臺灣博碩士論文加值系統

白樺酸（Betulinic acid）是一種萃取自白樺樹的三萜類化合物，具有抗病毒、抗瘧疾、抗腫瘤、抗發炎的活性。白樺酸具有治療當前醫藥界急欲克服的病症之潛力，然而白樺酸的細胞標的物質仍未知。為了要找尋白樺酸的細胞標的物質，本論文中將白樺酸固定在基質，製備成固定相白樺酸親和性管柱，並且研究此管柱的效用。經由前人的研究可知白樺酸可以抑制牛胰臟磷脂質分解酵素（bp-PLA2）的活性，在本論文證明固定相白樺酸可以與磷脂質分解酵素作交互作用。此交互作用可以改變磷脂質分解酵素的生物功能：經由酵素活性和細胞毒性的試驗顯示白樺酸可以抑制磷脂質分解酵素之活性並且可以抑制蛇毒磷脂質分解酵素的細胞毒性。白樺酸與磷脂質分解酵素的交互作用可望應用在磷脂質分解酵素的純化與毒性中和。本論文證明固定相白樺酸確實會與牛胰臟或蛇毒中之磷脂質分解酵素有交互作用，除此之外，固定相白樺酸管柱也可以與蛇毒中心臟毒，cardiotoxin gamma有交互作用，而cardiotoxin gamma的蛋白質三度空間結構與磷脂質分解酵素無相似之處，這表示在細胞中有機會找到非磷脂質分解酵素的白樺酸標的物質。本論文的貢獻即是設計此固定相白樺酸親和性管柱並測試其效用以應用來找尋白樺酸的細胞標的物質。

Betulinic acid (BA), a triterpenoid of white birch (Betula alba), exhibits anti-viral, anti-malarial, anti-tumor and anti-inflammatory activities. It has been shown to be an inhibitor of bovine pancreatic PLA2 (bp-PLA2); however, its cellular targets remain to be explored. As an effort of identifying the cellular target of BA, the BA immobilized matrix was made and the utility of the immobilized column was studied. Enzymatic activity assay and cytotoxicity test were used to confirm that the physical interaction between BA and PLA2 could change the PLA2 biological function. This interaction may be mediated by electrostatic interaction and hydrophobic interaction dependent on Ca2+. Eventually, the results identified a physical interaction of BA and bp-PLA2 or N-PLA2. It was suggested that this interaction could be applied to PLA2 purification and N-PLA2 toxin neutralization. In this thesis, the BA-immobilized column exhibited an affinity with bp-PLA2 or N-PLA2. Surprisingly, the column also binds cardiotoxin γ, which does not resemble to PLA2 in tertiary structure. The results represent the possibility of the cellular targets of BA besides PLA2. It was proved that BA-immobilized affinity column designed in this thesis might be applied to explore cellular targets of BA.

ABSTRACT 1
I. INTRODUCTION 3
1.1 Betulinic acid 3
1.2 Phospholipase A2 4
1.3 Affinity chromatography 5
1.4 Snake venom 6
1.5 The aims of this study 6
II. MATERIALS and METHODS 7
2.1 Materials 7
2.2 Preparation of betulinic acid immobilized affinity column 7
2.3 Immobilized betulinic acid affinity chromatography 8
2.4 SDS-PAGE and Western blotting 9
2.5 PLA2 enzymatic activity assay 10
2.6 Circular dichroism spectroscopy 11
2.7 Nodenauturing gel electrophoresis 12
2.8 High performance liquid chromatography 13
2.9 Cell culture 13
2.10 Cell viability analysis by trypan blue dye exclusion 13
2.11 Cell apoptosis analysis by flow cytometry using Hoechst and propidium
iodide (PI) double staining 14
III. RESULTS 15
Part A: Binding specificity of betulinic acid-immobilized affinity column.
3.1 Immobilization of betulinic acid to beads 15
3.2 Betulinic acid-immobilized affinity column could not bind BSA 16
3.3 Betulinic acid-affinity column could not bind to 0495
(small molecular weight of recombinant protein) 16
3.4 Uncoupled matrix could not interact with PLA2 from snake venom 17
Part B: Interaction between betulinic acid and bp-PLA2
3.5 The interaction between betulinic acid and bp-PLA2 17
3.6 Determining anti-bp-PLA2 enzymatic activity of betulinic acid 18
3.7 Ca2+-dependent Interaction between bp-PLA2 and immobilized BA 18
Part C: Identification between betulinic acid binding proteins from snake venom
by affinity chromatography
3.8 Analysis of purification progress by reverse native PAGE and HPLC 19
Part D: Interaction of betulinic acid and N-PLA2
3.9 The interaction of betulinic acid and N-PLA2 by affinity chromatography 20
3.10 Determining anti-N-PLA2 enzymatic activity of betulinic acid 20
3.11 Cytotoxicity of N-PLA2 to betulinic acid treated CHO.K1 cells 21
Part E: Interaction of betulinic acid and cardiotoxin γ
3.12 Determining the interaction of betulinic acid and cardiotoxin γ by
affinity chromatography 21
IV. DISCUSSION 22
V. REFERENCES 25
VI. FIGURES 29
VII. APPENDIX 50

Bringmann G, Saeb W, Assi LA, Francois G, Sankara Narayanan AS, Peters K, Peters EM. 1997. Betulinic acid: isolation from Triphyophyllum peltatum and Ancistrocladus heyneanus, antimalarial activity, and crystal structure of the benzyl ester. Planta Med. 63:255-7.
Chang, C.C. 1979. The action of snake venoms on nerve and muscle In: Snake venom, Handbook of Experimental Pharmacology. 52: 309-375, (Lee, C. Y. Ed) Berlin: Springer-Verlag.
Chi EY, Henderson WR, Klebanoff SJ. 1982. Phospholipase A2-induced rat mast cell secretion. Role of arachidonic acid metabolites. Lab Invest. 47(6):579-85.
Chien, K-H., Huang W-N., Jean J-H., and Wu, W. 1991. Fusion of sphingomyoelin vesicles induced by proteins from Taiwan cobra (Naja naja atra) venom. J. Biol. Chem. 266: 3252-3259.
Chowdhury AR, Mandal S, Mittra B, Sharma S, Mukhopadhyay S, Majumder HK. 2002. Betulinic acid, a potent inhibitor of eukaryotic topoisomerase I: identification of the inhibitory step, the major functional group responsible and development of more potent derivatives. Med Sci Monit. 8(7): BR254-65.
Dennis, E. A. 1997. Trends in Biochem. Sci. 22, 1-2
Fujioka T, Kashiwada Y, Kilkuskie RE, Cosentino LM, Ballas LM, Jiang JB, Janzen WP, Chen IS, Lee KH. 1994. Anti-AIDS agents, 11. Betulinic acid and platanic acid as anti-HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally related triterpenoids. J Nat Prod. 57:243-7.
Fulda S, Friesen C, Los M, Scaffidi C, Mier W, Benedict M, Nunez G, Krammer PH, Peter ME, Debatin KM. 1997. Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res. 57(21): 4956-64.
Harvey, A. L. and Hayashi. K. 1987. Depolarization of skeletal muscle cells in culture by a cardiotoxin-like basic polypeptide from the venom of Taiwan cobra (Naja naja atra), Toxicon. 25: 681-684.
Hack CE, Wolbink GJ, Schalkwijk C, Speijer H, Hermens WT, van den Bosch H. 1997. A role for secretory phospholipase A2 and C-reactive protein in the removal of injured cells. Immunol Today. 18:111-5.
Irvine, R. F. 1982. How is the level of free arachidonic acid controlled in mammalian cells? Biolchem. J. 204, 3-16.
Jack Taunton, Christian A. Hassig, Stuart L. Schreiber. 1996. A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science. 272: 408-411.
Kini RM, Evans HJ.1989. A model to explain the pharmacological effects of snake venom phospholipase A2. Toxicon. 27:613-35.
Leslie CC. 1997. Properties and regulation of cytosolic phospholipase A2. J Biol Chem. 272(27):16709-12.
Rosenberg, P. 1990. Phospholipase A2. Handbook of Toxicology (In Shier W.T., Mebs D. Ed), pp. 67-277, Marcel Dekker, New York
Lee, C.Y., Chang, C. C., Chiu, P. J. S. Tweng, T. C., and Lee S. Y. 1968. Pharmacological properties of cardiotoxin isolated from Formosan cobra venom. Naunyu-Schmiedbergs Arch. Pharmak. U. Exp. Path. 259: 360-374.
Lee, C.Y., and Lee, S. Y. 1979. Cardiovascular effect of snake venoms in: Snake venom, Handbook of Experimental Pharmacology. 52: 546-590, (Lee, C. Y. Ed) Berlin: Springer-Verlag.
Ma J, Starck SR, Hecht SM. DNA polymerase beta inhibitors from Tetracera boiviniana. 1999. J Nat Prod. 62(12): 1660-3.
Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, Beecher CW, Fong HH, Kinghorn AD, Brown DM, et al. 1995. Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat Med. 1(10): 1046-51.
Philippe B., Scior T, Didier B, Hibert M, Berthon JY. 2001. Ethnopharmacology and bioinformatic combination for leads discovery: application to phospholipase A(2) inhibitors. Phytochemistry. 58: 865-74.
Recio MC, Giner RM, Manez S, Gueho J, Julien HR, Hostettmann K, Rios JL. 1995. Investigations on the steroidal anti-inflammatory activity of triterpenoids from Diospyros leucomelas. Planta Med. 6:9-12.
R Manjunatha Kini. 2002. Molecular moulds with multiple missions: functional sites in three-finger toxins. Clinical and experimental pharmacology and physiology. 29:825-822
Scott, D.L, Sigler, P.B. 1994. Adv. Proteine Chem. 45, 53-88
S Fulda, C Friesen, M Los, C Scaffidi, W Mier, M Benedict, G Nunez, PH Krammer, ME Peter and KM Debatin. 1997. Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Research, 21:4956-4964.
Wu, W. 1997. Diversity of Cobra Cardiotoxin. J. Toxicol. —Toxin Reviews. 16: 115-134.
Yasukawa K, Takido M, Matsumoto T, Takeuchi M, Nakagawa S. 1991. Sterol and triterpene derivatives from plants inhibit the effects of a tumor promoter, and sitosterol and betulinic acid inhibit tumor formation in mouse skin two-stage carcinogenesis. Oncology. 48:72-6.
Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E, Gambacorti-Passerini C, Formelli F. 2002. Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett. 175(1): 17-25.