臺灣博碩士論文加值系統

蜂膠 (Propolis) 是一種複雜的混合物，其主要成份為多酚類物質 (polyphenolic compounds)。從本研究證明蜂膠酒精萃取物 (propolis ethanol extract; PEE) 具有抗自由基的作用：(1) 能清除由NBT-NADH-PMS系統所產生的超氧自由基 (O2-)；(2) 能穩定DPPH．上所帶有的自由基；(3) 能有效地抑制鼠肝微粒體膜上之脂質過氧化的發生；(4) 在總抗氧化能力 (total reactive antioxidant potential test; TRAP) 的實驗中，亦證明PEE具有抗氧化能力。此外，發現PEE能減低H2O2對肝細胞造成傷害的程度：只處理0.2 mM H2O2 6小時，細胞存活率為38.6%；若先處理不同濃度之PEE （25、50、100 g/ml）一小時再加入0.2 mM H2O2作用6小時，細胞之存活率分別提昇為58.7、63.8、72.3%。在本研究的第二部份，由細胞週期變化之分析發現PEE對肝癌細胞 (Hep G2) 之生長有抑制的作用。以PEE處理Hep G2 24小時，其G2/M期有增加之現象，處理Hep G2 48小時，細胞則停滯於G1期；而且這些現象均隨濃度越高而越顯著。本研究所得之結論為：蜂膠對自由基之清除作用與其對肝癌細胞生長的抑制作用，可能與其內所含的類黃素 (flavonoids) 有關。

Propolis is a complicated complex that is rich in phenolic compounds. In this study, the anti-free radical activity of propolis ethanol extract (PEE) was examined by four methods in comparison with pure compounds (caffeic acid, CA; ferulic acid, FA; protocatechuic acid, PA and quercetin, QCT), which can generally be found in propolis. We found that PEE had a high inhibitory effect on the rat liver microsomal membrane to process the NADPH-initiated lipid peroxidation. The results also showed that PEE could effectively stabilized DPPH free radical and had an ability to scavenge superoxide anion radical (O2-．) that generated from the NBT-NADH-PMS system. We also confirmed an antioxidant capacity of PEE in the experiment of TRAP (total reactive antioxidant potential test). The ability of PEE that could protect Chang Liver cells against H2O2 was also examined in our study. The cell viability was only 38.6% when cells were treated with 0.2 mM H2O2 for 6 hr. However, the cell viabilities were raised to 58.7, 63.8, and 72.3% when cells were treated with 25, 50, 100 g/ml of PEE for 1 hr before H2O2 was added, respectively. In the second part of this study, we found that PEE had an inhibitory effect on the growth of liver cancer cells (Hep G2). The cell cycle of Hep G2 was arrested at G1 or G2/M phase, after treating with PEE for 24 or 48hr. In conclusion, we considered that the anti-free radical and anti-cancer properties of PEE might be related to its phenolic constituents, especially the flavonoids.

中文摘要1
英文摘要2
緒論4
蜂膠酒精萃取物 (PEE) 的萃取方法 9
實驗材料與方法10
I. 蜂膠酒精萃取物 (PEE) 對自由基之清除作用
一． 超氧自由基之清除能力 11
二． DPPH自由基之清除能力13
三． 脂質過氧化作用之抑制14
四． 總抗氧化能力之測定16
五． PEE對肝細胞之毒性測試18
六． H2O2對肝細胞之毒性測試方法19
七． PEE對肝細胞之保護作用20
II. 蜂膠酒精萃取物 (PEE) 對肝癌細胞生長之抑制作用
一． PEE對肝癌細胞之毒性測試21
二． PEE對非肝癌細胞之毒性測試22
三． PEE對肝癌細胞生長之影響23
四． PEE對HepG2細胞週期變化之影響24
五． PEE對HepG2造成死亡之方式與程度的分析26
結果28
討論35
圖表45
參考文獻73
附錄一（英文縮寫表示）76
附錄二（實驗器材）77
附錄三（實驗材料來源）78
附錄四（試劑的配製）79
附錄五（類黃素之共同結構與
實驗中使用試劑之結構）80
附錄六（細胞週期調控之圖解）81

1. McCord, J. M. (1987) Oxygen-derived radicals: a link between reperfusion injury and inflammation. Fed. Proc. 46: 2402~2406
2. Munro, J. M. and Cotran, R. S. (1988) The pathogenesis of atherosclerosis: atherosclerosis and inflammation. Lab. Invest. 58: 249~261
3. Steinberg, D. (1991) Antioxidants and atherosclerosis — a current assessment. Circulation 84: 1420~1425
4. Gillery, P., Monboisse, J. C., Maqurt, J. C. and Borel, J. P. (1988) Glycation of protiens as a source of superoxide. Diabetes Metab. 14: 25~30
5. Hunt, J. V., Smith, C. C. T. and Wolff, S. P. (1990) Autoxidative glycosylation and possible involvement of peroxide and free radicals in LDL modification by glucose. Diabetes 39(11): 1420~1424
6. Floyd, R. A. (1990) Role of oxygen radicals in carcinogenesis and brain ischemia. FASEB J. 4: 2587~2597
7. Marnett, L. J. (1987) Peroxyl free radicals: potential mediators of tumor initiation and promotion. Carcinogenesis. 8: 1365
8. Dreher, D. and Junod, A. F. (1996) Role of oxygen free radicals in cancer development. Euro. J. Cancer 32(1): 30~38
9. Darley-Usmar, V., Wiseman, H. and Halliwell, B. (1995) Nitric oxide and oxygen radicals: a question of balance. FEBS Lett. 369: 131~135
10. Rotrosen, D. and Gallin, J. I. (1987) Disorders of phagocyte function. Ann. Rev. Immunol. 5: 127~150
11. Adelson, R., Saul, R. L. and Ames, B. N. (1988) Oxidative damage to DNA: relation to species metabolic rate and life span. Proc. Natl. Acad. Sci. USA. 85: 2706~2708
12. Culter, R. G. (1991) Recent progress in testing the longevity determinant and dysdifferentiation hypothesis of aging. Arch. Gerontol. Geriatr. 12: 75~98
13. Jacobson, M. D. (1996) Reactive oxygen apecies and programmed cell death. TIBS. 21: 83~86
14. Buttke, T. M. and Sandstrom, P. A. (1994) Oxidative stress as a mediator of apoptosis. Immunol. Today 15(1): 7~10
15. Karnovsky, M. J. (1994) Cytochemistry and reactive oxygen species: a retrospective. Histochem. 102: 15~27
16. Pryor, W. A. (1986) Oxy-radicals and related species: their formation, lifetimes, and reactions. Ann. Rev. Physiol. 48: 657~667
17. Janero, D. R. (1990) Malondialdehyde and thiobarbituric acid-reactive tissue injury. Free Radic. Bio. Med. 9: 515~540
18. Halliwell, B. and Gutteridge, J. M. C. (1990) The measurement and mechanism of lipid peroxidation in biological system. TIBS. 15: 129~135
19. Edwards, S. W. (1995) Cell signaling by integrins and immunoglobulin receptors in primed neutrophils. TIBS. 20: 362~367
20. Wang, J. F., Komarov, P. and Groot, H. (1993) Luminol-chemiluminescence in rat macrophages and granulocytes: the role of NO, O2-．/H2O2, and HOCl. Arch. Biochem. Biophys. 304: 189~196
21. Brunelli, L., Crow, J. P. and Beckman, J. S. (1995) The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch. Biochem. Biophys. 316: 327~334
22. Rodenas, J., Mitjavila, M. T. and Carbonell, T. (1995) Stimultaneous generation of nitric oxide and superoxide by inflammatory cells in rats. Free Radic. Bio. Med. 18(5): 869~875
23. 林天送 (1994) 自由基與健康。科學月刊25(7): 515~521
24. 林天送 (1994) 人體自由基知多少？健康世界雜誌 228: 20~24
25. Scheller, S., Wilczok, T., Imieiski, S., Krol, W., Gabrys, J. and Shani J. (1990) Free radical scavenging by ethanol extract of propolis. Int. J. Radiat. Biol. 57(3): 461~465
26. Krol, W., Czuba, Z., Scheller, S., Gabrys, J., Grabiec, S. and Shani, J. (1990) Anti-oxidant property of ethanolic extract of propolis (EEP) as evaluated by inhibiting the chemiluminescence oxidation of luminol. Biochem Intern. 21(4): 593~597
27. Pascual, C., Gonzalez, R. and Torricella, R. G. (1994) Scavenging action of propolis extract against oxygen radicals. J. Ethnopharm. 41: 9~13
28. Krol, W., Scheller, S., Czuba, Z., Matsuno, T., Zydowicz, G., Shani, J. and Mos, M. (1996) Inhibition of neutrophils'' chemiluminescence by ethanol extract of propolis (EEP) and its phenolic components. J. Ethnopharm. 55(1): 19~25
29. Burdock, G. A. (1998) Review of the biological properties and toxicity of bee propolis. Food & Chem. Toxico. 36(4): 347~363
30. Greenaway, W., May, J., Scaysbrook, T. and Whatley, F. R. (1991) Identification by gas chromatography-mass spectrometry of 150 compounds in propolis. Z. Natur. 46: 111~121
31. Rice-Evans, C. A., Miller, N. J. and Paganga, G. (1996) Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20(7): 933~956
32. Su, Z. Z., Lin, J., Grunberger, D. and Fisher, P. B. (1994) Growth suppression and toxicity induced by caffeic acid phenethyl ester (CAPE) in type 5 adenovirus-transformed rat embryo cells correlate directly with transformation progression. Cancer Res. 54(7): 1865~1870
33. Chen, J. H., Shao, Y., Huang, M. T., Chin, C. K. and Ho, C. T. (1996) Inhibitory effect of caffeic acid phenethyl ester on human leukemia HL-60 cells. Cancer Letters. 108(2): 211~214
34. Chiao, C., Carothers, A. M., Grunberger, D., Solomon, G., Preston, G. A. and Barrett, J. C. (1995) Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells. Cancer Res. 55(16): 3576~3583
35. Yoshida, M., Sakai, T., Hosokawa, N., Marui, N., Matsumoto, K., Fujioka, A., Nishino, H. and Aoike, A. (1990) The effect of quercetin on cell cycle progression and growth of human gastric cancer cells. FEBS Letters. 260(1): 10~13
36. Lepley, D. M. and Pelling, J. C. (1997) Induction of p21/WAF1 and G1 cell-cycle arrest by the chemopreventive agent apigenin. Mol. Carcin. 19(2): 74~82
37. Lepley, D. M., Li, B., Birt, D. F. and Pelling, J. C. (1996) The chemopreventive flavonoid apigenin induces G2/M arrest in keratinocytes. Carcinogenesis. 17(11): 2367~2375
38. Scheller, S., Krol, W., Swiacik, J., Owczarek, S., Gabrys, J. and Shani, J. (1989) Antitumoral property of ethanolic extract of propolis in mice-bearing Ehrlich carcinoma, as compared to bleomycin. Z. Natur. 44(11-12): 1063~1065
39. Fridovich, I. and Picker, S. D. (1984) On the mechanism of production of superoxide radical by reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium. Arch. Biochem. Biophys. 228(1): 155~158
40. Butacer, R. G. and Van Noorden, C. J. F. (1989) The involvement of superoxide anions in the nitroblue tetrazolium chloride reduction mediated by NADH and phenazine methosulfate. Anal. Biochem. 176: 170~174
41. Gryglewski, R. J. and Robak, J. (1988) Flavonoids are scavengers of superoxide anions. Biochem. Pharmacol. 375(5): 837~841
42. Blois, M. S. (1958) Antioxidaant determinations by the use of a stable free radical. Nature 181: 1199~1200
43. Das, N. P., Ratty, A. K. and Sunamoto, J. (1988) Interaction of flavonoids with ,1’-diphenyl--picryl-hydrazyl free radical, liposomal membranes and soybean lipoxygenase-1. Biochem. Pharmacol. 37(6): 989~995
44. Aust, S. D., Svingen, B. A., Buege, J. A. and O’Neal, F. O. (1979) The mechanism of NADPH-dependent lipid peroxidation. J. Biol. Chem. 254(13): 5892~5899
45. 呂鋒洲，徐展東 (1996) 發酵大麥清除超氧陰離子自由基能力的研究。食品科學 23(1): 10~19
46. Lissi, E., Salim-Hanna, M., Pascual, C. and Castillo, M.D.D. (1995) Evaluation of total antioxidant potential (TRAP) and total antioxidant reactivity from luminol-enhanced chemiluminescence measurements. Free Radic. Biol. Med. 18: 153~158
47. Chiba, Y., Kubota, T., Watanabe, M., Otani, Y., Teramoto, T., Matsumoto, Y., Koya ,K. and Kitajima, M. (1998) Selective antitumor activity of MKT-077, a delocalized lipophilic cation, on normal cells and cancer cells in vitro. J. Surgic. Onco. 69(2): 105~110
48. Matsui, T., Matsukawa, Y., Sakai, T., Nakamura, K. Aoike, A. and Kawai, K. (1995) Effect of ammonia on cell-cycle progression of human gastric cancer cells. Euro. J. Gastro. & Hepa. 7 Suppl 1: S79~81
49. Rani, C. S., Abe, A., Chang, Y., Rosenzweig, N., Saltiel, A. R., Radin, N. S. and Shayman, J. A. (1995) Cell cycle arrest induced by an inhibitor of glucosylceramide synthase. Correlation with cyclin-dependent kinases. J. Biol. Chem. 270(6): 2859~2867
50. Ishaque, A. and Al-Rubeai, M. (1998) Use of intracellular pH and annexin-V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture. J. Immu. Methods. 221(1-2): 43~57
51. Husain, S. R., Cillard, J. and Cillard, P (1987) Hydroxyl radical scavenging activity of flavonoids. Phytochem. 26(9): 2489~2491
52. Korkina, L. G. and Afanas, I. B. (1997) Antioxidant and chelating properties of flavonoids. Adv. in Pharmacol. (New York). 38: 151~163
53. Jovanovic, S. V., Steenken, S., Tosic, M., Marjanovic, B. and Simac, M. G. (1994) Flavonoids as Antioxidants. J. Am. Chem. Soc. 116: 4846~3851
54. Bravo, L. (1998) Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev. 56(11): 317~-333
55. Afanas''ev, I. B., Dorozhko, A. I,. Brodskii, A. V., Kostyuk, V. A. and Potapovitch, A. I. (1989) Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation. Biochem. Pharmacol. 38(11): 1763~1769
56. Kuthan, H. and Ullrich, V. (1982) Oxidase and oxygenase function of the microsomal cytochrome P450 monooxygenase system. Euro. J. Biochem. 126(3): 583~588
57. Hildebrandt, A. G., Speck, M. and Roots, I. (1973) Possible control of hydrogen peroxide production and degradation inmicrosomes during mixed function oxidation reaction. Biochemi. & Biophysic. Res. Comm. 54(3): 968~975
58. Kuthan, H., Tsuji, H., Graf, H. and Ullrich, V. (1978) Generation of superoxide anion as a source of hydrogen peroxide in areconstituted monooxygenase system. FEBS Letters. 91(2): 343~345
59. Scheller, S., Gazda, G., Pietsz, G., Gabrys, J., Szumlas, J. and Eckert, L. Shani, J. (1988) The ability of ethanol extract of propolis to stimulate plaque formation in immunized mouse spleen cells. Pharmacol. Res. Comm. 20(4): 323~328
60. Graziani, Y. and Chayoth, R. (1979) Regulation of cyclic AMP level and synthesis of DNA, RNA and protein by quercetin in Ehrlich ascites tumor cells. Biochem. Pharmacol.28(3):397~403
61. Hladon, B., Bylka, W., Ellnain-Wojtaszek, M., Skrzypczak, L., Szafarek, P., Chodera, A. and Kowalewski (1988) In vitro studies on the cytostatic activity of propolis extracts. Arz. Forsc.30(11): 1847~1848
62. Hirano, T., Gotoh, M. and Oka, K. (1994) Natural flavonoids and lignans are potent cytostatic agents against human leukemic HL-60 cells. Life Sciences. 55(13): 1061~1069
63. Avila, M. A., Velasco, J. A., Cansado, J. and Notario, V. (1994) Quercetin mediates the down-regulation of mutant p53 in the human breast cancer cell line MDA-MB468. Cancer Res.. 54(9): 2424~2428
64. Bai, F., Matsui, T., Ohtani-Fujita, N., Matsukawa, Y., Ding, Y. and Sakai, T. (1998) Promoter activation and following induction of the p21/WAF1 gene by flavone is involved in G1 phase arrest in A549 lung adenocarcinoma cells. FEBS Letters. 437(1-2): 61~-64
65. Plaumann, B., Fritsche, M., Rimpler, H., Brandner, G. and Hess R D. (1996) Flavonoids activate wild-type p53. Oncogene. 13(8): 1605~14