討論 - 台灣粗榧樹葉中具生物活性成分之分離與鑑定

4'，5，7－trihydroxyflavone又名apigenin(芹菜素)，此一化合物在台灣粗榧中是第一次被發現，亦是三尖杉屬植物中第一次發現，經由此次實驗測試，

apigenin對HeLa cell進行MTT assay且經由計算，其ED₅₀為3.08 μg/mL（11.4 μ M），此數值與之前文獻所提供之數值（IC₅₀為9.8 μM或 2.64 μg/mL）⁵⁵差異不大。

在實驗中，除apigenin 對 HeLa cell 做 MTT assay 外，並以 BEAS-2B cell、

AGS cell、COLO-205 cell 作為受測的細胞，發現 COLO-205 cell 有生長抑制作用，而BEAS-2B cell、AGS cell 無明顯生長抑制作用。

（1）HeLa cell

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0

0 . 7 8 1 . 5 6 3 . 1 3 6 . 2 5 1 2 . 5 2 5 5 0 μ g / m l

The rate of HeLa's survive (%)

圖3-22 由圖 3-11 中正丁醇第五層 HPLC 分析收得的單一峰對 HeLa cell 之 MTT assay

（2）AGS cell

The rate of AGS's survive (%)

圖3-23 由圖 3-11 中正丁醇第五層 HPLC 分析收得的單一峰對 AGS cell 之 MTT assay

（3）COLO 205 cell

0 The rate of COLO 205's survive (%)

圖3-24 由圖 3-11 中正丁醇第五層 HPLC 分析收得的單一峰對 COLO 205 之 MTT assay

（4）BEAS-2B cell

0 20 40 60 80 100 120

0.78 1.53 3.13 6.25 12.5 25 50 μg/ml

The rate of BEAS-2B's survive (%)

數列1

圖3-25 由圖 3-11 中正丁醇第五層 HPLC 分析收得的單一峰對 BEAS-2B 之 MTT assay

HeLa cell 之 MTT assay 結果可由圖 3-22 觀察得知，在未知物濃度 1.56 μg/ml 時，細胞存活率已有下降趨勢，在未知物濃度 6.25 μg/ml 時，其細胞存活率已低於20%；AGS cell 之 MTT assay 結果可由圖 3-23 觀察得知，即使在最大濃度 50 μg/ml 之未知物測試下，細胞存活率仍有 80%，亦無明顯之生長抑制作用；COLO 205 cell 之 MTT assay 結果可由圖 3-24 觀察得知，在未知物濃度3.13 μg/ml 時，細胞存活率已有下降趨勢，在最大濃度 50.0 μg/ml 之未知物測試時，細胞存活率已低於20%，所以未知物對 COLO 205 cell 有明顯的生長抑制作用。BEAS-2B cell 之 MTT assay 結果可由圖 3-25 觀察得知，在最大濃度50.0 μg/ml 之未知物測試下，依然無明顯生長抑制作用之現象。

ED50

CELL μg/ml μM

BEAS-2B x x

AGS x x

COLO-205 8.20 30.4

HeLa 3.08 11.4

在本實驗中，所受測之四種細胞，僅BEAS-2B cell為正常細胞，AGS cell、

COLO-205 cell、HeLa cell為癌細胞，在三種癌細胞中，apigenin僅對COLO-205 cell（ED₅₀ 8.20 μg/ml或 30.4 μM）和HeLa cell（ED₅₀ 3.08 μg/ml或 11.4μM）有明顯的生長抑制作用，故apigenin對細胞的生長作用中，對正常細胞是不具影響，而對特定的癌細胞具有生長抑制的作用。

Apigenin屬於類黃酮（Flavonoids），目前已知的類黃酮衍生物超過 9000 種，實驗中分離得到的apigenin是其中一種，皆以黃酮（flavone）為其基本結構，黃酮對於癌症的預防、治療和心臟冠狀動脈疾病都具有成效，此外，黃酮也被拿來做為藥物設計的基本結構。⁵⁴

Apigenin主要存在蔬菜水果^43-46，例如：芹菜、豆類、柑橘類水果及一些堅果類之中。此類成分具有抗氧化作用⁴⁷，目前已經有許多研究報導指出此類成分也具有抗癌的效果，例如：大腸直腸癌、血癌、皮膚癌、乳癌和前列腺癌

44-46,48-51，主要是引起癌細胞的細胞週期停止而造成細胞凋亡使癌細胞增生受

到抑制^50,52,53。Apigenin 也具有防癌的效果，因為 apigenin 可以抑制 TPA

（12-O-tetradecanoyl-phorbol-13-acetate）所調節的癌促進作用而達到防止癌症的產生⁵²。目前已經有將此類藥物的結構加以修飾，使其穩定性增加後，已經

應用在臨床上，例如：Flavopiridol⁵¹（圖3-26）。

Apigenin對子宮頸癌細胞（HeLa cell）之研究，除生物活性測試外，已有文獻指出apigenin使子宮頸癌細胞造成細胞凋亡之原因與p53、p21、Bcl-2 及Fas 有關（圖 3-27）⁵⁵；腫瘤抑制基因p53 在細胞生長和死亡當中扮演著維持平衡的角色，也是抑制或誘導腫瘤細胞凋亡的關鍵；p21 在細胞中的濃度受到p53 之影響，會因為p53 濃度的上升使得p21 亦隨之提高，並且p21 能夠在細胞循環中去抑制細胞G1 及S狀態之間的轉換，使細胞循環停滯在G1 狀態；Bcl-2 在細胞凋亡途徑中扮演著關鍵調控者的一種蛋白，對細胞凋亡的發生有著很強的抑制效果；Fas在細胞中是一種接收細胞凋亡訊號的接收者分子，當p53 過量表現時將會引起Fas的表現，Fas接收細胞凋亡訊號進而誘導癌細胞的死亡。

最後，本研究已架構由粗榧樹葉中分離出apigenin 之方法，其原理是依靠各化合物間有著不同極性之特性將之分離，故在其它亦含apigenin 的廣泛植物中，也能運用相同分離方法分離出apigenin。

O O

圖3-26 Flavopiridol 圖3-27 Apigenin 在子宮頸癌細胞凋亡途徑中所造成之影響

四、未來展望

Apigenin 在自然界中普遍存在於植物當中，相關文獻也已證實 apigenin 對許多不同癌細胞有抑制生長的作用，但目前的研究仍侷限於人類及老鼠的細胞測試，對於動物活體實驗甚是缺乏。

雖然在植物中所包含各種不同的類黃酮（Flavonoids），當中許多成份亦可能涵蓋apigenin 的結構在其中，卻甚少發現比 apigenin 之生物活性更強的衍生物，勢必有許多未知的各種apigenin 衍生物存在於植物裡，其中可能有更適合對抗癌細胞的apigenin 衍生物，但要從植物中分離出來，並不符合經濟效益且費時，所以apigenin 衍生物的取得，可從化學結構合成及修飾來著手，逐一找出對癌細胞有更好抑制作用的成份，甚至可找出適宜存在於動物活體內的成份，例如在 apigenin 接上一醣基,試圖延長 apigenin 衍生物在動物活體內的滯留時間，以充分在動物活體內發揮長時間的藥效。

雖然過去文獻中apigenin 對子宮頸癌細胞的研究已有初步探討，僅知道細胞凋亡途徑中是由少數蛋白質所造成的現象，而細胞凋亡的相關途徑卻是相當複雜而多樣的，故日後仍需再進一步探討apigenin 對子宮頸癌所造成細胞凋亡途徑的相關研究。

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