單寧酸抑制黃嘌呤氧化酵素及細胞凋亡之研究
The inhibiton of xanthine oxidase and cell apoptosis by tannic acid
中文摘要
痛風(gout)在生化上的標誌及診斷的必要條件是高尿酸血症，尿酸 (uric acid)的來源是由內生性及外生性的嘌呤鹼基經由黃嘌呤氧化酵 素(xanthine oxidase)氧化成亞黃嘌呤(hypoxanthine)，再連續氧化成黃 嘌呤(xanthine)，最後產生尿酸；在其氧化的過程中會同時產生活性 態氧(ROS)，包括過氧化氫(hydrogen peroxide)及超氧自由基
(superoxide anion radical)，造成細胞內氧化壓力的增加。在許多研究 中已證實過多的 ROS 在生物系統是有破壞性的。在本研究中，我們 經由定量 uric acid 產生的速度測量 xanthine oxidase 的活性，探討自 然界中廣泛存在的 tannic acid、gallic acid 及 pyrogallol 對 xanthine oxidase 活性的抑制作用。與 xanthine oxidase 抑制劑 allopurinol (IC50
=1.61 uM)做比較，我們發現 pyrogallol (IC50 =0.79 uM)與 tannic acid (IC50 =3.54 uM)對 xanthine oxidase 有明顯的抑制效果，而 gallic acid (IC50 =109.09 uM)則較不明顯。從酵素動力學我們發現 pyrogallol 對 xanthine oxidase 為競爭型抑制(competitive inhibition)，其 Km 值由 15.79uM 增加為 49.81uM，Ki 值為 0.28uM；而 tannic acid 對 xanthine oxidase 的抑制則為混合型之非競爭型抑制(noncompetitive-
uncompetitive inhibition)，其 Km 值由 15.79uM 降為 9.30uM，而 Vmax 值由 8.06uM/min 降為 3.65uM/min，KI 值為 2.48uM、Ki 值為 9.95uM。
Xanthine 與 xanthine oxidase 的反應會造成 ROS 的增加而誘發細胞凋 亡(apoptosis)，因此我們建立一細胞實驗模式，分別從細胞型態及 DNA 片段化(DNA fragmentation)等細胞凋亡特徵當作 xanthine oxidase 活性的指標，探討 tannic acid 等相關化合物對經由 xanthine / xanthine oxidase 所引發的細胞凋亡之抑制作用。結果顯示 tannic acid 確實可以經由抑制 xanthine oxidase 的活性而達到保護細胞的作用。
然而 tannic acid 對於經由過氧化氫所引發的細胞凋亡也具有抑制的 作用，說明了 tannic acid 同時具有抗氧化的活性。綜合以上結果我 們認為 tannic acid 可藉由對 xanthine oxidase 之抑制作用，應有潛力 被應用於痛風之預防及病症之減輕；並藉由其抗氧化活性降低與 ROS 有關疾病之危險性。
英文摘要
The major biochemical feature of gout is an elevated level of uric acid in the serum. In humans, uric acid is the final product of purine degradation.
Xanthine oxidase converts hypoxanthine to xanthine and then to uric
acid, and also causes generation of the reactive oxygen species (hydrogen peroxide and superoxide anion radical). In this report, we demonstrate that tannic acid and its metabolites, such as gallic acid and pyrogallol, inhibit xanthine oxidase. Determination of IC50 of various inhibitors of xanthine oxidase revealed that tannic acid (IC50= 3.54 uM) and pyrogallol (IC50= 0.79 uM) possessed significant inhibitory effect in comparison to allopurinol (IC50= 1.61 uM), whereas gallic acid (IC50=109.09 uM) has less effect. Pyrogallol was a competitive inhibitor; the Km increased from 15.79 to 49.81uM while Vmax did not change (Ki = 0.28 uM). Tannic acid exhibited mixed-noncompetitive inhibition in which Km decreased from 15.79 to 9.30uM and Vmax decreased from 8.06 to 3.65uM/min (KI = 2.48 uM; Ki = 9.95 uM). We established a cell model to examine inhibitors which protect programmed cell death (apoptosis) triggerd by xanthine / xanthine oxidase. Characteristics during apoptosis including
morphological changes and DNA fragmentation can serve as an index of xanthine oxidase activity with respect to a variety of inhibitors. The results showed that tannic acid protected HL-60 cells from apoptosis triggered by superoxide anion radicals. Also, tannic acid protected the HL-60 cells from apoptosis induced by hydrogen peroxide through its antioxidant activity. Based on our results, we conclude that tannic acid could be utilized for gout prevention and therapy due to its inhibitory effect against xanthine oxidase as well as antioxidant effect on reactive oxygen species (ROS).
