| 摘要: | 第一部分以萃取物經不同極性分離後，其抗氧化、抑制黑色素生成與保護質體DNA作用，並篩選出促進細胞增生之萃取物進行對皮膚細胞保護與修復作用之評估。RCE之 n-Butanol萃取物具有良好清除DPPH自由基(91.7%)與富含黃酮類與酚類含量，其Ethyl acetate萃取物具有清除NO自由基(27.6%)效能；COE之Ethyl acetate萃取物具有良好清除ABTS自由基(83.7%)及抑制脂質過氧化能力(98%)；LLE具有優異的抑制HaCaT細胞生成活性氧物質(reactive oxygen species, ROS) (74.8%)與提升GSH (32.1%)含量之能力，其n-Hexane萃取物具有明顯抑制細胞內酪胺酸?(92%)、多巴氧化?(100%)與黑色素生成(69%)之能力；於保護質體DNA試驗發現RCE較有明顯保護效果。以RCE進行對皮膚細胞保護與修復之評估與SKH-1小鼠經UVB照射後其紅斑表現量試驗，結果顯示經RCE作用後的SKH-1小鼠紅斑表現量明顯下降，推斷具有修復經UVB照射後所引起的發炎反應。綜合所有試驗發現RCE最具有發展之潛力。第二部分本研究由海洋生物中純化出之化合物(1)與(2)，作用在人類皮膚癌細胞，探討其對皮膚癌之毒殺機制。癌細胞株經(1)與(2)反應72小時後，發現(1)與(2)對人類上皮癌細胞株(Human epithelial carcinoma cell, A431)與人類皮膚鱗狀上皮癌細胞株(Human squamous carcinoma cell, SCC25)具有明顯毒殺性且具濃度依存性(Dose-dependent)，觀察細胞死亡型態變化，發現細胞呈現皺縮、空泡等細胞凋亡(Apoptosis)典型特徵，再經Annexin V/Propidium iodide染色試驗證實細胞凋亡區域比例提升；分析細胞週期變化發現(1)與(2)使A431與SCC25細胞之細胞週期停滯於G0/G1期；在活性氧(Reactive oxygen species, ROS)及穀胱甘?(Glutathione, GSH)含量試驗，發現兩個化合物皆隨著作用時間與濃度上升，增加A431與SCC25癌細胞之ROS與降低GSH含量；免疫螢光分析與西方墨點法試驗發現，A431與SCC25細胞經(1)與(2)作用後細胞凋亡相關蛋白質p53、p21、bax、cytochrome c、Fas、FasL、TRAIL、TRAILR1、TRADD、FADD、Caspase-8、Caspase-9和Caspase-3表現量增加。故推測(1)與(2)可能透過粒線體與死亡受體途徑誘發細胞凋亡，可能具有潛力作為研究皮膚癌之用藥。
Part1The bioactivity-guided fractionation isolation the extract of traditional herbs of RCE, COE, LLE and LPE in ethanol, n-hexane, ethyl acetate and n-butanol phase was investigated the anti-oxidation, anti-melanogenesis, and protection of plasmid DNA. We selected the extracts for improving skin cell proliferation, and then the protection and repair of skin cells of the extracts were examined. The extract of RCE in n-butanol phase expressed 91.7% DPPH free radical scavenging activity and rich polyphenols and flavonoids. The extract of RCE in ethyl acetate phase inhibited 27.6% NO free radical activities. The extract of COE in ethyl acetate phase had great effect of ABTS free radical scavenging activity (83.7%) and suppressed 98% lipid peroxidation production. Furthermore, LLE decreased intracellular ROS (74.8%) and increased GSH (32.1%) production in H2O2-treated human keratinocyte HaCaT cells. The extract of LLE in n-hexane phase could significantly suppress the cellular tyrosinase activity (92%), DOPA oxidase activity (100%) and melanin content (69%). Additionally, we also found that RCE evidently had a greater effect to keep DNA integrity and protect skin cells against UVB. RCE obviously decreased erythema formation after UVB-induced acute inflammation in male SKH-1 hairless mice. Therefore, we suggested that RCE has ability to prevent inflammation reaction from UVB irradiation. Base on all above these experiments, we concluded that RCE has strong potential to be developed.Part2In this study, we investigated the mechanism of anti-skin cancer though two pure compounds (1) and (2), which from marine lives. The cell viability in human epithelial carcinoma cell (A431) and human squamous carcinoma cell (SCC25) had been demonstrated by MTT assay. We found that treatment with (1) and (2) had apparently cytotoxicity and dose-dependent effects on A431 and SCC25 cells after 72 h. Morphological observation on cell death induced by (1) and (2) in A431 and SCC25 cells. Following treatment with (1) and (2) in both cancer cells, we found the typical features such as cell shrink, dynamic membrane blebbing and chromatin condensation. Analyzing the periodically changed of cell cycle, we showed that (1) and (2) led both cells arrest in G0/G1 phase with a concomitant significantly increased of sub-G1 population. In the intracellular reactive oxygen species (ROS) and glutathione (GSH) experiments, we found both compounds reduced the GSH and increased ROS generation. Further, we investigated the expression of apoptosis-associated factors by immunofluorescence and western blotting, results showed that (1) and (2) upregulated the expression of p53, p21, bax, cytochrome c, Fas, FasL, TRAIL, TRAILR1, TRADD, FADD, Caspase-8, Caspase-9 and Caspase-3. Therefore we deducted that (1) and (2) possibly induced apoptosis through bath of mitochondrion and death receptor pathway. These observations show that (1) and (2) have an anti-cancer effect which may be related with the prevention of cell proliferation, and promotion the activities of oxidative stress and cell apoptosis-related proteins in the skin cancer cells. |
