臺灣博碩士論文加值系統

栝樓實為葫蘆科(Cucurbitaceae)栝樓屬植物栝樓(Trichosanthes kirilowii Maxim.)之果實，為傳統中藥具有鎮咳、化痰，潤腸通便的作用。近年來的研究發現，栝樓實的粗萃物具有顯著的抗氧化、抗菌、抗腫瘤、抗糖尿病、抗發炎、抗動脈粥樣硬化、免疫調節、抗心律不整、保護胃壁黏膜、抑制血小板凝集及擴張冠狀動脈等作用。
本研究取用栝樓實(33.00 kg)以酒精抽取得到(9.48 kg)酒萃物，再以正己烷、甲醇及水分配萃取後，得到正己烷層(2.96 kg)，甲醇層(0.19 kg)及水層(6.33 kg)。將甲醇層以移動相為乙酸乙脂/甲醇濃度梯度的矽膠層析管住分離後得到七個分層(D1-D7)。將其中對肌肉細胞(C2C12)有促進葡萄糖攝取的五個分層(D-2.3.4.5.7)進行逆相高效液相層析及Sephadex LH-20管柱層析，分離純化後的到一個新化合物D5-L422343233(3)，一個已知化合物2-O-β-D-Glucopyranosyl cucurbitacin B (1)，及兩個未知化合物。
在葡萄糖攝取試驗中，化合物(1)增強肌肉細胞的葡萄糖攝取。而其他栝樓實有效成分仍在分離純化中。

The fruit of Trichosanthes kirilowii Maxim. (Cucurbitaceae) is a traditional Chinese medicine used as an antipyretic and expectorant, and constipation treatment. Previous study on the crude extract showed significant anti-oxidation, antibacterial, antitumor, antidiabetic, anti-inflammatory, anti-atherosclerosis, immunoregulatory, antiarrhythmic and protection of the gastric mucosa, platelet aggregation inhibition, and expansion of the coronary artery.
The ethanolic extracts (9.48 kg) of T. kirilowii fruits(33.00 kg) were partitioned into n-hexane layer (2.96 kg), methanol layer (0.19 k g), and H2O layer (6.33 kg). The methanol layer was fractionated by silica gel chromatography with ethyl acetate/methanol system gradient to obtain seven fractions (D1-D7). According to the results of glucose uptake assay in C2C12 muscle cell, the active fraction (D-2.3.4.5.7) were subjected to reversed phase chromatography and Sephadex LH-20 chromatography, and yielded one new compound, D5-L422343233 (2) , one known compound, 2-O-β-D-Glucopyranosyl cucurbitacin B (1), and two unknown compounds.
In the glucose uptake assay, Compound 1 enhanced glucose uptake in C2C12 cells. The active compounds of this plant remain to be clarified.

第一章 緒論 1
第一節 研究背景 1
第二節 研究目的 13
第三節 植物及藥材之概述 14
第四節 化學成分回顧 17
第五節 藥理活性回顧 44
第二章 化學成分抽取與分離 50
第一節 抽取與部分分層 50
第二節 初步分離與純化 52
第三節 大量分離與純化 54
第三章 化學成分結構鑑定 61
第一節 2-O-β-D-Glucopyranosyl cucurbitacin B之結構鑑定 61
第二節 2-Hydroxy-(22-27)-hexanorcucurbita-5,16-diene-3,11,20-trione 2-O-β-D-glucopyranoside之結構鑑定 65
第四章 藥理活性試驗結果與討論 69
第一節 栝樓實粗萃物之肌肉細胞葡萄糖攝取試驗 69
第二節 部分分層之肌肉細胞與脂肪細胞葡萄糖攝取試驗 70
第五章 實驗材料與方法 72
第一節 材料與儀器 72
第二節 藥理活性測試 81
第六章 結論 89
化合物之物理與光譜數據 91
參考文獻 92

1.楊仕哲; 張賢哲; 張恒鴻, 消渴疾病史的研究進展. 中醫藥雜誌 2009, 20, (3&4), 153-162.
2.World Health Organization, Global report on diabetes. 2016.
3.Say, L.; Chou, D.; Gemmill, A.; Tunçalp, Ö.; Moller, A.-B.; Daniels, J.; Gülmezoglu, A. M.; Temmerman, M.; Alkema, L., Global causes of maternal death: a WHO systematic analysis. The Lancet Global Health 2014, 2, (6), e323-e333.
4.行政院衛生署中央健康保險局, WHA 臺灣發表非傳染病防治成果,全民健康保險雙月刊. 2011, 92, 14-15.
5.何俊傑,蔡慧盈, 淺談第二型糖尿病之近期發展. 成醫藥誌 2010, 21, (1), 6-10.
6.Huang, S.; Czech, M. P., The GLUT4 glucose transporter. Cell metabolism 2007, 5, (4), 237-252.
7.Gao, Z.; Zhang, X.; Zuberi, A.; Hwang, D.; Quon, M. J.; Lefevre, M.; Ye, J., Inhibition of insulin sensitivity by free fatty acids requires activation of multiple serine kinases in 3T3-L1 adipocytes. Molecular endocrinology 2004, 18, (8), 2024-2034.
8.Coll, T.; Eyre, E.; Rodríguez-Calvo, R.; Palomer, X.; Sánchez, R. M.; Merlos, M.; Laguna, J. C.; Vázquez-Carrera, M., Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells. Journal of biological chemistry 2008, 283, (17), 11107-11116.
9.Erion, D. M.; Shulman, G. I., Diacylglycerol-mediated insulin resistance. Nature medicine 2010, 16, (4), 400-402.
10.Schenk, S.; Saberi, M.; Olefsky, J. M., Insulin sensitivity: modulation by nutrients and inflammation. The journal of clinical investigation 2008, 118, (9), 2992-3002.
11.Paz, K.; Hemi, R.; LeRoith, D.; Karasik, A.; Elhanany, E.; Kanety, H.; Zick, Y., A Molecular Basis for Insulin Resistance elevated serine/threonine phosphorylation of irs-1 and irs-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation. Journal of biological chemistry 1997, 272, (47), 29911-29918.
12.Shoelson, S. E.; Lee, J.; Goldfine, A. B., Inflammation and insulin resistance. The journal of clinical investigation 2006, 116, (7), 1793-1801.
13.Nguyen, M. A.; Satoh, H.; Favelyukis, S.; Babendure, J. L.; Imamura, T.; Sbodio, J. I.; Zalevsky, J.; Dahiyat, B. I.; Chi, N.-W.; Olefsky, J. M., JNK and tumor necrosis factor-α mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes. Journal of biological chemistry 2005, 280, (42), 35361-35371.
14.Jové, M.; Planavila, A.; Sánchez, R. M.; Merlos, M.; Laguna, J. C.; Vázquez-Carrera, M., Palmitate induces tumor necrosis factor-α expression in C2C12 skeletal muscle cells by a mechanism involving protein kinase C and nuclear factor-κB activation. Endocrinology 2006, 147, (1), 552-561.
15.Jové, M.; Planavila, A.; Laguna, J. C.; Vázquez-Carrera, M., Palmitate-induced interleukin 6 production is mediated by protein kinase C and nuclear-factor κB activation and leads to glucose transporter 4 down-regulation in skeletal muscle cells. Endocrinology 2005, 146, (7), 3087-3095.
16.Moller, D. E., Potential role of TNF-α in the pathogenesis of insulin resistance and type 2 diabetes. Trends in endocrinology & metabolism 2000, 11, (6), 212-217.
17.Olsen, G. S.; Hansen, B. F., AMP kinase activation ameliorates insulin resistance induced by free fatty acids in rat skeletal muscle. American journal of physiology-endocrinology and metabolism 2002, 283, (5), E965-E970.
18.Kahn, B. B.; Alquier, T.; Carling, D.; Hardie, D. G., AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell metabolism 2005, 1, (1), 15-25.
19.Niu, W.; Bilan, P. J.; Ishikura, S.; Schertzer, J. D.; Contreras-Ferrat, A.; Fu, Z.; Liu, J.; Boguslavsky, S.; Foley, K. P.; Liu, Z., Contraction-related stimuli regulate GLUT4 traffic in C2C12-GLUT4myc skeletal muscle cells. American journal of physiology-endocrinology and metabolism 2010, 298, (5), E1058-E1071.
20.Röckl, K. S.; Witczak, C. A.; Goodyear, L. J., Signaling mechanisms in skeletal muscle: acute responses and chronic adaptations to exercise. IUBMB life 2008, 60, (3), 145-153.
21.American Diabetes Association, 6. Obesity management for the treatment of type 2 diabetes. Diabetes care 2016, 39, (Supplement 1), S47-S51.
22.高本釗, 新編中藥大辭典（上）新文豐出版公司,臺北,. 1981, 2817-2820,.
23.Tan, M.J.; Ye, J.M.; Turner, N.; Hohnen-Behrens, C.; Ke, C.Q.; Tang, C.P.; Chen, T.; Weiss, H.C.; Gesing, E.-R.; Rowland, A., Antidiabetic activities of triterpenoids isolated from bitter melon associated with activation of the AMPK pathway. Chemistry & biology 2008, 15, (3), 263-273.
24.Leung, L.; Birtwhistle, R.; Kotecha, J.; Hannah, S.; Cuthbertson, S., Anti-diabetic and hypoglycaemic effects of Momordica charantia (bitter melon): a mini review. British journal of nutrition 2009, 102, (12), 1703-1708.
25.Kraegen, E. W.; Cooney, G. J., Free fatty acids and skeletal muscle insulin resistance. Current opinion in lipidology 2008, 19, (3), 235-241.
26.趙中振; 蕭培根, 當代藥用植物典. 香港賽馬會中藥研究院,香港 ,2006, 483-487.
27.賴瑞聲; 鄭統隆; 高瑞隆; 蕭翌柱; 吳明哲, 具有血糖調節功能之藥用植物簡介, 行政院農委會,台北, 2007, 62,
28.行政院衛生署中華藥典中藥集編修小組, 中華中藥典，第一版. 行政院衛生署, 臺灣. 2004, 138-139.
29.Rahman, M.; Aziz, A.; Moon, S.-S., Isoetin 5'-methyl ether, a cytotoxic flavone from Trichosanthes kirilowii. Bulletin of the korean chemical society 2007, 28, (8), 1261-1264.
30.Dat, N. T.; Jin, X.; Hong, Y. S.; Lee, J. J., An isoaurone and other constituents from Trichosanthes kirilowii seeds inhibit hypoxia-inducible factor-1 and nuclear factor-kappaB. Journal of natural products, 2010, 73, (6), 1167-9.
31.Li, A.; Sun, A.; Liu, R.; Zhang, Y.; Cui, J., An efficient preparative procedure for main flavonoids from the peel of Trichosanthes kirilowii Maxim. using polyamide resin followed by semi-preparative high performance liquid chromatography. Journal of chromatography. B, analytical technologies in the biomedical and life sciences, 2014, 965, 150-7.
32.Li, A. F.; Sun, A. L.; Liu, R. M.; Zhang, Y. Q., Chemical constituents of Trichosanthes kirilowii peels. Zhong Yao Cai 2014, 37, (3), 428-31.
33.Sun, X. Y.; Wu, H. H.; Fu, A. Z.; Zhang, P., Chemical constituents of Trichosanthes kirilowii Maxim. Yao Xue Xue Bao 2012, 47, (7), 922-5.
34.李愛峰; 張永清; 山東中醫藥大學藥學院, 栝樓的化學成分研究進展. 安徽農業科學 2012, 14713-14716.
35.Minh, C. V.; Nhiem, N. X.; Yen, H. T.; Kiem, P. V.; Tai, B. H.; Le Tuan Anh, H.; Hien, T. T.; Park, S.; Kim, N.; Kim, S. H., Chemical constituents of Trichosanthes kirilowii and their cytotoxic activities. Archives of pharmacal research, 2015, 38, (8), 1443-8.
36.陳勝發; 黃時偉; 王昌利; 陝西中醫學院, 栝樓屬植物化學成分的研究進展. Chinese traditional patent medicine 2006, (2002年 01), 11-13.
37.Kumagai, M. H.; Turpen, T. H.; Weinzettl, N.; della-Cioppa, G.; Turpen, A. M.; Donson, J.; Hilf, M. E.; Grantham, G. L.; Dawson, W. O.; Chow, T. P., Rapid, high-level expression of biologically active alpha-trichosanthin in transfected plants by an RNA viral vector. Proceedings of the national academy of sciences, 1993, 90, (2), 427-30.
38.Zhang, L.; Jia, Y. H.; Zhao, X. S.; Zhou, F. H.; Pan, Y. Y.; Wan, Q.; Cui, X. B.; Sun, X. G.; Chen, Y. Y.; Zhang, Y.; Cheng, S. B., Trichosanatine alleviates oxidized low-density lipoprotein induced endothelial cells injury via inhibiting the LOX-1/p38 MAPK pathway. American journal of translational research, 2016, 8, (12), 5455-5464.
39.Wu, T.; Cheng, X.M.; Bligh, S. A.; Chou, G.X.; Wang, Z.T.; Bashall, A.; Branford-White, C., Multiflorane triterpene esters from the seeds of Trichosanthes kirilowii. Helvetica chimica acta 2005, 88, (10), 2617-2623.
40.Kimura, Y.; Akihisa, T.; Yasukawa, K.; Takase, S.i.; Tamura, T.; Ida, Y., Cyclokirilodiol and isocyclokirilodiol: Two novel cycloartanes from the seeds of Trichosanthes kirilowii Maxim. Chemical and pharmaceutical bulletin 1997, 45, (2), 415-417.
41.Akihisa, T.; Kokke, W. C.; Krause, J. A.; Eggleston, D. S.; Katayama, S.-i.; Kimura, Y.; Tamura, T., 5-Dehydrokarounidiol [D: C-Friedo-oleana-5, 7, 9 (11)-triene-3α, 29-diol], a Novel Triterpene from Trichosanthes kirilowii Maxim. Chemical and pharmaceutical bulletin 1992, 40, (12), 3280-3283.
42.Akihisa, T.; Yasukawa, K.; Kimura, Y.; Takido, M.; Kokke, W. C.; Tamura, T., Five D:C-friedo-oleanane triterpenes from the seeds of Trichosanthes kirilowii Maxim. and their anti-inflammatory effects. Chemical and pharmaceutical bulletin, 1994, 42, (5), 1101-5.
43.Akihisa, T.; Yasukawa, K.; Kimura, Y.; Takido, M.; Kokke, W. C.; Tamura, T., 7-Oxo-10α-Cucurbitadienol from the seeds of Trichosanthes kirilowii and its anti-inflammatory effect. Phytochemistry 1994, 36, (1), 153-157.
44.何祥久; 邱峰, 栝樓屬植物化學成分. 國外醫藥: 植物藥分冊 2002, 17, (1), 11-13.
45.陳勝發; 黃時偉; 王昌利, 栝樓屬植物化學成分的研究進展. 中成藥 2006, 28, (8), 1187-1192.
46.Ryu, S. Y.; Lee, S. H.; Choi, S. U.; Lee, C. O.; No, Z.; Ahn, J. W., Antitumor activity of Trichosanthes kirilowii. Archives of pharmacal research 1994, 17, (5), 348-353.
47.Kondo, T.; Inoue, M.; Mizukami, H.; Ogihara, Y., Cytotoxic activity of bryonolic acid isolated from transformed hairy roots of Trichosanthes kirilowii var. japonica. Biological and pharmaceutical bulletin, 1995, 18, (5), 726-9.
48.Moon, S. S.; Rahman, A. A.; Kim, J. Y.; Kee, S. H., Hanultarin, a cytotoxic lignan as an inhibitor of actin cytoskeleton polymerization from the seeds of Trichosanthes kirilowii. Bioorganic & medicinal chemistry, 2008, 16, (15), 7264-9.
49.李愛峰; 孫愛玲; 柳仁民; 張永清, 栝樓果皮化學成分研究. 中藥材 2014, (3), 428-431.
50.徐禮英; 張小平; 蔣繼宏, 栝樓子揮發油的成分分析及其生物活性的初步研究. 中國實驗方劑學雜誌 2009, (8), 38-43.
51.易亞喬; 李鑫輝; 張炳填; 葛立新; 趙釗; 楊麗; 郭雁; 鄧璽瑋, 栝樓藥理作用研究輯要. 中醫藥導報 2015, 21, (11), 80-83.
52.顏正華, 中藥學(上). 知音出版社,台灣, 2003, 123-125.
53.Lee, H. S.; Huang, P. L.; Kung, H. F.; Li, B. Q.; Huang, P. L.; Huang, P.; Huang, H. I.; Chen, H. C., TAP 29: an anti-human immunodeficiency virus protein from Trichosanthes kirilowii that is nontoxic to intact cells. Proceedings of the national academy of sciences, 1991, 88, (15), 6570-4.
54.Gong, Q.; Deng, D.; Ding, J.; Wang, C.; Bian, Z.; Ye, Z.; Xu, J., Trichosanthin, an extract of Trichosanthes kirilowii, effectively prevents acute rejection of major histocompatibility complex-mismatched mouse skin allograft. Transplantation proceedings, 2008, 40, (10), 3714-8.
55.Ni, L.; Zhu, X.; Gong, C.; Luo, Y.; Wang, L.; Zhou, W.; Zhu, S.; Li, Y., Trichosanthes kirilowii fruits inhibit non-small cell lung cancer cell growth through mitotic cell-cycle arrest. The american journal of Chinese medicine, 2015, 43, (2), 349-64.
56.萬麗娟; 盧金清; 許俊潔; 黎強; 蔡君龍; 郭勝男, 瓜蔞子化學成分和藥理作用的研究進展. 中醫藥導報, 2008, 7, (20), 10-15.
57.Chen, D. K. F.; Mak, J. W.; Ooi, S. S.; Mak, K. F.; Chong, K. H., Effect of traditional chinese medicine complex for diabetes (TCM-D™) on experimentally induced diabetic mice. Tumour Cells with CERB2 Expression 2013, 42-52.
58.林哲瑋. 栝蔞皮之抑菌與抗氧化活性分析. 義守大學碩士論文, 高雄市, 2007.
59.Maraganore, J. M.; Joseph, M.; Bailey, M. C., Purification and characterization of trichosanthin. Homology to the ricin A chain and implications as to mechanism of abortifacient activity. Journal of biological chemistry, 1987, 262, (24), 11628-33.
60.Thoennissen, N. H.; Iwanski, G. B.; Doan, N. B.; Okamoto, R.; Lin, P.; Abbassi, S.; Song, J. H.; Yin, D.; Toh, M.; Xie, W. D., Cucurbitacin B induces apoptosis by inhibition of the JAK/STAT pathway and potentiates antiproliferative effects of gemcitabine on pancreatic cancer cells. Cancer research 2009, 69, (14), 5876-5884.
61.Ma, J.; Zi Jiang, Y.; Shi, H.; Mi, C.; Li, J.; Xing Nan, J.; Wu, X.; Joon Lee, J.; Jin, X., Cucurbitacin B inhibits the translational expression of hypoxia-inducible factor-1alpha. European journal of pharmacology, 2014, 723, 46-54.
62.Tang, Y.; Li, W.; Cao, J.; Zhao, Y., Bioassay-guided isolation and identification of cytotoxic compounds from Bolbostemma paniculatum. Journal of ethnopharmacology , 2015, 169, 18-23.
63.Yang, L.; Wu, S.; Zhang, Q.; Liu, F.; Wu, P., 23, 24-Dihydrocucurbitacin B induces G 2/M cell-cycle arrest and mitochondria-dependent apoptosis in human breast cancer cells (Bcap37). Cancer letters, 2007, 256, (2), 267-278.
64.Takahashi, N.; Yoshida, Y.; Sugiura, T.; Matsuno, K.; Fujino, A.; Yamashita, U., Cucurbitacin D isolated from Trichosanthes kirilowii induces apoptosis in human hepatocellular carcinoma cells in vitro. International immunopharmacology, 2009, 9, (4), 508-13.
65.Shu, S. H.; Xie, G. Z.; Guo, X. L.; Wang, M., Purification and characterization of a novel ribosome-inactivating protein from seeds of Trichosanthes kirilowii Maxim. Protein expression and purification, 2009, 67, (2), 120-5.
66.Gong, Q.; Deng, D.; Ding, J.; Wang, C.; Bian, Z.; Ye, Z.; Xu, J. In Trichosanthin, an extract of Trichosanthes kirilowii, effectively prevents acute rejection of major histocompatibility complex–mismatched mouse skin allograft, Transplantation proceedings, 2008; 3714-3718.
67.Wei, B.; Huang, Q.; Huang, S.; Mai, W.; Zhong, X., Trichosanthin-induced autophagy in gastric cancer cell MKN-45 is dependent on reactive oxygen species (ROS) and NF-kappaB/p53 pathway. Journal of pharmacological sciences, 2016, 43-54.
68.Li, J.; Li, H.; Zhang, Z.; Wang, N.; Zhang, Y., The anti-cancerous activity of recombinant trichosanthin on prostate cancer cell PC3. Biological research, 2016, 49-58.
69.Ma, J.; Mi, C.; Wang, K. S.; Lee, J. J.; Jin, X., 4',6-Dihydroxy-4-methoxyisoaurone inhibits TNF-alpha-induced NF-kappaB activation and expressions of NF-kappaB-regulated target gene products. Journal of pharmacological sciences, 2016, 130, (2), 43-50.
70.Lee, H. S.; Huang, P. L.; Kung, H.F.; Li, B.Q.; Huang, P.; Huang, H.; Chen, H., TAP 29: an anti-human immunodeficiency virus protein from Trichosanthes kirilowii that is nontoxic to intact cells. Proceedings of the national academy of sciences 1991, 88, (15), 6570-6574.
71.Escandell, J. M.; Recio, M.C.; Máñez, S.; Giner, R.M.; Cerdá-Nicolás, M.; Ríos, J.L., Dihydrocucurbitacin B, isolated from Cayaponia tayuya, reduces damage in adjuvant-induced arthritis. European journal of pharmacology 2006, 532, (1), 145-154.
72.Gatbonton-Schwager, T. N.; Letterio, J. J.; Tochtrop, G. P., Bryonolic acid transcriptional control of anti-inflammatory and antioxidant genes in macrophages in vitro and in vivo. Journal of natural products 2012, 75, (4), 591-598.
73.Song, Y.; Ding, N.; Kanazawa, T.; Yamashita, U.; Yoshida, Y., Cucurbitacin D is a new inflammasome activator in macrophages. International immunopharmacology, 2013, 17, (4), 1044-50.
74.Oh, H.; Mun, Y. J.; Im, S. J.; Lee, S. Y.; Song, H. J.; Lee, H. S.; Woo, W. H., Cucurbitacins from Trichosanthes kirilowii as the inhibitory components on tyrosinase activity and melanin synthesis of B16/F10 melanoma cells. Planta medica, 2002, 68, (9), 832-3.
75.Perez G, R.; Zavala S, M.; Perez G, S.; Perez G, C., Antidiabetic effect of compounds isolated from plants. Phytomedicine 1998, 5, (1), 55-75.
76.Kawahara, N.; Kurata, A.; Hakamatsuka, T.; Sekita, S.; Satake, M., Two new cucurbitacin glucosides, opercurins A and B, from the Brazilian folk medicine" Buchinha"(Luffa operculata). Chemical and pharmaceutical bulletin , 2004, 52, (8), 1018-1020.
77.Gan, M.; Liu, M.; Liu, B.; Lin, S.; Zhang, Y.; Zi, J.; Song, W.; Ye, F.; Chen, X.; Shi, J., Cucurbitane glucosides from the root of Machilus yaoshansis. Journal of natural products 2011, 74, (11), 2431-2437.