天然抗氧化劑保健功能之研究

Studies on the health-protective functions of nature antioxidants

莊文貞(Wen-Chen Chuang)

指導教授 ： 呂鋒洲 ； 柯俊良

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2009年)

摘要

第一部份
杜莎藻是一個具有雙鞭毛的單細胞綠藻並且含有大量的β胡蘿蔔素，一些文獻指出杜莎藻具有抗氧化及保肝的功能。在本研究中我們發現杜莎藻萃取物對不同血癌細胞株HL-60, K-562, KG-1, MOLT-4, Jurkat細胞之毒殺作用具有劑量及時間效應，並且不會對正常周邊血液單核細胞(PBMC) 產生顯著的毒殺作用。在細胞株的測試中，HL-60細胞具有最高的敏感性，於是我們選擇HL-60細胞進一步分析其可能的相關機制。由細胞型態的變化、去氧核醣核酸片段化及流式細胞儀的實驗結果顯示杜莎藻萃取物藉由誘發HL-60細胞進行細胞凋亡而顯著地減少細胞的存活率。我們發現杜莎藻萃取物所誘導的細胞凋亡與caspase 9 和caspase 3的活化有關，並且廣效性caspase抑制劑(z-vad-fmk)的共同處理可以部分的阻斷杜莎藻萃取物所誘導的細胞凋亡，另外，粒線體膜電位的降低與Bax/Bcl-2 及 Bad/Bcl-xL 的比例提高有關。此外，杜莎藻萃取物可能是透過促進p21、p27蛋白表現，而抑制CyclinA、CyclinD1、CyclinE、cdk2、cdk4、cdk6的表現，使得細胞週期停滯於G1期。β胡蘿蔔素的處理會誘導HL-60死亡並產生H2O2 ，而杜莎藻萃取物的處理則會降低H2O2以及增加NO的含量。值得注意的是，低劑量杜莎藻萃取物的處理便足以促進三氧化二砷的毒殺作用。本研究為首篇杜莎藻萃取物針對抗血癌作用的機制探討，證實杜莎藻萃取物抑制HL-60細胞生長是經由粒線體路徑之caspase依賴性的細胞凋亡及藉由CDK抑制劑使細胞週期停滯於G1期並且具有輔助三氧化二砷作用的潛能。未來我們將持續做更進一步的探討，以釐清杜莎藻萃取物抗癌的分子機制。
第二部分
本研究旨在探討負氫離子對誘導小鼠化學性慢性肝炎的保護作用。每週兩次經口投予四氯化碳(20 %, 0.l mL/mice) 以誘導肝損傷及肝硬化，為期八週，小白鼠隨機分為七組，分別為二次水組、橄欖油組、四氯化碳組、四氯化碳+低劑量負氫離子組(1x, 104mg/kg)、四氯化碳+中劑量負氫離子組(2x, 208mg/kg)、四氯化碳+高劑量負氫離子組(5x, 520mg/kg)、四氯化碳+silymarin組，每週利用餵管的方式給予負氫離子或silymarin六次 。肝功能的評估分別有ALT、AST、鹼性磷酸酶(SALP)、總蛋白(total protein)、白蛋白(albumin)、三酸甘油脂(TG)、膽固醇(Cholesterol)，以及抗氧化評估：超氧歧化酶(SOD)、過氧化氫酶(Catalase)、GSH-Px、GSH-Rd、谷胱甘肽(GSH)、MDA-TBA，並且測量小鼠體重及肝重。與單獨四氯化碳處理相較之下，發現負氫離子或silymarin的給予，可減少血清中ALT、AST、三酸甘油脂、膽固醇的表現，並提升超氧歧化酶(SOD)、過氧化氫酶(Catalase)、GSH-Px的活性，增加谷胱甘肽(GSH)的含量、抑制MDA-TBA的產生。結果顯示負氫離子可能透過提升抗氧化酵素的活性及抑制脂質的過氧化而達到保肝的效果。

關鍵字

杜莎藻 ； 細胞凋亡 ； 細胞週期停滯 ； 活性氧 ； 負氫離子 ； 保肝 ； 四氯化碳

並列摘要

Dunaliella salina (D. salina) is a unicellular biflagellate green alga and contains abundant β-carotene. Some papers reported that D. salina exhibited antioxidant activity and liver protection. In this study, D. salina extract showed dose- and time- dependent cytotoxic effects on various human leukemia cell lines, including HL-60, KG-1, K-562, MOLT-4 and Jurkat T cells but not normal peripheral blood mononuclear cells (PBMC). Among cell lines tested, HL-60 showed the highest sensitivity to D.salina extract and was so chosen for studying on the mechanism of action. D. salina extract could dramatically reduce the viability by inducing the apoptosis of HL-60 cells as demonstrated by morphological changes, DNA fragmentation and flow cytometry. The D. salina extract-induced apoptosis of HL-60 cells was associated with activation of caspase-9 and caspase-3 and the addition of a pen caspase inhibitor, Z-VAD-FMK, partially inhibited D. salina extract-induced apoptosis. In addition, mitochondrial membrane potential was decreased which was associated with a shift in Bax/Bcl-2 and Bad/Bcl-xL ratio. Moreover, treatment of HL-60 cells with D. salina extract resulted G1 arrest in cell cycle progression which was associated with decreasing the expression of protein of cyclin A, D1, and E and their activating partner CDK2, 4 and 6 with concomitant induction of WAF/p21 and KIP1/p27. The delay in cell growth by β-carotene was coincident with the increased H2O2 .Treatment with D. salina extract oppositely led to the HL-60 reducing H2O2 and increasing nitric oxide. Notably, a low dose of D. salina extract was sufficient to aggravate As2O3-induced cytotoxicity in HL-60 cells. This is the first report of the D.salina extract that had anti-leukemia effects through caspase-dependent apoptosis of mitochondrial pathway and also blocking cell cycle progression via CDK inhibitors and had the therapeutic potential by being an adjunct to As2O3. The further molecular mechanisms of anti-cancer effect of D. salina extract will be continuing study in future.
part2
The purpose of this study was to investigate the hepato-protecitve effects of negative hydrogen ion on chemical-induced chronic hepatotixicity in mice. Liver damage and cirrhosis were induced by carbon tetrachloride (CCl4) (20 %, 0.l mL/mice, by gavage) twice a week for 8 weeks. Carbon tetrachloride-induced mice were randomly assigned to seven groups: ddH2O, oliver oil, CCl4 , CCl4 plus silymarin, CCl4 plus low dose of negative hydrogen ion (1x, 104mg/kg) and medium dose of negative hydrogen ion (2x, 208mg/kg) and high dose of negative hydrogen ion (5x, 520mg/kg), each given (negative hydrogen ion or silymarin) by gavage 6 times a week for 8 weeks. Liver function tests (biochemical marker: GOT, GPT, SALP, total protein, total albumin, TG, Cholesterol; antioxidative enzymes: SOD, catalase GSH, GSH-Px, GSH-Rd, MDA-TBA) were performed, and liver and body weights were measured. Treatment with negative hydrogen ion or silymarin could significantly (p < 0.05) decrease the GOT, GPT, TG and Cholesterol levels in serum and increase the activities of SOD, catalase, GSH-Px and GSH content and decrease the MDA content in liver when compared with CCl4-treated group. The results suggest that negative hydrogen ion exhibits the hepatoprotective effects on CCl4-induced liver damages in mice, and that the hepatoprotective effects of negative hydrogen ion may be due to both the increase of antioxidant enzymes activities and inhibition of lipid peroxidation

並列關鍵字

Dunaliella salina ； apoptosis ； cell cycle arrest ； ROS ； Negative hydrogen ion ； Hepatoprotective effect ； Carbon tetrachloride

參考文獻

Aasen, A. J., Eimhjellen, K. E., and Liaaen-Jensen, S. (1969). An extreme source of beta-carotene. Acta Chem Scand 23, 2544-2545.

Aharon, O. (2005). A hundred years of Dunaliella research: 1905–2005. Saline Syst. 1, 1-14.

Alves-Rodrigues, A., and Shao, A. (2004). The science behind lutein. Toxicology Letters 150, 57-83.

Amir, H., Karas, M., Giat, J., Danilenko, M., Levy, R., Yermiahu, T., Levy, J., and Sharoni, Y. (1999). Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of cell cycle progression and induction of differentiation in HL-60 leukemic cells. Nutr Cancer 33, 105-112.

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