. The effect of quercetin on different viruses. Acta. Microbiologica. Academiae Scientiarum Hungaricac. 13: 113-118, 1968.
63. Zhao, H., Neamati, N., Mazumder, A., Sunder, S., Pommier, Y., Burke, T. R. Jr. J. Med. Chem. 40: 1186, 1997.
64. Alley, M. C., Scudiero, D. A., Monks, A., Hursey, M. L., Czerwinski, M. J., Fine, D. L., Abbott, B. J., Mayo, J. G., Shoemaker, R. H., and Boyd, M. R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res., 48(3): 589-601, 1988.
65. Berger, M. L., and Sozeri, T. Rapid halogenated hycrocarbon toxicity in isolated hepatocytes is mediated by direct solvent effect. Toxicology, 45: 319-330, 1987.
66. Kamchonwongpaisan, S., Paitayatat, S., Thebtaranonth, Y., Wilairat, P., and Yuthavong, Y. Mechanism-based development of new antimalarials: synthesis of derivatives of aryemisinin attached to iron chelators. J. Med. Chem., 38: 2311-2316, 1995.
67. Tanaka, K., Matsuo, K., Nakanishi, A., Hatano, T., Izeki, H., Ishida, Y., and Mori, W. Synthesis and anti-inflammatory and aalgesic activities of hydroxamic acids and acid hydrazides, Chem. Pharm. Bull., 31(8): 2810-2819, 1983.
68. Tschesche, R., Diederich, A., and Jha, H. C., Caffeic acid 4-Rutirioside from Leonurus Cardiaca. Phytochemistry, 19: 2783, 1980.
69. Glover, S. A., Rowbottom, A., Scott, A. P., and Schoonraad, J. L. Alkoxynitrenium ion cyclisations: evidence for differet mechanisms in the formation of benzoxazines and benzoxazepines. Tetrahedron, 46: 7247-7262, 1990.
70. Sekiya, T., Hiranuma, H., Hata, S., Mizogami, S., Hanazuka, M., and Yamada, S. I. Pyrimidine derivatives. 4. Synthesis and antihypertensive activity of 4-amino-2-(4-cinnamoylpiperazino)-6, 7-dimethoxyquinazoline derivatives. J. Med. Chem., 26: 411-416, 1983.
71. Fukuoka, M. Chemical and toxicological studies on Bracken Fern, Petridium aquilinum var. latiusculum. VI. Isolation of 5-O-Caffeoylshikimic acid as an antithiamine factor. Chem. Parm. Bull., 30(9): 3219-3224, 1982.
72. 莊壽洺: 舌癌之病理學研究. 台灣醫學會雜誌. 80: 479-492, 1981.
73. Pindborg, J. J., and Sirsat, S. M. Oral submucous fibrosis. Oral Surg., 2: 764, 1966.
74. Pillai, R., Balaram, P. and Reddiar, K. S. Pathogensis of oral submucous fibrosis. Cancer, 69 (8): 2011-2020, 1992.
75. Pindborg, J. J. Oral cancer and precancer. Bristal: John Wright. 107-113, 1980.
76. Meghij, S., Scutt, A., Harvey, W., Canniff, J. P. An in vitro comparison of human fibroblasts from normal and oral submucous fibrosis tissue. Archs. Oral Biol., 32: 213-215, 1987.
77. Meghji, S., Scutt, A., Harvey, W., Canniff, J. P. An in vitro comparison of human fibroblasts from normal and oral submucous fibrosis tissue. Archs. Oral. Biol., 32: 213-215, 1987.
78. Schneider, E. L., Mitsui, Y. The relationship between in vitro cellular aging and in vivo human age. Proc. Natl. Acad. Sci. USA., 73: 3584-3588, 1976.
79. Ursini, F., Maiorino, M., Morazzoni, P., Roveri, A., and Pifferi, G. A novel antioxidant flavonoid (IdB 1031) affecting molecular mechanisms of cellular activation. Free Radical Biol. Med., 16: 547-553, 1994.
80. Helferich, B., and Vorsatz, J. Esters of caffeic acid, J. Prakt. Chem., 142: 191-192, 1935.
81. Puskas, I., Fields, E. K. 4-Esters of trimellitic anhydride. Ind. Eng. Chem. Prod. Res. Develop., 9: 403-407, 1970.
82. Jiang JD; Wang Y; Roboz J; Strauchen J; Holland JF; Bekesi JG. Inhibition of microtubule assembly in tumor cells by 3-bromoacetylamino benzoylurea, a new cancericidal compound.Cancer Res 58: 2126-2133, 1998.