以活性導向方式分離中國橄欖果實內具抑制癌細胞增生之成分

Abstract

中國橄欖（Canarium album）為橄欖科、橄欖屬。在中醫觀點中，因其具有抗菌、抗病毒、抗發炎與解毒之效果而被用以治療感染、痢疾及解蛇毒。本實驗室先前的研究發現中國橄欖甲醇萃取物正丁醇區分層（（Methanol-butanol olive extract（MBO））在小鼠大腸癌細胞（CT26）中具有顯著抑制癌細胞增生之效果，因此將進一步利用人體細胞平台的檢測結果，分析主要抗癌細胞增生活性的成分，最後將討論其抗癌細胞增生的機制。先前文獻指出，許多天然物會影響粒線體活性，而影響依賴粒線體酵素活性的細胞存活率測試方法之吸光值（如：MTT assay）；因此透過與IN Cell Analyzer 2200確認不同細胞存活率方法學的準確性，該儀器透過螢光染劑（DAPI）將細胞核中染色質染色後，由儀器計數得到準確細胞數。根據實驗結果，選擇MTT assay做為抗癌細胞增生活性測試的方法。樣品的部分，本實驗選用新竹縣寶山鄉種植之中國橄欖，收集水萃後之殘渣以甲醇萃取後減壓濃縮，再利用液-液萃取（liquid-liquid partition）的方式，以正己烷、乙酸乙酯及正丁醇等與水不互溶之有機溶劑，將此中國橄欖甲醇萃取物進行極性切割。再將中國橄欖甲醇-正丁醇區分層（MBO）在不同人類細胞平台進行活性測試，發現其在人類之多種大腸癌 （HCT116、HCT116 p53-/-、CaCO2）、舌癌（Ca922）、肺癌（A549）、血癌（HL-60）…等細胞中亦具有良好之抗癌細胞增生的效果。進一步將中國橄欖甲醇萃取物正丁醇區分層（MBO）利用酸、中、鹼三種不同pH值的緩衝溶液處理，藉此改變物質溶解度後，再以正丁醇反萃出不同酸鹼值的物質進行抗癌細胞增生活性篩選；發現中國橄欖甲醇-正丁醇區分層（MBO）經酸處理後具有最佳抗癌細胞增生效果。因此將此酸處理後的物質利用HP20疏水樹脂逆相管柱層析，以五種不同濃度的甲醇依序進行流洗，發現E4之次區分層保有最好的活性。而進一步以HPLC檢測，發現其含有高達72 %的鞣花酸。故推測鞣花酸為中國橄欖甲醇萃取物正丁醇區分層抗癌細胞增生之主要有效成分之一，至於其他波峰為何則需要進一步的鑑定分析。

Chinese olive (Canarium album.), which belongs to family of Burseraceae and genus of Canarium, is a traditional Chinese medicine with antibiotic, anti-virus, anti-inflammatory, as well as detoxification effects, and is used for curing infection, malaria, venom poinsoning. Former results in our lab indicated that the methanol-butanol olive extraction (MBO) significantly repressed the viability of murine colon cancer cells (CT26); however, more investigations for verifying the bioactive compounds in human cells model are needed. In the present study, Chinese olives debris was collected after water extraction, and extracted again with methanol. The methanol extract of Chinese olives was then fractioned by liquid-liquid partition with hexane, ethyl acetate, and butanol. The MBO was collected for further anti-cancer analysis. On the other hand, previous studies in our lab or other lab shows that some phytochemicals increase the activity of enzymes in mitochondria, making mitochondria-dependent viability assays, such as MTT become imprecise. To ascertain the accuracy of methodology, comparison of WST-1 and MTT with the control of cells counting by IN Cell Analyzer 2200 were conducted, and MTT assay was chosen to be the optimal way to determine the cancer cell viability. Results showed that MBO possessed anti-proliferative capacity against several cancer cells including colon cancer cells of HCT116 and HCT116 p53-/-, tongue cancer cell (Ca922), lung cancer cells (A549), and blood cancer cells (HL-60). Besides, MBO didn’t repress the proliferation of normal cell, including Int407 and CaCO2. After processing MBO with acidic buffer solution, MBO-2B fraction possessed enhancing the anti-tumor ability. Furthermore, fractionation the MBO-2B by HP20 resin, obtaining E4 sub-fraction with enriched anti-proliferative effect. By HPLC, compound of Ellagic acid accounted for 72 % of this elution. Hence, Ellagic acid was believed as the functional compound for the antitumor property in Chinese olives.