臺灣博碩士論文加值系統

本研究利用本實驗室所建立之細胞毒性試驗及促進類人類巨噬細胞 U937 細胞株吞噬作用篩選平台，分離台灣草藥中有效成分。選用台灣產的厚葉捕魚木莖部及葉部 (GBS, GBL)、山胡椒莖部及葉部 (LCS, LCL) 以及南嶺前胡莖部 (PLS)，進行水及 95 % 乙醇萃取，分別得到這些草藥之水萃取物及乙醇萃取物。再將萃取物利用 cell counting kit-8 方法進行細胞毒性試驗，及促進 U937 細胞株吞噬標示綠色螢光蛋白之克雷白氏肺炎桿菌 (Klebsiella pneumoniae-gfp) 試驗進行篩選，選擇對 U937 細胞株無毒性及具有最佳促進 U937 細胞株吞噬作用之草藥萃取物。結果顯示南嶺前胡乙醇萃取物 (PLSE) 在 200 �慊/ml 濃度下，對 U937 細胞具有 104.2 % 存活率，及具有最佳 (44.67 %) 之促進吞噬能力。進而利用乙醇進行南嶺前胡之大量 (965g) 萃取，所得萃取物再以 CH2Cl2、EtOAc、n-BuOH 及 H2O 等溶媒進行分配萃取。共得到五個萃取層：二氯甲烷層 (PLSEC)、乳化層 (PLSEE) 、乙酸乙酯層 (PLSEA)、正丁醇層 (PLSEB) 及水層 (PLSEW)。分別以篩選平台進行篩選，結果顯示其中無毒性及最佳促進吞噬能力之萃取層為 PLSEA，並將其利用矽膠管柱層析分離，可獲得六個分層 (PLSEA 1∼6)。其中 PLSEA 5 對 U937 細胞不具細胞毒性，且促進 U937 細胞吞噬能力最佳 (40.27%)。再次利用矽膠管柱層析法分離 PLSEA 5 之成分，共獲得六個分層 (PLSEA 5-1 ∼ PLSEA 5-6) 。六個分層中 PLSEA 5-2 及 PLSEA 5-5 對 U937 細胞不具細胞毒性，且促進 U937 細胞吞噬能力，分別為 53.46 % 及 51.57 %。其有效成分有待進一步分離鑑定。

The purpose of this study is to isolate the active constituents from Taiwanese herbal medicines (THMs) by using a screening platform for immunity-enhancing properties. Three THMs, namely, the leaves and stems of Grewia biloba (GBL, GBS), the leaves and stems of Litsea cubeba (LCL, LCS), the stems of Peucedanum longshengense (PLS), were collected in Taiwan, and H2O and 95% EtOH were used to produce two different extracts of each THM.
U937 cells, a kind of macrophage-like cells, were used for the cytotoxicity test by the cell counting kit-eight method. The number of Klebsiella pneumoniae-gfp phagocytosed by U937 cells was used as a phagocytosis-enhancing assay by THMs. The results showed that EtOH extract of PLS (PLSE) at 200�n�慊/ml gave 104.2% viability of U937 cells, and was the most effective (44.67%) in enhancing phagocytosis of U937 cells. A large amount (965g) of PLS was extracted with EtOH.
The large scale extract was partitioned with CH2Cl2, EtOAc, n-BuOH and H2O. Five layer of extracts, namely, CH2Cl2 layer (PLSEC), emulsion layer (PLSEE), EtOAc layer (PLSEA), n-BuOH layer (PLSEB) and H2O layer (PLSEW), were obtained, and then they were examined by cytotoxicity and phagocytosis tests. PLSEA had the lowest cytotoxicity and the most effective, and it was subjected on a silica gel column chromatography and eluted with CH2Cl2: MeOH (25 : 1). Six fractions, PLSEA 1~6, were collected. PLSEA 5 had non-cytotoxicity and the strongest enhancing phagocytosis (40.27 %) of U937 cells among the six fractions.
PLSEA 5 was further subjected on a silica gel column chromatography and eluted with CHCl3: MeOH: EtOAc (5:1:1). Six fractions, PLSEA 5-1 ~ 5-6, were collected and were evaluated by cytotoxicity and phagocytosis tests. The results demonstrated that PLSEA 5-2 and 5-5 exhibited non-cytotoxicity and effective enhancing phagocytosis of U937 cell, 53.46 % and 51.57 %, respectively. The active constituents of enhancing phagocytosis of U937 cell of PLSEA 5-2 and 5-5 will further need to be isolated and identified.

中 文 摘 要 I
Abstract III
謝 誌 V
目 錄 VI
圖 目 錄 VIII
表 目 錄 XI
第一章 緒論 1
第一節 中草藥與免疫 1
第二節 免疫與疾病 2
第三節 免疫活性物質之評估方法 5
第四節 類人類白血病細胞株 U937 細胞株特性 9
第五節 流式細胞技術 10
第六節 研究動機及目的 10
第七節 厚葉捕魚木、南嶺前胡及山胡椒簡介與其相關之研究 11
第二章 材料與方法 15
第一節 厚葉捕魚木、山胡椒及南嶺前胡之萃取方法 15
第二節 GB，LC 及 PL 之水及 95 % 乙醇萃取物對 U937細胞株之
細胞毒性試驗 17
第三節 GB，LC 及 PL 之水及 95 % 乙醇萃取物促進 U937 細胞株
之吞噬能力試驗 23
第四節 南嶺前胡莖部有效成分大量萃取與分離方法 29
第三章 結果與討論 32
第一節 厚葉捕魚木、山胡椒、南嶺前胡萃取結果 32
第二節 GB，LC 及 PL 之水及 95 % 乙醇萃取物對 U937 細胞株之
細胞毒性試驗結果 33
第三節 GB，LC 及 PL 之水及 95 % 乙醇萃取物促進 U937 細胞株
吞噬能力試驗結果 41
第四節 大量之南嶺前胡莖部乙醇萃取及分離結果 43
第四章 結論 56
參 考 文 獻 58

1.Lin YL, Liang YC, Tseng YS, et al. An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF-{kappa}B and MAPK pathways. J Leukoc Biol. Jun 4 2009.
2.Chan WK, Cheung CC, Law HK, Lau YL, Chan GC. Ganoderma lucidum polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function. J Hematol Oncol. 2008;1:9.
3.Jeurink PV, Noguera CL, Savelkoul HF, Wichers HJ. Immunomodulatory capacity of fungal proteins on the cytokine production of human peripheral blood mononuclear cells. Int Immunopharmacol. Aug 2008;8(8):1124-1133.
4.Ho YW, Yeung JS, Chiu PK, et al. Ganoderma lucidum polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblast. Mol Cell Biochem. Jul 2007;301(1-2):173-179.
5.Lin YL, Lee SS, Hou SM, Chiang BL. Polysaccharide purified from Ganoderma lucidum induces gene expression changes in human dendritic cells and promotes T helper 1 immune response in BALB/c mice. Mol Pharmacol. Aug 2006;70(2):637-644.
6.Wang SY, Hsu ML, Hsu HC, et al. The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. Int J Cancer. Mar 17 1997;70(6):699-705.
7.Rajput ZI, Hu SH, Xiao CW, Arijo AG. Adjuvant effects of saponins on animal immune responses. J Zhejiang Univ Sci B. Mar 2007;8(3):153-161.
8.Spelman K, Burns J, Nichols D, Winters N, Ottersberg S, Tenborg M. Modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators. Altern Med Rev. Jun 2006;11(2):128-150.
9.Lee DC, Yang CL, Chik SC, et al. Bioactivity-guided identification and cell signaling technology to delineate the immunomodulatory effects of Panax ginseng on human promonocytic U937 cells. J Transl Med. 2009;7:34.
10.Kuo CF, Chen CC, Luo YH, et al. Cordyceps sinensis mycelium protects mice from group A streptococcal infection. J Med Microbiol. Aug 2005;54(Pt 8):795-802.
11.Kuo YC, Weng SC, Chou CJ, Chang TT, Tsai WJ. Activation and proliferation signals in primary human T lymphocytes inhibited by ergosterol peroxide isolated from Cordyceps cicadae. Br J Pharmacol. Nov 2003;140(5):895-906.
12.Koh JH, Yu KW, Suh HJ, Choi YM, Ahn TS. Activation of macrophages and the intestinal immune system by an orally administered decoction from cultured mycelia of Cordyceps sinensis. Biosci Biotechnol Biochem. Feb 2002;66(2):407-411.
13.Yang X, Zhao Y, Wang H, Mei Q. Macrophage activation by an acidic polysaccharide isolated from Angelica sinensis (Oliv.) Diels. J Biochem Mol Biol. Sep 30 2007;40(5):636-643.
14.Baumann S, Fas SC, Giaisi M, et al. Wogonin preferentially kills malignant lymphocytes and suppresses T-cell tumor growth by inducing PLCgamma1- and Ca2+-dependent apoptosis. Blood. Feb 15 2008;111(4):2354-2363.
15.Hsieh CJ, Hall K, Ha T, Li C, Krishnaswamy G, Chi DS. Baicalein inhibits IL-1beta- and TNF-alpha-induced inflammatory cytokine production from human mast cells via regulation of the NF-kappaB pathway. Clin Mol Allergy. 2007;5:5.
16.Zeng Y, Song C, Ding X, Ji X, Yi L, Zhu K. Baicalin reduces the severity of experimental autoimmune encephalomyelitis. Braz J Med Biol Res. Jul 2007;40(7):1003-1010.
17.Nanjundaiah SM, Annaiah HN, S MD. Gastroprotective Effect of Ginger Rhizome (Zingiber officinale) Extract: Role of Gallic Acid and Cinnamic Acid in H+, K+-ATPase/H. pylori Inhibition and Anti-oxidative Mechanism. Evid Based Complement Alternat Med. Jul 1 2009.
18.Abbas AK, Lichtman AH, 謝 文欽. 細胞與分子免疫學. 台北市: 合記; 2004.
19.Roitt IM, 楊 沂淵, 陳 建和, 呂 思潔. 實用免疫學. 1 ed. 台北縣: 藝軒; 2000.
20.李 朝品. 臨床免疫學. 1 ed. 北京: 人民軍醫; 2004.
21.Goldsby RA, 王 政光. 免疫學. 台北市: 九州圖書; 2004.
22.Zhang Y, Cheng G, Yang K, et al. A novel function of granulocyte colony-stimulating factor in mobilization of human hematopoietic progenitor cells. Immunol Cell Biol. Mar 3 2009.
23.Donayre-Torres AJ, Esquivel-Soto E, Gutierrez-Xicotencatl Mde L, Esquivel-Guadarrama FR, Gomez-Lim MA. Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli. Virol J. 2009;6:17.
24.Chang YC, Hsu TL, Lin HH, et al. Modulation of macrophage differentiation and activation by decoy receptor 3. J Leukoc Biol. Mar 2004;75(3):486-494.
25.Fuqua CF, Akomeah R, Price JO, Adunyah SE. Involvement of ERK-1/2 in IL-21-induced cytokine production in leukemia cells and human monocytes. Cytokine. Oct 2008;44(1):101-107.
26.Brekke OL, Christiansen D, Fure H, et al. Combined inhibition of complement and CD14 abolish E. coli-induced cytokine-, chemokine- and growth factor-synthesis in human whole blood. Mol Immunol. Aug 2008;45(14):3804-3813.
27.Chen YY, Chang HM. Antiproliferative and differentiating effects of polysaccharide fraction from fu-ling (Poria cocos) on human leukemic U937 and HL-60 cells. Food Chem Toxicol. May 2004;42(5):759-769.
28.Potts BE, Chapes SK. Functions of C2D macrophage cells after adoptive transfer. J Leukoc Biol. Mar 2008;83(3):602-609.
29.Lei HY, Chang CP. Lectin of Concanavalin A as an anti-hepatoma therapeutic agent. J Biomed Sci. 2009;16:10.
30.Kucharska AM, Gorska E, Wasik M, Demkow U. Altered expression of T lymphocyte surface markers in children with chronic autoimmune thyroiditis. J Physiol Pharmacol. Dec 2008;59 Suppl 6:375-382.
31.Skendros P, Sarantopoulos A, Tselios K, Boura P. Chronic brucellosis patients retain low frequency of CD4+ T-lymphocytes expressing CD25 and CD28 after Escherichia coli LPS stimulation of PHA-cultured PBMCs. Clin Dev Immunol. 2008;2008:327346.
32.Secor ER, Jr., Singh A, Guernsey LA, et al. Bromelain treatment reduces CD25 expression on activated CD4+ T cells in vitro. Int Immunopharmacol. Mar 2009;9(3):340-346.
33.Apte SH, Baz A, Groves P, Kelso A, Kienzle N. Interferon-gamma and interleukin-4 reciprocally regulate CD8 expression in CD8+ T cells. Proc Natl Acad Sci U S A. Nov 11 2008;105(45):17475-17480.
34.Baker EJ, Gerard DA, Bamberger EG, Lozzio CB, Ichiki AT. HL-60 cell growth-conditioned medium is an effective inducer of myeloperoxidase expression in K-562 human leukemia cells. Leuk Res. Nov 2002;26(11):1017-1025.
35.Gardiner CM, Reen DJ. Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity. Immunology. Apr 1998;93(4):511-517.
36.Blanchard DK, Friedman H, Stewart WE, 2nd, Klein TW, Djeu JY. Role of gamma interferon in induction of natural killer activity by Legionella pneumophila in vitro and in an experimental murine infection model. Infect Immun. May 1988;56(5):1187-1193.
37.Yang LP, Cheng P, Peng XC, et al. Anti-tumor effect of adenovirus-mediated gene transfer of pigment epithelium-derived factor on mouse B16-F10 melanoma. J Exp Clin Cancer Res. 2009;28:75.
38.Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. Dec 16 1983;65(1-2):55-63.
39.Lu YY, Chen TS, Qu JL, Pan WL, Sun L, Wei XB. Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells. J Biomed Sci. 2009;16:16.
40.Matsuda J. [Phagocytosis, nitroblue tetrazolium dye reduction test]. Rinsho Byori. 1982;Suppl 48:150-166.
41.Liu YQ, You S, Zhang CL, Tashiro S, Onodera S, Ikejima T. Oridonin enhances phagocytosis of UV-irradiated apoptotic U937 cells. Biol Pharm Bull. Mar 2005;28(3):461-467.
42.Rothe G, Kellermann W, Valet G. Flow cytometric parameters of neutrophil function as early indicators of sepsis- or trauma-related pulmonary or cardiovascular organ failure. J Lab Clin Med. Jan 1990;115(1):52-61.
43.Chen YJ, Shiao MS, Lee SS, Wang SY. Effect of Cordyceps sinensis on the proliferation and differentiation of human leukemic U937 cells. Life Sci. 1997;60(25):2349-2359.
44.Steinckwich N, Frippiat JP, Stasia MJ, et al. Potent inhibition of store-operated Ca2+ influx and superoxide production in HL60 cells and polymorphonuclear neutrophils by the pyrazole derivative BTP2. J Leukoc Biol. Apr 2007;81(4):1054-1064.
45.Greenberg ME, Sun M, Zhang R, Febbraio M, Silverstein R, Hazen SL. Oxidized phosphatidylserine-CD36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells. J Exp Med. Nov 27 2006;203(12):2613-2625.
46.Yazdanparast R, Mahdavi M, Moosavi MA. Induction of differentiation and apoptosis in three human leukemia cell lines by a new compound from Dendrostellera lessertii. Acta Biochim Biophys Sin (Shanghai). Jul 2006;38(7):477-483.
47.Sundstrom C, Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer. May 15 1976;17(5):565-577.
48.李 美璇. Studies on enhancing U937 cells phagocytosis from the leaves of Chloranthus glabra [碩士論文]. 台南: 藥物科技研究所, 嘉南藥理科技大學; 2007.
49.Gille C, Leiber A, Mundle I, et al. Phagocytosis and postphagocytic reaction of cord blood and adult blood monocyte after infection with green fluorescent protein-labeled Escherichia coli and group B Streptococci. Cytometry B Clin Cytom. Mar 13 2009.
50.Hatano Y, Taniuchi S, Masuda M, et al. Phagocytosis of heat-killed Staphylococcus aureus by eosinophils: comparison with neutrophils. APMIS. Feb 2009;117(2):115-123.
51.Shen YD, Shao XT, Ni YD, Xu H, Tong XM. [Cordyceps sinensis polysaccharide enhances apoptosis of HL-60 cells induced by triptolide.]. Zhejiang Da Xue Xue Bao Yi Xue Ban. Mar 2009;38(2):158-162.
52.Elbim C, Lizard G. Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations. Cytometry A. Apr 8 2009.
53.Han J, Talorete TP, Yamada P, Isoda H. Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells. Cytotechnology. Apr 8 2009.
54.Vrba J, Dolezel P, Vicar J, Ulrichova J. Cytotoxic activity of sanguinarine and dihydrosanguinarine in human promyelocytic leukemia HL-60 cells. Toxicol In Vitro. Feb 3 2009.
55.Hsiao YP, Huang HL, Lai WW, Chung JG, Yang JH. Antiproliferative effects of lactic acid via the induction of apoptosis and cell cycle arrest in a human keratinocyte cell line (HaCaT). J Dermatol Sci. Mar 30 2009.
56.Lee SY, Kim GT, Roh SH, et al. Proteomic Analysis of the Anti-Cancer Effect of 20S-Ginsenoside Rg(3) in Human Colon Cancer Cell Lines. Biosci Biotechnol Biochem. Apr 7 2009.
57.Urmaliya VB, Church JE, Coupar IM, Rose''meyer RB, Pouton CW, White PJ. Cardioprotection Induced by Adenosine A1 Receptor Agonists in a Cardiac Cell Ischemia Model Involves Cooperative Activation of Adenosine A2A and A2B Receptors by Endogenous Adenosine. J Cardiovasc Pharmacol. Mar 28 2009.
58.劉 和義, 楊 遠波, 施 炳霖, 呂 勝由. 臺灣維管束植物簡誌 第參卷. 台北市: 行政院農業委員會; 1991.
59.廖 江川. 藥用植物圖錄. 台中市: 台中市藥用植物研究會; 1999:36.
60.Liu J-Q, Wu J-M, Kou X-L, Hong Q. Studies on chemical constituents from Grewia biloba. Zhong yao cai = Zhongyaocai = Journal of Chinese medicinal materials. 2008;31(10):1505-1507.
61.Liu J, Wu J, Kou X, Hong Q, Liu J, Wu J, Kou X, Hong QLiu J, Wu J, Kou X, Hong Qs; (Jiangxi University of Traditional Chinese Medicine, Peop. Rep. China). assignee. Preparation method for extract of grewia biloba bark, and use of the extract in preparation of antitumor drugs. US patent 2008-10070691
101244100. 20080227., 2008.
62.邱 年永, 張 光雄. 原色臺灣藥用植物圖鑑. Vol 1. 2 ed. 台北市: 南天書局有限公司; 1987.
63.Lalko J, Api AM. Investigation of the dermal sensitization potential of various essential oils in the local lymph node assay. Food Chem Toxicol. May 2006;44(5):739-746.
64.Hwang JK, Choi EM, Lee JH. Antioxidant activity of Litsea cubeba. Fitoterapia. Dec 2005;76(7-8):684-686.
65.Choi EM, Hwang JK. Effects of methanolic extract and fractions from Litsea cubeba bark on the production of inflammatory mediators in RAW264.7 cells. Fitoterapia. Mar 2004;75(2):141-148.
66.Luo M, Jiang LK, Huang YX, Xiao M, Li B, Zou GL. Effects of citral on Aspergillus flavus spores by quasi-elastic light scattering and multiplex microanalysis techniques. Acta Biochim Biophys Sin (Shanghai). Apr 2004;36(4):277-283.
67.Liu ZL, Goh SH, Ho SH. Screening of Chinese medicinal herbs for bioactivity against Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst). Journal of Stored Products Research. 2007;43(3):290-296.
68.Wang CY. [The active principles of Litsea cubeba in the treatment of coronary heart disease]. Zhong Yao Tong Bao. Sep 1985;10(9):30-32.
69.Yang YZ. [Treatment of cerebral thrombosis with Litsea cubeba (Zour) pers]. Zhong Xi Yi Jie He Za Zhi. Dec 1984;4(12):740-741.
70.Shan R-h, Sheh M-l, Wang T-s, Pu F-t, Shen K-m, Chang H-t. NEW TAXA OF THE CHINESE UMBELLIFERAE(2). Acta Phytotaxonomica Sinica. 1986;24(4):304-316.
71.石 贇容, 孔 令義. 以前胡為例，探討中藥活性成分研究的思路與方法. 世界科學技術-中醫藥現代化. 2005;1:38-46.
72.黃 平, 鄭 學忠, 西 正敏, 中 西勤. 南嶺前胡素A的結構鑑定. Acta phamaceutics Sinica 1997;32(1):62-64.
73.高 安平. The consitituents of the leaves of Peucedanum longshengense and whole plant of Ampelopsis cantoniensis [碩士論文]. 台南: 化學研究所, 國立成奶j學; 2006.