中國橄欖之抗增生活性因子的純化及鑑定 暨其生理活性之研究

Analyze and identify the anti-tumor compounds from Chinese olive extracts and its physiological activity

葉昱德(Yu-Te Yeh)

指導教授 ： 謝淑貞

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.02063

摘要

中國橄欖具有抗菌、抗病毒、抗發炎與解毒功效。先前的研究顯示其含有豐富的多酚類物質與體外抗氧化功效成分。因此，本研究目的為探討中國橄欖抗癌功能之有效成分。本實驗利用WST-1 assay作為抗癌功能之篩選平台，結果顯示，以甲醇-乙酸乙酯區分層(OM-EtOAc)進行Diaion HP-20大孔樹脂管柱層析後，所得次區分層OM-EtOAc-C和OM-EtOAc-D的抗腫瘤效果明顯優於其它次區分層。再經由90% 的Hexane/Ethyl acetate 進行沉澱， 得到上清液與沉澱後， 發現
OM-EtOAc-C的上清液對於細胞抑制效果效果優於沉澱部分，而OM-EtOAc-D的上清液與沉澱部分皆有細胞抑制效果，透過HPLC與TLC找出適當分離條件後，最後以silica gel 進行管柱層析，所得OM-EtOAc-C2b抑制細胞增生效果明顯優於OM-EtOAc-C的上清液，然而OM-EtOAc-D的上清液的次區分層之抑制細胞增生效果並無優於OM-EtOAc-D 的上清液。最後選用OM-EtOAc-C2b 進行LC-SPE-NMR與LC-MS的化學結構鑑定，共鑑定出共9個化合物，其中有2個化合物須更進一步確認分子式為何，並進行細胞存活率分析。在動物實驗方面，利用大腸癌細胞轉殖於Balb/c 6週齡公鼠，餵食高劑量(300mg/kg bw/day) 的OM-EtOAc與低劑量(150mg/kg bw/day) 的OM-EtOAc，4週後發現腫瘤細胞與腫
瘤組相比有明顯變小，推測可能進行細胞凋亡或壞死途徑。綜合上述，中國橄欖確實具有抗腫瘤增生效果，其具有功效的成分為gallic acid, methyl gallate, ellagic acid, esculetin, scopoletin。

關鍵字

中國橄欖 ； 抗癌 ； 總多酚

並列摘要

Chinese olive is a traditional medicine material, exhibiting beneficial functions in
anti-bacterium, anti-virus, anti-inflammation and detoxification. Previous studies have
displayed Chinese olive a phenolic compounds rich resource for providing
antioxidative activity. However, the role of Chinese olive in anti-cancer remains
unknown. Therefore, our aim is to search for compounds with anti-proliferation effect
in Chinese olive. In this study, we used WST-1 assay to examine the anti-proliferation
function of compounds within Chinese olive. Firstly, we obtained the ethyl acetate
fraction from Chinese olive methanolic extract and named it as OM-EtOAc. We then
found that the subfraction of OM-EtOAc-C and OM-EtOAc-D from OM-EtOAc
through Diaion HP-20 column chromatography exhibited better antitumor effect than
the other subfractions. These two subfractions were then precipitated by 90%
Hexane / Ethyl acetate. The supernatant of OM-EtOAc-C revealed superior anti-tumor
activity to the precipitate, whereas both the supernatant and precipitate of
OM-EtOAc-D displayed comparable effect. We then used HPLC and TLC spot to
titrate appropriate separation conditions for separating the supernatants of
OM-EtOAc-C and OM-EtOAc-D (OM-EtOAc-C2 and OM-EtOAc-D2), followed by
chromatography on silica gel. The OM-EtOAc-C2b revealed better effect thanOM-EtOAc-C2, however, no fraction exhibited higer anti-tumor activity than OM-EtOAc-D2. Finally, we choose OM-EtOAc-C2b to identify functionalcompounds using LC-SPE-NMR and LC-MS, and 12 compounds were identified.Among the compounds identified, three remained to be further analyzed to confirm
the chemical structure. In animal experiments, We also implanted mouse CT26 colon cancer cells to BALB/c mice and allowed them to form tumors. The tumor-bearing mice were treated with either high dose of OM-EtOAc (300mg/kg bw/day) or lowdose of (150mg/kg bw/day) OM-EtOAc. Compared with control mice, OM-EtOActreated mice beared significantly smaller tumor after four-week treatment. In conclusion, Chinese olive has anti-tumor proliferative effect, and the functional compounds includee gallic acid, methyl gallate, ellagic acid, esculetin, scopoletin.

並列關鍵字

Chinese olive ； anti-tumor ； total polyphenol

參考文獻

邱詩婷。 2010。建立並評估生物技術平台篩選具有抗發炎效果的食品。國立台

陳奕安。2012。利用細胞平台的評估進行中國橄欖萃取物中具有抗發炎或抗腫瘤

total phenolic contents of tonic Chinese medicinal herbs. Inflammopharmacology

2008, 16, 201-7.

3. Zhang, L. L.; Lin, Y. M., Tannins from Canarium album with potent antioxidant