| 摘要: | ((Part 1)本研究以L及R水萃及乙醇萃取物進行。試驗證實L和R乙醇萃取物(LE和RE)之清除DPPH∙及ABTS∙+自由基能力、還原力、螯合鐵離子及抑制脂質過氧化物效能；抑制酪胺酸酶活性；清除一氧化氮(NO)自由基、抑制脂氧合酶lipoxygenase-1 (LOX-1)活性；總酚、總黃銅、總花青素及總多醣含量，均優於水萃取物(LW和RW)，且LE和RE作用於人類皮膚角質株化細胞(HaCaT)及人類皮膚纖維母細胞(Hs68)無明顯的細胞毒性。因此本研究進一步探討LE和RE之抗氧化、抑制黑色素生成、抗發炎及細胞修復等機制作用。LE和RE及其化合物betanin具有降低因H2O2刺激而引起的HaCaT細胞內氧化壓力，提高細胞內glutathione (GSH)含量，及提高抗氧化酵素superoxide dismutase (SOD)、catalase和glutathione peroxidase (GPx)的表現量。經α-melanocyte-stimulating hormone (α-MSH)刺激老鼠黑色素瘤細胞(B16F10)生成黑色素，LE和RE具抑制細胞內酪胺酸酶(tyrosinase)活性及降低黑色素生成量。以免疫螢光試驗及西方墨點法試驗證實，經LE和RE及其化合物betanin作用B16F10細胞後，LE和RE及其化合物betanin具抑制黑色素生合成途經中接受器MC1R (melanocortin-1 receptor)和MITF (microphthalmia-associated transcription factor)之表現，並影響酪胺酸酶、TRP-2 (tyrosinase-related proteins-2)和TRP-1 (tyrosinase-related proteins-1)的表現，進而抑制黑色素生成。在抗發炎能力試驗中，以人類皮膚角質株化細胞(HaCaT)經lipopolysaccharide (LPS)刺激後，發現LE和RE具抑制細胞內發炎因子(TNF-α、IL-1β及IL-6)的含量。經西方墨點法試驗證實LE和RE及其化合物betanin具有降低發炎相關路徑的p38、IL-6、IL-1β和TNF-α蛋白質表現。動物試驗SKH-1小鼠經UVB照射後以免疫組織化學染色法immunohistochemistry (IHC)及西方墨點法試驗再次證實LE和RE及其化合物betanin具有降低發炎相關路徑的p38、IL-6、IL-1β和TNF-α蛋白質表現。DNA修復及保護能力試驗中，證實LE和RE具調節經UVB照射後，人類皮膚角質株化細胞(HaCaT)中之細胞週期(cell cycle)、傷口癒合(wound healing)、彗星試驗(comet assay)及以免疫螢光進行UV光產物cyclobutane pyrimidine dimer (CPD)、6-4 pyrimidine-pyrimidone photoproducts (6-4PPs)和pyrimidine-pyrimidone photoproducts (PPs)含量試驗，最後以西方墨點法試驗證實LE和RE及化合物betanin都具有調節nucleotide excision repair (NER)修復系統之相關因子來修復由UV照射引起的DNA之損傷。而在動物試驗中以SKH-1小鼠經UVB照射後以immunohistochemistry (IHC)及西方墨點法試驗再次證實LE和RE及betanin具有調節p53及NER修復系統之相關因子來修復由UV照射引起的DNA之損傷。本研究證實LE和RE及betanin分別具有清除活性氧和抑制黑色素生成，並具有皮膚表皮細胞之DNA保護、修復能力以及具有抑制發炎反應的產生，具發展成為美白、抗氧化及抗發炎的保養品或保健食品添加劑的潛力。
(Part 2)本研究以P作用於正常細胞(HaCaT、DOK)及口腔癌細胞(SCC25、OECM-1)之抗氧化、抗發炎及抑制遷移的機制探討。將P作用於四種不同的細胞株進行毒性評估，發現在1及24小時的作用後，正常細胞對於P的耐受性明顯高於癌細胞，而後續試驗將以P對細胞之非致死濃度進行後續試驗。發現經P作用後，正常細胞及癌細胞中超氧陰離子(superoxide anion radical，.O2-)、活性氧物質(reactive oxygen species, ROS)及榖胱苷肽(glutathione, GSH)的含量隨著P濃度的增加而有不同的趨勢，進一步利用西方墨點法證實，與細胞中抗氧化酵素的含量有關。P對於正常細胞及癌細胞之遷移能力有不同的變化，以西方墨點法證實，P影響正常及癌化細胞中基質金屬蛋白酶(matrix metallopeptidase, MMP)之表現。發炎因子會促進癌細胞的遷移能力，再次以西方墨點法證實，P藉由降低細胞內發炎因子MAPK之表現，進而減少癌細胞遷移變化。因此證實P在適當的濃度下具有抑制癌細胞增生、抗發炎及降低腫瘤細胞之遷移能力。
(Part 1) This study evaluated anti-oxidation, anti-melanogenesis, anti-inflammatory and DNA protection properties of water (LW) and ethanol (LE) extrects from L and water (RW) and ethanol (RE) extrects from R. Firstly, the preliminary tests confirmed that the ethanol extracts both better than water extracts. In anti-oxidant experiments, LE and RE had excellent abilities on DPPH? and ABTS?+ free radicals scavenging, reducing power, metal ferrous ion chelating and inhibit of lipid peroxidantion, We determined the total phenolic content (TPC), total flavonoids content (TFC) and total anthocyanin content (TAC) as well as inhibition of the mushroom tyrosinase ability; NO free radical scavenging and inhibition of LOX-1 ability. The bioactivities of the ethenol extracts were better than the water extracts. And we found that LE and RE showed non-cytotoxicity obviouly in keratinocyte (HaCaT), melanoma cells (B16) and human fibroblast (Hs68). LE, RE and betanin also decreased cellular oxidative stress, increased intracellular GSH content and enhanced the antioxidant enzyme activities, such as superoxide dismutase (SOD), catalaseand glutathione peroxidase (GPx). Ultraviolet-B (UVB) and oxidants may cause inflammatory response and skin pigmentation, so we assessed properties of LE, RE and betanin on anti-inflammatory and anti-melanogenesis as well. The data showed that LE and RE inhibited the melanin secretion and intracellular tyrosinase activity. LE, RE and betanin also decreased the melanogenesis related protein expression of melanocortin-1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-2 (TRP-2) and tyrosinase-related protein-1 (TRP-1) as determined by western blotting assay. Besides, LE, RE and betanin inhibited inflammatory factors (TNF-α, IL-1β and IL-6) release in HaCaT cells by lipidpolysaccharide (LPS) stimulation, and it decreased level of inflammatory-related protein expression as determined by western blotting assay. Animal experiments confirmed again that the LE, RE and betanin inhibited inflammatory factors (p38, IL-6, IL-1β and TNF-α) release in SKH-1 mice skin tissue by UVB stimulation and it decreased level of inflammatory-related protein expression as determined by immunohistochemistry (IHC) and western blotting assay. Moreover, we also assessed that LE and RE whether protected DNA form UVB and oxidants damage. LE and RE inhibited activities of hyaluronidase (HAase) and Matrix metalloproteinases (MMPs), DNA fragmentation, UVB Photoproducts, such as cyclobutane pyrimidine dimer (CPD), 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs) and pyrimidine-pyrimidone photoproducts (PPs) formation, and it promoted cellular collagen formation, healing and regulated protein expression of the nuclear excision repair (NER) system as determined by western blotting assay. Animal experiments confirmed again that the LE, RE and betanin regulated protein expression of the nuclear excision repair (NER) system reduced UVB induced DNA damage. This study demonstrated that LE and RE had antioxidant, inhibiting of melanin formation, protecting and repairing of DNA as well as decreasing inflammatory factors production. LE and RE had a potential to become novel health care products or food or cosmetic additives on whitening, antioxidant and anti-inflammatory.(Part 2) This study evaluated the anti-oxidation, anti-migration and anti-inflammatory properties in normal cells and oral cancer cells. P treated in four different cell lines for toxicity assessment. We found normal cells have higher tolerance than cancer cells after treated with P for 1 h and 24 h. We found the normal cell and cancer cell intracellular superoxide anion radical (.O2-), reactive oxygen species (ROS) and glutathione (GSH) contents along with P concentration had different trends, after treated with P for 24 h. We further used western blotting analysis to confirm related intracellular antioxidant enzyme activity. Migration activity in normal cells and cancer cells had different changes. We used western blotting analysis to confirm that P regurates MMPs expression in normal cells and cancer cells. Besides, that pro-inflammation factor, can promot cancer cells migration also confirmed that P reduced of migration in cancer cells by downregurated of intracellular pro-inflammation factor MAPK expression. In this study, we confirmed that P had effects in inhibiting proliferation, anti-inflammation and anti-migration in oral cancer. |
