摘要

1. 第一部份：牡丹皮抑制Matrix Metalloproteinases ( MMPs) 活性之探討 第二部分：牡丹皮成份、Pyrimidine類、Benzotriazole類及其類似物對黃嘌呤氧化酵素之抑制作用 • 第一部份 ： 牡丹皮抑制MMPs活性之有效成份分離與探討牡丹皮 (Paeonia suffruticosa Andrews)為毛茛科(Ranunculaceae)植物。根據中醫文獻計載，牡丹皮有和血、破積血、通經脈等之功效，而西醫文獻則計載其有止血、解熱、抗菌、抗發炎、降血壓、中樞抑制、抗乙醯膽鹼等作用，近來臨床上應用於肺癌、肝癌、子宮頸癌方面有抑制作用。自民國七十一年以來癌症一直都是國內十大死因的首位，其中百分之八十的癌症病人死於癌細胞的轉移 (一般具侵入性)，所以抑制癌細胞的轉移將可大幅減低癌症死亡率。依文獻指出，癌細胞的侵入性轉移和細胞間質蛋白質裂解酵素 (matrix metallproteinases ; MMPs) 有密切關係，本實驗以高度表現 MMP-2 和 MMP-9 的A431 為模式，探討牡丹皮成份對其活性的影響。初步發現牡丹皮以 butanol 萃取，經 sephadex LH-20 所分離出的fraction 6具較佳之活性(IC50=4.6±0.2μg / ml)，而oleanolic acid其IC50為50.38±6.58μM。第二部份 ： 牡丹皮成份、Pyrimidine類、Benzotriazole類及其類似物對黃嘌呤氧化酵素之抑制作用黃嘌呤受黃嘌呤氧化酵素催化轉變成尿酸，過程中亦產生了許多超氧化陰離子自由基和過氧化氫，根據文獻記載黃嘌呤氧化酵素、超氧化陰離子自由基、過氧化氫與痛風、缺血後組織在灌流之傷害、腦瘤、急性胰臟炎及肝炎等疾病有關。本實驗探討牡丹皮成份、pyrimidine類、benzotriazole類化合物對黃嘌呤氧化酵素(xanthine oxidase)之抑制百分比、抑制百分之五十濃度 (IC50)、抑制型態、抑制常數 (Ki)，以期對上述疾病有所改善。黃嘌呤氧化酵素催化黃嘌呤轉變成尿酸，而尿酸在295 nm處有最大吸收，故利用紫外線光譜測定法測定尿酸吸光值變化速率，藉此得知黃嘌呤氧化酵素之活性是否改變。抑制型態、抑制常數可依據　Lineweaver - Burk plot 公式求得。結果顯示牡丹皮及bnzotriazole類抑制作用不明顯。Pyrimidine類中以2-mercaptopyrimidine (4)對黃嘌呤氧化酵素具較強的抑制作用，抑制型態屬於非競爭型抑制劑(IC50＝ 82.85μΜ, Ki = 6.07μΜ)。其他有效化合物以2-mercapto-4(3H)-quinazolinone (2) 和sulfasalazine (3) 抑制作用較強，抑制型態分別屬於混合型(不競爭型-非競爭型)與不競爭型抑制劑(IC50 ＝ 108.85、10.59μΜ, Ki = 67.59、32.12μΜ)。

2. Part I: Studies on the Matrix Metalloproteinases (MMPs) Inhibitory Effect of Paeonia suffruticosa Part II: Inhibition of Xanthine Oxidase by the Constituents of Paeonia suffruticosa, Pyrimidines, Benzotriazoles and Analogues • Part Ⅰ. Studies on the Matrix Metalloproteinases (MMPs) Inhibitory Effect of Paeonia suffruticosaPaeonia suffruticosa is a plant of Ranunculaceae. According to Chinese herb literature, Paeonia suffruticosa has been used as regulate blood, remove blood stasis and promote blood circulation. Paeonia suffruticosa has been reported to be used in hemostasis, antipyretic, anti-microbial, anti-inflammatory, anti-hypertention, anti-cholingergic and CNS inhibiton. In clinical, it also be used in treatment of lung cancer, hepatoma and cervical carcinoma, recently.Cancer has been the most serious cause of death in our country since 1982, the metastasis (invasion) of cancer cell leads 80% patients dead, and inhibition of the metastasis of cancer cell will decrease markly the death rate caused by cancer. According to the literature, the invasion of cancer is relative to matrix metalloproteinases (MMPs). This study will evaluate the inhibition of metastasis of Paeonia suffruticosa constituents on the A431 model. We found fraction 6 of butanol layer, which chromatographed from Sephadex LH-20, had high potential (IC50 = 4.6±0.2μg / ml), and another effective constitutent is oleanolic acid (IC50 = 50.4±6.6μM).Part Ⅱ. Inhibition of Xanthine Oxidase by the Constituents of Paeonia suffruticosa, Pyrimidines, Benzotriazoles and AnaloguesXanthine oxidase catalyses the oxidation of xanthine to uric acid, and yields superoxide anion and hydrogen peroxide. According to the literature, xanthine oxidase, superoxide anion and hydrogen peroxide is related to several diseases such as gout, ischemia-reperfusion injury, brain tumors, acute pancreatitis and hepatitis. In this study, the constitutents of Paeonia suffruticosa, pyrimidines, benzotriazoles and analogues have been tested for their inhibitory activities as percent inhibition, IC50, inhibitory type and inhibition constant (Ki). It is expected useful for the treatment of these dieases.The activity of xanthine oxidase was detected as the rate of uric acid production which has λmax of 295 nm. According to the Lineweaver-Burk plot equation, inhibition type and inhibition constant (Ki) were calculated. The constituents of Paeonia suffruticosa and benzotriazoles showed weak inhibitory effect on xanthine oxidase. Among pyrimidines, 2-mercaptopyrimidine (4) (IC50 = 82.5μM, Ki = 6.07μM) displayed potent activity on xanthine oxidase inhibition, and showed noncompectitive type inhibition which respect to the substrate xanthine. Among the analogues, 2-mercapto-4(3H)-quinazolinone (2) (IC50 = 108.85μM, Ki = 67.59μM) and sulfasalazine (3) (IC50 = 67.59μM, Ki = 32.12μM) induced mix type (non-competitive-uncompetitive) and noncompetitive type inhibition respectively.