AP-1 是一個很特別的蛋白質，它會受到一些外來訊息的刺激，
例如 TPA、生長因子及紫外線等，促使基因轉錄，進而造成腫瘤的發 生(Angel and Karin, 1991)。報告指出能抑制 AP-1 活性的物質即能抑 制細胞轉形或腫瘤的產生(Barthelman et al., 1998; Dong et al., 1997;
Domann et al., 1994)，因此我們以 98 種中藥作為材料，探討中藥是否 可以透過 AP-1 的方式來抑制肝細胞轉形的產生，其抑制 AP-1 的機 制又是如何。
我們選擇麻黃、防己、吳茱萸、知母、厚朴、檳榔、大黃、苦參、
皂莢、甘草等十種中藥作為下一步所要接下來的實驗。
麻黃、防己、吳茱萸、知母、厚朴、檳榔、大黃、苦參、皂莢、
甘草在 Chang/AP-1 中是抑制由 TPA 所誘發 AP-1 活性的前十名中藥，
另外在 HepG2/AP-1 細胞中抑制由 TPA 所誘發 AP-1 活性的前十名中 藥則分別為吳茱萸、天花粉、赤芍藥、烏梅、皂莢、大黃、青蒿、苦 參、黃蓮、黃柏，若將在兩細胞株中抑制 AP-1 活性的前十名藥物做 相互比較，可以發現吳茱萸、皂莢、大黃、苦參等四種中藥，在 Chang/AP-1 及 HepG2/AP-1 兩細胞中都同時可以抑制 AP-1 的活性，
有效的抑制 AP-1 活性。但是從 soft agar 的結果發現抑制 Chang/AP-1 細胞最明顯的中藥是吳茱萸和防己。而其他的報告指出吳茱萸萃取物 可 以 抑 制 老 鼠 大 腸 癌 細 胞 的 轉 移 且 吳 茱 萸 在 五 大 類 (Alkaloids, Phenylpropanoids, Terpenoids, Flavonoids, Steroids)共 23 種藥物中對抑 制腫瘤轉移的效果最顯著(Ogasawara et al., 2001)，所以我們選擇吳茱 萸來進一步探討其抑制腫瘤的分子機轉。
在分析吳茱萸之前，我們想了解是否市售的吳茱萸都有相同抑 制 AP-1 活性的能力，因此我們比較了兩個來源不同批號的吳茱萸，
經過相同萃取及實驗結果發現不同來源的吳茱萸確實在抑制 AP-1 的 活性上有不同的結果，特別是在吳茱萸甲醇萃取物的 25 μg/ml 及 125 μg/ml 兩個濃度上，而這剛好也是吳茱萸 EC50的濃度區間。會 造成這種差異可能的原因是市售吳茱萸在乾燥時的過程不同而導致 有效成分在加工處理的過程中流失。
吳茱萸的甲醇萃取物可以抑制 AP-1 活性及抑制 Chang/AP-1 細 胞轉形，並且隨著劑量的增加效果越顯著。透過五種萃取吳茱萸的方 式(酸性乙醇、乙醇、甲醇、水、沸水)可以發現水及熱水的吳茱萸萃 取物對於抑制 AP-1 活性沒有顯著的效果，而透過統計的計算，酸性 乙醇、乙醇及甲醇三種溶劑的吳茱萸萃取物則對 AP-1 有明顯的抑制
細胞轉形的物質是屬於非極性，並且有效濃度在 5 μg/ml 及 25 μ g/ml 之間。乙醇可以萃取出非極性的成分，若在乙醇中加入 0.5%~1%
的酸，則可以利用生物鹼溶於酸性溶液的特性，而過濾除去不溶於酸 性溶液的非極性成分(肖, 1987)。報告指出吳茱萸的生物鹼成分之 一：Evocarpine，可以促進 HL-60 血癌細胞的細胞凋亡(Apoptosis)( Kim NY et al., 2001)；另一種生物鹼：Evodiamine 則可以抑制 26-L5 大腸 癌細胞的轉移(Ogasawara et al., 2001)，這與我們的實驗有相似的結 果，因此我們的結果也可以推測出吳茱萸具抑制 AP-1 的活性及抗肝 細胞轉形的物質為生物鹼，因此值得進一步利用動物試驗驗證其治療 肝癌的效果。
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附表、本研究所使用中藥之 1/1000 所代表之濃度
Abstract
Activator protein 1(AP-1), a nuclear transcription factor, plays important roles in cell proliferation, differentiation, and transformation.
Previous study indicated that blocking of AP-1 activity is able to inhibit the cellular transformation. We therefore develop the anti-human hepatocellular compounds using AP-1 on the target in this study. Effects of 98 herbs on 12-
O
-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activity in Chang/AP-1 and HepG2/AP-1 recombinant cells were investigated. Among the herbs, 10 herbs, which showed inhibition abilities on AP-1 activity, were selected. These herbs include Ephedrasinica, Evodia rutaecarpa, Magnolia officinallis, Rheum officinale, Arcea
catechu, Stephanica tetrandra , Anemarrhena asphodeloids, Gledititsia
sinensis, Sophora flavescens, and Glycyrrhiza uralensis. These herbs
were further analyzed for their anti-AP-1 activities and anti-hepatocellular transformation activities by luciferase assay and anchorage-independent transformation assay. Results showed that Evodiarutaecarpa exhibited the significant inhibition ability on AP-1 activity
and hepatocellular transformation in a dose-dependent manner. Moreover, the methanol, acid alcoholic, and alcoholic extracts of E. rutaecarpa were responsible for their anti-hepatocellular transformation abilities.作者簡歷
姓名：吳泰賢
民國65年6月24日生
出生地：台北市
學歷：
台北縣立板橋國小 台北縣立重慶國中 國立板橋高級中學
私立中國醫藥學院公共衛生學系
私立中國醫藥學院醫學研究所碩士
博碩士論文授權書
本授權書所授權之論文為本人在 私 立 中 國 醫 藥＿學院＿醫學研究＿所 基 礎 醫 學 組＿ 九 十 ＿學年度第＿二＿學期取得＿碩＿士學位之論文。
論文名稱：＿＿以 AP-1 為 標 的 開 發 抗 肝 細 胞 轉 形 藥 物＿＿＿＿＿＿＿
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指導教授姓名:
研究生簽名: 學號:8966009
日期:民國 91 年 7 月 30 日