大葉山欖果實萃取物之抗氧化活性及對肝癌細胞HepG2之抑制活性探討

Antioxidant activity and biological activity against HepG2 cell of the extract from Palaquium formosanum Hayata fruits

陳品宏(Chen Pin-Hung)

指導教授 ： 廖遠東

國立屏東科技大學/農學院/食品科學系所/碩士(2020年)

https://doi.org/10.6346/NPUST202000299

摘要

大葉山欖(Palaquium formosanum Hayata)為山欖科(Sapotaceae)膠木屬(Palaquium)植物，主要分布於熱帶低海拔地區，其果實可食。文獻指出，多種山欖科植物之果實及種子具有抗氧化及多種生物活性，經研究證實大葉山欖種子之三萜類皂苷具有抗腫瘤效果。目前大葉山欖全果之研究探討相當有限，因此本研究將其果實分為三部分，以不同溶劑進行萃取，分別探討其抗氧化能力及對人類肝癌細胞HepG2之抑制活性。結果顯示，種子之總酚含量(183.91-233.19 mg GAE/g)及總類黃酮含量(169.06-196.28 mg quercetin/g)皆顯著高於果肉及果皮。於DPPH自由基清除能力及總抗氧化能力(TEAC)試驗皆以種子之50%乙醇萃取物為最佳，IC50值分別為7.60±0.53 μg/mL及62.54±1.50 μg/mL；還原能力以1000 ppm BHA作為基準(100%)與實驗組做對照，種子之50%乙醇萃取物具最佳還原能力，於300 μg/mL濃度下達到88.24±0.58 %。經高效液相層析儀(HPLC)分析，種子萃取物含有沒食子酸及兒茶素等酚類化合物，進一步以(LC-MS)分析，推測萃取物中未知化合物為表沒食子兒茶素沒食子酸酯(EGCG)。抗癌活性試驗結果顯示，種子萃取物對於人類肝癌細胞HepG2具有顯著抑制能力，尤以種子水萃取物最佳，於作用48及72小時後，半抑制濃度分別為180.50±15.94及99.08±6.81 μg/mL，藉由流式細胞儀分析，HepG2之細胞週期明顯有SubG1期增加之趨勢且呈現濃度及時間依賴性。經Annexin V及PI雙染之細胞凋亡試驗評估，種子水萃取物具有顯著誘導HepG2細胞走向凋亡之效果。最後利用FITC-DEVD-FMK染劑並以流式細胞儀分析，證實經種子萃取物作用，HepG2之下游凋亡蛋白Caspase-3受到活化，導致細胞失去修補DNA之能力進而誘導細胞凋亡。綜合上述結果，種子萃取物之抗氧化活性優於果肉及果皮，並且能夠促進HepG2細胞之細胞凋亡，未來能更完整地分析凋亡相關蛋白表現量以釐清抑癌作用之完整機制，期待能增加其輔助癌症治療之可能性。

關鍵字

大葉山欖 ； 抗氧化 ； 人類肝癌細胞(HepG2)

並列摘要

Palaquium formosanum Hayata belong to Sapotaceae family, distributes to tropical and its fruit is edible. In some literature, fruits of Sapotaceae family possess antioxidant and various biological activities. Moreover, the triterpenoid saponins from seeds of P. formosanum which were indicated that having antitumor effect. Currently, the researches on the fruit of P. formosanum are quite limited. As a result, in this study, the parts of fruit (seed, flesh and peel) were extracted with water and different concentration of ethanol (25%, 50% and 75%), respectively, to explore antioxidant and anticancer activities. The results of antioxidant assay revealed that the total phenolic (183.91-233.19 mg GAE/g) and total flavonoid (169.06-196.28 mg quercetin/g) content of the seed showed the greater content than that of the pulp and peel extracts. In the DPPH free radical scavenging activity and Trolox Equivalent Antioxidant Capacity(TEAC), the 50% ethanol extract of seed presented the best effects, and the IC50 values were 7.60±0.53 μg/mL and 62.54±1.50 μg/mL, respectively. The reducing power ratio compared as BHA at 1000 ppm, the 50% ethanol of seed extract had the greatest reducing power and reaches 88.24±0.58% at 300 μg/mL. The bioactive components were analysed by HPLC, the seed extracts were found gallic acid and catechin. Further analysis by (LC-MS) revealed that the unknown compound in the extract was (3)-epigallocatechin gallate (EGCG). In anticancer assay, through the MTT cell viability assay, the seed extracts presented significant inhibitory effect against HepG2 (hepatocellular carcinoma cell). In addition, after water extract of seed was treated for 48 and 72 hours, the IC50 of inhibitory ability were 180.50±15.94 and 99.08±6.81 μg / mL, respectively. The cell cycle distribution was analyzed by flow cytometry, the cell cycle through the treated of water extract of seed obviously revealed an increasing trend of SubG1 phase in dose and time-dependent manner. By annexin V-PI apoptosis analysis, the significant effect of inducing apoptosis in HepG2 cell can be found treated with water extract of seed. It was
confirmed that the downstream apoptosis protein caspase-3 of HepG2 which treated with seed extract was activated, causing the cells to lose the ability to repair DNA and induce apoptosis. These results suggest the antioxidant activity of the crude extract of seed was greater than that of flesh and peel, and possess significant inhibitory effect on the HepG2 cell. In the future, the expression level of apoptosis-related proteins can be analyzed more completely to clarify the complete mechanism of anticancer, and it is expected to increase the possibility of cure for cancer.

並列關鍵字

Palaquium formosanum Hayata ； Antioxidant activity ； Human hepatocellular carcinoma cell (HepG2)

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