熊果酸（ursolic acid）是存在於天然植物中的一種三萜類化合物，具有鎮静、抗炎、抗菌、抗糖尿病、降低血糖等多種生物學效應；近年來發現，也具有抗癌作用。過去研究指出，熊果酸具有可減輕由四氯化碳誘導大鼠肝臟損傷與抗氧化酵素的下降。然而其真正的保肝的機轉仍然有必要進一步去探討。本研究以4％四氯化碳誘導小鼠產生急性及慢性肝臟損傷之模式，探討熊果酸之療效及作用機轉。急性模式為每天腹腔注射熊果酸一次，在第五天測定血中麩丙酮酸轉氨（glutamate pyruvate transaminase, GPT）之含量；慢性模式為每天口服熊果酸一次，在第一、三、六、八週測定血中GPT含量，並在第八週犧牲小鼠後，發現抗氧化酵素的表現，指標包含過氧化氫（catalase）、超氧化岐化酶（superoxide dismutase, SOD）、穀胱甘肽過氧化脢（glutathione peroxidase）、穀胱甘肽過還原脢（glutathione reductase），以及脂質過氧化的現象均有改善的效果。接著利用免疫組織染色切片，觀察肝纖維化的指標—α-smooth muscle actin（α-SMA）, connective tissue growth factor（CTGF）以及羥脯氨酸（hydroxyproline）—的含量，均發現纖維化的程度有改善。此外利用western blot也發現，經過熊果酸處理之後，CTGF含量比起四氯化碳組有減少的現象。Reactive oxygen species（ROS）氧化壓力的存在，是促進肝臟星狀細胞（HSC）活化的一個重要因子。在細胞模式中，我們以利用流式細胞儀及免疫螢光方法，證明熊果酸具有清除ROS的能力，並且具有減低HSC分泌metalloproteinase-2, 9（MMP-2, 9）的活性，以及清除脂質過氧化的裂解產物丙二酸（malondialdehyde, MDA）的能力。同時，利用western blot也發現，熊果酸可以抑制HSC活化時所分泌的CTGF含量。結果指出熊果酸具有對抗小鼠急性及慢性肝臟損傷的作用，其可能是透過清除自由基，免於肝臟受到ROS攻擊，且熊果酸可以減輕CCl4所引起的病理組織的改變。熊果酸同樣的可以降低CTGF的表現在體外及體內實驗，所以或許熊果酸可以成為治療抗纖維化的藥物。

Ursolic acid (UA), a pentacyclic triterpenoid exists widely in natural plants, has been shown to have a variety of pharmacological effects: as anti-inflammatory, anti-arthritic, anti-carcinogenic, anti-ulceric, anti-hyperlipidemic and anti-hyper- glycemic activities. Previous researches indicate UA possesses some antioxidant activities, capable of reducing toxic effect of carbon tetrachloride (CCl4). The present investigation aims at studying the hepato-protective activity of UA, with special emphasis placed on knowing the anti-oxidation and anti-fibrosis effects of UA. Using an acute animal model with a 5-d and a chronic animal model with an 8w-treatment schedules of CCl4 and enzyme assays at 1, 3, 6 and 8 w, we found that UA indeed can protect liver from damage exerted by CCl4 as judged by serum enzyme assay of glutamate pyruvate transaminase and hepatic enzyme assays of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. One of the major indicators of liver fibrosis is the activation of hepatic stellate cell (HSC), which is activated by the oxidative stress exerted by reactive oxygen species (ROS). We hence used ROS flow cytometry assay and lipid peroxidation assay as indicated by malondialdehyde level to see if UA has anti-oxidant activity. UA indeed has free radical scavenging activities. Immunofluorescence assay of metalloproteinase-2, 9 (MMP-2, MMP-9), the most relevant MMPs in hepatic fibrosis, are found to be reduced by UA treatment. Furthermore, three other indicators of liver fibrosis: (1) collagen as assayed by hydroxyproline content in the liver, (2) α-smooth muscle actin (α-SMA) in HSC, and (3) connective tissue growth factor (CTGF) assayed by histochemical staining and western blot, were found to be decreased by UA treatment. The results taken together indicate that UA can effectively protect against chemical-induced hepatic injury in vivo, suggesting that UA could become a candidate drug for the amelioration of liver fibrosis.

1. Abou-Shady M, Friess H, Zimmermann A, di Mola FF, Guo XZ, Baer HU, Büchler MW. Connective tissue growth factor in human liver cirrhosis. Liver 20(4): 296-304, 2000
2. Aparecida Resende F, de Andrade Barcala CA, da Silva Faria MC, Kato FH, Cunha WR, Tavares DC. Antimutagenicity of ursolic acid and oleanolic acid against doxorubicin-induced clastogenesis in Balb/c mice. Life Sci. 79(13): 1268-73, 2006
3. Balanehru S, Nagarajan B. Protective effect of oleanolic acid and ursolic acid against lipid peroxidation. Biochem. Int. 24(5): 981-90, 1991
4. Bataller R, Brenner DA. Liver fibrosis. J. Clin. Invest. 115: 209-218, 2005
5. Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 46(4): 443-6, 2000.
6. Blumenkrantz N, Asboe-Hansen G. A quick and specific assay for hydroxyproline. Anal Biochem. 55(1): 288-91, 1973
7. Castilla A, Prieto J, Fausto N. Transforming growth factors beta 1 and alpha in chronic liver disease. Effects of interferon alfa therapy. N. Engl. J. Med. 324(14): 933-40, 1991
8. Chen MM, Lam A, Abraham JA, Schreiner GF, Joly AH. CTGF expression is induced by TGF- beta in cardiac fibroblasts and cardiac myocytes: a potential role in heart fibrosis. J. Mol. Cell Cardiol. 32(10): 1805-19, 2000
9. Chung MI, Gan KH, Lin CN, Chen IJ. Studies on the constituents of Formosan gentianaceous plants. Part VII. Constituents of Swertia randaiensis Hayata and pharmacological activity of norswertianolin. Gaoxiong Yi Xue Ke Xue Za Zhi 2(2): 131-5, 1986
10. Eng FJ, Friedman SL. Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex. Am J Physiol Gastrointest Liver Physiol. 279(1): G7- G11, 2000
11. Erba D, Riso P, Criscuoli F, Testolin G. Malondialdehyde production in Jurkat T cells subjected to oxidative stress. Nutrition 19(6): 545-8, 2003
12. Fraga, C.g., Leibovitz, B.E. and Tappel, A.L. Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes. Free. Radic. Biol. Med. 4: 155-61, 1988
13. Friedman SL, Bansal MB. Reversal of hepatic fibrosis -- fact or fantasy? Hepatology 43(2 Suppl 1): S82-8, 2006
14. Friedrichsen S, Heuer H, Christ S, Winckler M, Brauer D, Bauer K, Raivich G. CTGF expression during mouse embryonic development. Cell Tissue Res. 312(2): 175-88, 2003
15. Gabele E, Brenner DA, Rippe RA. Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell. Front. Biosci. 1(8): 69-77, 2003
16. Gao J, Tang X, Dou H, Fan Y, Zhao X, Xu Q. Hepatoprotective activity of Terminalia catappa L. leaves and its two triterpenoids. J. Pharm .Pharmacol. 56(11): 1449-55, 2004
17. Gressner AM, Bachem MG. Molecular mechanisms of liver fibrogenesis--a homage to the role of activated fat-storing cells. Digestion 56(5): 335-46, 1995
18. Gressner AM, Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J. Cell Mol. Med. 10(1): 76-99, 2006
19. Halliwell B. Oxidants and human disease: some new concepts. FASEB. J. 1(5): 358-64, 1987
20. Han DW, Ma XH, Zhao TC, Yin L, Ji CX. Studies on the preventive action of oleanolic acid on experimental cirrhosis. J .Tradit. Chin. Med. 2(2): 83-90, 1981
21. Horn T, Junge J, Christoffersen P. Early alcoholic liver injury. Activation of lipocytes in acinar zone 3 and correlation to degree of collagen formation in the Disse space. J. Hepatol. 3(3): 333-40, 1986
22. Hunan Med. Inst. Pharmacological studied of hepatoprotective compounds from Swertia mileensis. Traditional Medicine 6: 47-62, 1975
23. John L. Brubacher, Niels C. Bols. Chemically de-acetylated 2, 7- dichlorodihydro- fluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes. J. Immunol. Methods. 251: 81-91, 2001
24. Jung HA, Park JC, Chung HY, Kim J, Choi JS. Antioxidant flavonoids and chlorogenic acid from the leaves of Eriobotrya japonica. Arch. Pharm. Res. 22(2): 213-8, 1999
25. Kim KA, Lee JS, Park HJ, Kim JW, Kim CJ, Shim IS, Kim NJ, Han SM, Lim S. Inhibition of cytochrome P450 activities by oleanolic acid and ursolic acid in human liver microsomes. Life Sci. 74(22): 2769-79, 2004
26. Kim, K.H., Chang, M.W., Kim, E.Y., Park, K.Y. and Sunwoo, Y.I., 1993. Effects of linoleic acid and ursolic acid on T subsets in sarcoma 180-transplated mice. Chemical Abstracts 119 262145x
27. Lieber CS, Leo MA, Cao Q, Ren C, DeCarli LM Silymarin retards the progression of alcohol-induced hepatic fibrosis in baboons. J. Clin. Gastroenterol. 37(4): 336-9, 2003
28. Lin CN, Chung MI, Gan KH. Novel antihepatotoxic principles of Solanum incanum. Planta. Med. 54(3): 222, 1988
29. Liu J. Pharmacology of oleanolic acid and ursolic acid. J. Ethnopharmacol. 49(2): 57-68, 1995
30. Liu J, Liu Y, Mao Q, Klaassen CD. The effects of 10 triterpenoid compounds on experimental liver injury in mice. Fundam. Appl. Toxicol. 22(1): 34-40, 1994
31. Liu J, Liu Y, Klaassen CD. The effect of Chinese hepatoprotective medicines on experimental liver injury in mice. J. Ethnopharmacol. 42(3): 183-91, 1994
32. Liu J, Liu Y, Klaassen CD. Protective effect of oleanolic acid against chemical- induced acute necrotic liver injury in mice. Zhongguo Yao Li Xue Bao 16(2): 97-102, 1995
33. Maher JJ, McGuire RF. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J. Clin. Invest. 86(5): 1641-8, 1990
34. Manns MP, Cornberg M, Wedemeyer H.Current and future treatment of hepatitis C. Indian J. Gastroenterol. Suppl 1: C47-51,2001
35. Marra F, Gentilini A, Pinzani M, Choudhury GG, Parola M, Herbst H, Dianzani MU, Laffi G, Abboud HE, Gentilini P. Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells. Gastroenterology 112(4): 1297-306, 1997
36. Martin-Aragón S, de las Heras B, Sanchez-Reus MI, Benedi J. Pharmacological modification of endogenous antioxidant enzymes by ursolic acid on tetrachloride- induced liver damage in rats and primary cultures of rat hepatocytes. Exp. Toxicol. Pathol. 53(2-3): 199-206, 2001
37. Masini A, Ceccarelli D, Giovannini F, Montosi G, Garuti C, Pietrangelo A. Iron-induced oxidant stress leads to irreversible mitochondrial dysfunctions and fibrosis in the liver of chronic iron-dosed gerbils. The effect of silybin. J. Bioenerg. Biomembr. 32(2): 175-82, 2000
38. Mathurin P, Moussalli J, Cadranel JF, Thibault V, Charlotte F, Dumouchel P, Cazier A, Huraux JM, Devergie B, Vidaud M, Opolon P, Poynard T. Slow progression rate of fibrosis in hepatitis C virus patients with persistently normal alanine transaminase activity. Hepatology 27(3): 868-72, 1998
39. Ma XH, Zhao YC, Yin L, Xu RL, Han DW, Wang MS. Studies on the preventive and therapeutic effects of ursolic acid (UA) on acute hepatic injury in rats. Yao Xue Xue Bao 21(5): 332-5, 1986
40. Milani S, Herbst H, Schuppan D, Hahn EG, Stein H. In situ hybridization for procollagen types I, III and IV mRNA in normal and fibrotic rat liver: evidence for predominant expression in nonparenchymal liver cells. Hepatology 10(1): 84-92, 1989
41. Mourelle M, Muriel P, Favari L, Franco T. Prevention of CCL4-induced liver cirrhosis by silymarin. Fundam. Clin. Pharmacol. 3(3): 183-91, 1989
42. Murakami S, Takashima H, Sato-Watanabe M, Chonan S, Yamamoto K, Saitoh M, Saito S, Yoshimura H, Sugawara K, Yang J, Gao N, Zhang X. Ursolic acid, an antagonist for transforming growth factor (TGF)-beta1. FEBS Lett. 566(1-3): 55-9, 2004
43. Paradis V, Dargere D, Bonvoust F. Effects and regulation of connective Tissue growth factor on hepatic stellate cells. Lab Invest. 82: 767-774, 2002
44. Paradis V, Dargere D, Vidaud M, De Gouville AC, Huet S, Martinez V, Gauthier JM, Ba N, Sobesky R, Ratziu V, Bedossa P. Expression of connective tissue growth factor in experimental rat and human liver fibrosis. Hepatology 30(4): 968-76, 1999
45. Pinzani M. Liver fibrosis. Springer. Semin. Immunopathol. 21(4): 475-90, 1999
46. Price KR, Johnson IT, Fenwick GR. The chemistry and biological significance of saponins in foods and feedingstuffs. Crit. Rev. Food. Sci. Nutr. 26(1): 27-135, 1987
47. Reeves HL, Friedman SL. Activation of hepatic stellate cells--a key issue in liver fibrosis. Front. Biosci. 1(7): 808-26, 2002
48. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine UI, Liotta LA, Falanga V, Kehrl JH, et al. Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. U.S.A. 83(12): 4167-71, 1986
49. Rockey DC. Hepatic blood flow regulation by stellate cells in normal and injured liver. Semin. Liver Dis. 21(3): 337-49, 2001
50. Sadlier DM, Connolly SB, Kieran NE, Roxburgh S, Brazil DP, Kairaitis L, Wang Y, Harris DC, Doran P, Brady HR. Sequential extracellular matrix-focused and baited- global cluster analysis of serial transcriptomic profiles identifies candidate modulators of renal tubulointerstitial fibrosis in murine adriamycin-induced nephropathy. J. Biol. Chem. 279(28): 29670-80, 2004
51. Safadi R, Friedman SL. Hepatic fibrosis-role of hepatic stellate cell activation. Med. Gen. Med. 4: 27, 2002
52. Saller R, Meier R, Brignoli R. The use of silymarin in the treatment of liver diseases. Drugs 61(14): 2035-63, 2001
53. Saraswat B, Visen PK, Agarwal DP. Ursolic acid isolated from Eucalyptus tereticornis protects against ethanol toxicity in isolated rat hepatocytes. Phytother. Res. 14(3): 163-6, 2000
54. Saravanan R, Viswanathan P, Pugalendi KV. Protective effect of ursolic acid on ethanol-mediated experimental liver damage in rats. Life Sci. 78(7): 713-8, 2006
55. Schuppan D. Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins. Semin. Liver Dis. 10(1): 1-10, 1990
56. Sedlaczek N, Jia JD, Bauer M, Herbst H, Ruehl M, Hahn EG, Schuppan D. Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis. Am. J. Pathol. 158(4): 1239-44, 2001
57. Shek FW, Benyon RC. How can transforming growth factor beta be targeted usefully to combat liver fibrosis? Eur. J. Gastroenterol. Hepatol.16(2): 123-6, 2004
58. Shukla B, Viser S, Patnaik G.K, Tripathi SC, Srimal RC, Day S and Dobhal P.C Hepatoprotective activity in the rat of ursolic acid isolated from Eucaltptus hybrid. Phytother. Res. 6(2) 74–79, 1992
59. Tang X, Gao J, Chen J, Fang F, Wang Y, Dou H, Xu Q, Qian Z. Inhibition by [corrected] ursolic acid of [corrected] calcium-induced mitochondrial permeability transition and release of two proapoptotic proteins. Biochem. Biophys. Res. Commun. 337(1): 320-4, 2005
60. Tyagi SC, Ratajska A, Weber KT. Myocardial matrix metalloproteinases: localization and activation. Mol. Cell Biochem. 126(1): 49-59, 1993
61. Wang JF, Olson ME, Ball DK, Brigstock DR, Hart DA. Recombinant connective tissue growth factor modulates porcine skin fibroblast gene expression. Wound Repair Regen. 11(3): 220-9, 2003
62. Watanabe T, Niioka M, Ishikawa A, Hozawa S, Arai M, Maruyama K, Okada A, Okazaki I. Dynamic change of cells expressing MMP-2 mRNA and MT1-MMP mRNA in the recovery from liver fibrosis in the rat. J. Hepatol. 35(4): 465-73, 2001
63. Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Critical Rev Toxicol. 33: 105-36, 2003
64. Xu GF, Li PT, Wang XY, Jia X, Tian DL, Jiang LD, Yang JX. Dynamic changes in the expression of matrix metalloproteinases and their inhibitors, TIMPs, during hepatic fibrosis induced by alcohol in rats. World J. Gastroenterol. 10(24): 3621-7, 2004
65. Yang C, Zeisberg M, Mosterman B, Sudhakar A, Yerramalla U, Holthaus K, Xu L, Eng F, Afdhal N, Kalluri R. Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology 124(1): 147-59, 2003
66. 高潤平 齊曉豔. 肝纖維化的發生機制與治療進展. 世界華人消化雜誌. 14(23): 2263-2269, 2006
67. 熊筱娟 陳武 鎮國安 張俊明 易增興 郭晟. 烏索酸的一般藥理學實驗研究. 江西師範大學學報. 29(1): 81-84, 2005
68. 熊筱娟 肖小華 陳武 易增興. 烏索酸對D-氨基半乳糖致小鼠急性肝損傷的影響. 宜春學院學報. 27(4): 1-9, 2005
69. 岳蘭萍 馬紅 賈繼東. 基質金屬蛋白酶及其抑制因子與肝纖維化. 胃腸病學和肝病學雜誌. 15(6): 627-629, 2006
70. 行政院衛生署十大死亡原因統計
71. 財團法人肝病防治學術基金會