|摘要: ||(PART 1)本研究以水萃物CYY1PW和CYY1FW、酒萃物CYY1PE和CYY1FE；水萃物CYY2W、CYY2SW和CYY2LW、酒萃物CYY2E、CYY2SE和CYY2LE進行抗氧化、美白及DNA修復之機制探討。
抗氧化實驗以自由基清除能力、總抗氧化成分含量、脂質過氧化及還原力來評估。細胞內實驗以HaCaT和B16F10細胞進行毒性試驗，結果顯示水萃物較酒萃物無毒。所以選擇對細胞無毒之水萃物，利用HaCaT cells經H2O2刺激後，測試萃取物對ROS、GSH、Catalase及GPx之影響。美白實驗以測定B16F10 cells酪胺酸脢活性、黑色素含量及分析黑色素生成相關蛋白質之表現。發現CYY1PW、CYY2W對於MITF、MC1R、Tyrosinase、TRP2及TRP1及蛋白質表現上皆有明顯的抑制效果。在DNA修復以CYY1PW、CYY1FW及CYY2W進行其對修復系統相關機制的研究。藉由UV照射HaCaT cells引起細胞損傷發現CYY1PW、CYY2W可以促使HaCaT cells修復，並在調控核苷酸修復系統(nucleotide excision repair, NER)之相關蛋白質亦具有明顯表現。結果證實由水萃的CYY1P及整株CYY2W具有抗氧化、美白及DNA修復之功效，具發展成為美白及修復性保養品之添加劑。
(PART 2)嚼食檳榔為台灣早期文化之一種型態，依據衛生福利部國民健康署公告數據，民國99年至民國101年，口腔癌皆位居國人癌症發生人數第五名，罹癌人數也自99年的6,560人上升至7,047人。國際癌症研究中心( IARC )亦發表公告，「檳榔子本身即是第一類致癌物」，亦即檳榔不加任何添加物，也會導致癌症發生。
本研究利用由海洋生物獲得的純化合物¬–SP3作用於本土口腔癌細胞，探討其抗癌機制。細胞毒性證實其對人類頭部鱗狀上皮癌細胞(SCC4、SCC9和SCC25)、人類黑色素癌細胞(A375)及本土口腔癌細胞(OECM-1)皆具毒殺作用，由光學顯微鏡觀察，發現20 uM濃度在細胞作用48 h皆呈現明顯細胞自噬特徵。之後利用免疫螢光染色、流式細胞儀及西方墨點法探討經SP3作用於OECM-1之細胞自噬及凋亡相關蛋白質表現，發現SP3可提升癌細胞內ROS、降低GSH及粒線體膜電位下降、造成細胞自噬和凋亡相關蛋白質之表現，並降低OECM-1細胞遷移能力。綜合以上結果顯示SP3會造成口腔癌細胞自噬，促使細胞凋亡讓細胞死亡，推測其具有發展為治療本土口腔癌之抗癌藥物。
(PART 1)This study investigated the antioxidantation, antimelanogenesis and DNA repair mechanisms of CYY1P and CYY1F; CYY2, CYY2S, CYY2L in water and ethanol extraction. The antioxidantation activities were evaluated by measuring activities of free-radical scavenging, total antioxidantation contents, thiobarbituric acid reactive substances and reducing power. Cytotoxicity measurement was demonstrated by keratinocytes (HaCaT cells) and melanoma (B16F10 cells) and we found that the water extract had less cytotoxicity than ethanol extracts. Thus, we accessed the in vivo test by water extracts, such as the reactive oxygen species (ROS), glutathione (GSH), catalase and GPx formation in H2O2-treated in HaCaT cells were tested. The antimelanogenesis assay was analyzed by measuring tyrosinase activities, melanin contents and regulated-protein expressions in B16F10 cells. CYY1PW and CYY2W could downregulated-protein expressions of MITF, MC1R, tyrosinase, TRP-2 and TRP-1. Additionally, the DNA repair mechanism of CYY1PW and CYY2W were studied. These results showed that CYY1PW and CYY2W could promote the HaCaT cells to repair after UVB-irradiated and they increased significantly of protein expression on NER (nucleotide excision repair) system. These results demonstrated that water extracts of CYY1PW and CYY2W had antioxidandation, antimelanogenesis and DNA repair properties. Thus, the nature products can be used as whitening and repair skincare cosmetics additive.(PART 2)Betel nut chewing practice is widespread in Taiwan. According to the statistics of the standardized death rate of major cancers by the Health Promotion Administration, Ministry of Health and Welfare, oral cancer is the fifth leading cause of cancer and the number of 6,560 patients grew up to 7,407 patients between year 2010 to 2012. The International Agency for Research on Cancer (IARC) had declared that betel nut with or without additive is carcinogenic to human. In this study, we investigated the anticancer mechanism of SP3 from marine animal in human oral cancer. The cell cytotoxicity in human head and neck squamous cell carcinoma (SCC4, SCC9 and SCC25), human melanoma cells (A375) and keratinocytes of human gingival squamous carcinoma cell (OECM-1) were determined by MTT assay and the cell autophagy were observed after treatment with SP3 for 48 h. Thus, we also determined the autophagy and apoptosis related protein expression by immunofluorescence, flow cytometer and western blot. Besides, SP3 can increase ROS generation, reduce GSH level and mitochondria membrane potential (MMP) and anti-migration in OECM-1 cell. These results indicated SP3 may cross-link the pathways between autophagy and apoptosis, therefor we suggested that it has potential on oral cancer treatment.