| 摘要: | 葡萄科植物是具清熱效果的生藥，一般民間作為消炎、保肝、治療肝病或搗敷治癰腫。本論文以台灣特有種小本山葡萄全草之丙酮粗萃物及不同極性層析層進行抗氧化能力評估，分離出的純化合物則進行細胞毒殺試驗、B 型肝炎病毒抑制、DNA 保護作用及抑制 MMPs 活性測試。希望藉由研究小本山葡萄植物的抗氧化能力，來評估其添加於化粧品上可能性；同時未來能進一步開發成為抗肝腫瘤、抗肺腫瘤及抗B型肝炎病毒的天然資源。
在小本山葡萄的莖部粗萃物部分，針對 PLC/PRF/5 cell、HepG2 cell 兩株人類肝癌細胞及表達 HBsAg、HBeAg 的人類肝癌细胞系 HepG2.2.15 細胞株測試其抑制活性。其中以小本山葡萄莖部丙酮萃取物 (VTSA) 抑制 PLC/PRF/5 cell 的活性最好，在濃度 100 μg/mL 時達到 81 % 的抑制率。針對 HepG2 cell，所有粗萃物在 100 μg/mL 抑制率皆在 50 % 以下。在抗 B 型肝炎病毒 HBsAg 和 HBeAg 試驗中，都以小本山葡萄根部丙酮萃取物 (VTRA) 最好，在濃度 100 μg/mL 第九天時抑制率分別為 64.15 %和 59.11 %。
抗氧化試驗中，包含清除 DPPH 自由基、清除 ABTS 自由基、總酚含量的測定及脂質過氧化等幾項。在清除 DPPH 自由基及 ABTS 自由基方面結果顯示，小本山葡萄莖部丙酮萃取 (VTSA) 及其配比分離的乙酸乙酯層 (VTSAE)、水層 (VTSAW) 和粗分的 Fraction 7~10，具有相當好的抗氧化效果，總酚含量的結果則以 Fraction 7 和 8 最好。接著以其中效果較好的 VTSA、VTSAE、VTSAW 及 Fraction 7~10 做亞麻油酸過氧化物試驗，發現 7 個樣品抑制氧化物的效果均具相當於標準品 BHT的抗氧化能力，且其抗氧化作用，優於 Ascorbic acid 和α-Tocophperol，深具做為抗氧化化粧品之潛力。
由粗分的 Fraction 2 和 Fraction 3 分離出 Lupeol；Fraction 4-2、4-3、4-4 及 Fraction 5 分離出 Betulin。兩化合物的活性試驗，分別檢測了 DNA 修復試驗、A549人類肺腺癌細胞MTT試驗及Huh7、HepG2、Hep3B、PLC/PRF/5 等四株人類肝癌細胞株毒殺試驗。兩化合物在 DNA 保護方面都具有效果，其中 Betulin 對於 A549 細胞株具有細胞毒性，IC50 小於 15 ppm；在四株人類肝癌細胞中，以 Betulin 在 24 hr時毒殺 Huh7細胞的 CC50 = 31.7 ± 2.07 μg/mL 作用較好；而HepG2 的 CC50>60 μg/mL 不具明顯毒殺活性，其餘細胞株則都在 60 μg/mL 以下。Lupeol 的部分除了 Huh7 在 48 hr 的 CC50 = 50.2 ± 1.54 μg/mL 之外，其餘細胞株的 CC50皆大於 60 μg/mL，顯示不具明顯的癌細胞毒殺作用。
由以上實驗結果得知，小本山葡萄的抗氧化活性成分集中在偏高極性，僅需簡單步驟即可獲得優異之抗氧化萃取物。而在低極性層分離出的純化合物部分，Lupeol對細胞不具有毒性且具有優異的DNA保護效果，非常適合用於化粧品的添加；Betulin則對於人類肺腺癌A549 細胞株具有優異的毒殺作用，對於人類肝癌Huh7、PLC/PRF/5兩細胞株亦有毒殺效果，因此，在未來抗肺腺癌、抗肝癌的發展上，具有良好的應用價值。
本研究為第一次檢測Lupeol及Betulin對於Huh7、HepG2、Hep3B、PLC/PRF/5 等四株人類肝癌細胞株的毒殺作用，而結果亦顯示Betulin對Huh7、PLC/PRF/5兩細胞株具有毒殺效果，因此推斷，Betulin可能為小本山葡萄中抗Huh7、PLC/PRF/5兩癌細胞株的活性成分之一。
Vitaceae is a kind of material for cleaning-heat in medicine, normally, we use it for anti-flammatory, treating for liver trouble or trauma dressing. In this study, we evaluated the the ability of antioxidant from the stems of Vitis thunbergii were extracted with acetone and the different polar layers, then the pure compounds also evaluated cytotoxicity assay, inhibition of hepatitis B virus ( HBV ), DNA protectant activity and Inhibition of MMPs activity. We hope to studied by the antioxidant ability of Vitis thunbergii, to evaluate the possibility of application on development of new drugs and the practicality of adding to cosmetics. At the same time, it can develop to become the natural resources of anti-liver cancer, lung cancer and anti- hepatitis B virus in the future.
In the part of crude extracts of Vitis thunbergii, we used two different kinds of human liver cancer cells ( PLC/PRF/5 cell, Huh7 cell ) and the HepG2.2.15 cell line that express hepatitis B virus ( HBsAg and HBeAg ) for the experiment. Among these, stems of Vitis thunbergii were extracted with acetone, which had best inhibitory activity to PLC/PRF/5 cell, at a concentration of 100 μg/mL, the inhibition percentage was 81 % ; while all the crude extracts concentration were 100 μg/mL, the inhibition of HepG2 cells was less than 50 %. In the HBV of HBsAg and HBeAg experiment, VTRA had the best effects, in the ninth day, at a concentration of 100 μg/mL, the inhibition percentage were 64.15 % and 59.11 %.
The antioxidant tests were including DPPH free radical scavenging, ABTS free radical scavenging, total phenol content and lipid peroxidation test. For the VTSA , VTSAE, VTSAW and coarse isolation of the fraction 7-10, the DPPH scavenging and total antioxidant capacity had better effects. The result of total phenol content showed that fraction 7 and 8 had the best effect. Then choose the better results of VTSA, VTSAE, VTSAW and fraction 7~10 to determination of antioxidant activity using linolenic acid, the results found that there were seven samples which the inhibit effect was similar to the standard of BHT, and better than ascorbic acid and α-tocophperol.
The pure compounds : Lupeol was isolated from fraction 2 and fraction 3. Betulin was isolated from fraction 4-2, 4-3, 4-4 and fraction 5. For the activity test of two compounds, which were evaluated with DNA protection test, MTT test ( A549 cell ) and cytotoxicity test ( Huh7 cell, HepG2 cell, Hep3B cell, PLC/PRF/5 cell ), respectively. Among these, Betulin had cytotoxicity on A549 cells, the IC50 value was less than 15 μg/mL. On four human liver cancer cells, the Betulin had better cytotoxicity effect for Huh7 at 24 hr that the CC50 value were 31.7 ± 2.07 μg/mL. the CC50 value of HepG2 was higher than 60 μg/mL exhibited no significant cytotoxicity. While the CC50 value of Lupeol were 50.2 ± 1.54 μg/mL except 48 hr, the others were higher than 60 μg/mL, exhibited no significant cytotoxicity.
Above the results indicated that the antioxidant activity composition of Vitis thunbergii centralized on the high-polar, only needed the simple steps that can obtain the excellent antioxidant of extract. At the part of the pure compounds on the low-polar layer, Lupeol had not only cytotoxicity but also the excellent DNA protection effect, it was very suitable for adding to cosmetic. Betulin had excellent cytotoxicity onhuman lung adenocarcinoma cell line A549, two kinds of human liver cancer cells ( PLC/PRF/5 cell, Huh7 cell ) had the same cytotoxicity effect. Therefore, in the future, thedevelopment of anti-lung cancer and anti-liver cancer had the best value of application.
In this study, the result showed that the first time cytotoxicity test of Lupeol and Betulin for Huh7, HepG2, Hep3B, PLC/PRF/5 all had the cytotoxicity effect, Therefore, extrapolated that Betulin was the one of the activity composition of Vitis thunbergii to resist Huh7, and PLC/PRF/5 cell lines. |
