| 摘要: | 本研究主要是評估PP殼(shell, PPS)及PP葉(leaf , PPL)的水萃(PPSW)及乙醇萃取物抑制黑色素生成、抗氧化、抗發炎及DNA修復效能。首先利用人類皮膚角質株化細胞(HaCaT)、老鼠黑色素瘤細胞(B16F10)進行細胞毒性試驗，再以對細胞不造成毒性的安全濃度進行抗氧化、抑制黑色素生成、抗發炎及細胞修復等試驗。抗氧化的部分，在體外試驗中發現，PP殼(PPS)及PP葉(PPL)乙醇萃取物之清除DPPH∙及ABTS∙+自由基能力、螯合鐵離子效能；總酚、總黃銅、總花青素及總多醣含量，均優於水萃取物，在細胞內試驗中發現，PP殼(PPS)及PP葉(PPL)乙醇萃取物具有降低因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。美白試驗的部分，利用促黑激素(α-melanocyte-stimulating hormone ; α-MSH)刺激老鼠黑色素瘤細胞(B16F10)生成黑色素，PPSW及PPLW具抑制細胞內酪胺酸酶(tyrosinase)活性及降低黑色素生成量。以流式細胞儀及免疫螢光試驗證實，經PPSW及PPLW作用B16F10細胞後，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)經脂多醣lipopolysaccharide (LPS)刺激後，發現PPSW及PPLW具抑制細胞內發炎因子(TNF-α、IL-1β及IL-6)的含量。經流式細胞儀試驗證實PPSW及PPLW具有降低發炎相關路徑的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)試驗再次證實PPSW及PPLW具有降低發炎相關路徑的p38、IL-6、IL-1β和TNF-α蛋白質表現。在DNA修復及保護能力試驗中，以PPSW及PPLW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PPSW及PPLW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。本研究證實PPSW及PPLW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。
This study evaluated anti-oxidation, anti-melanogenesis, anti-inflammatory and DNA protection properties of water (PPSW) and ethanol (PPSE) extrects from PPP shell and water (PPLW) and ethanol (PPLE) extrects from PP leaf. Cytotoxicity measurement was demonstrated by keratinocytes (HaCaT cells) and melanoma (B16F10 cells). Firstly, the preliminary tests confirmed that the ethanol extracts are both better than water extracts. In anti-oxidant experiments, PPSE and PPLE had excellent abilities on DPPH? and ABTS?+ free radicals scavenging and metal ferrous ion chelating as well as it had high antioxidant contents, including total phenolic content (TPC), total flavonoids content (TFC) and total anthocyanin content (TAC). PPSE and PPLE also decreased cellular oxidative stress, increased intracellular GSH content and enhanced the antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx).The anti-melanogenesis testments were analyzed by measuring tyrosinase activities, melanin contents and melanogenesis related-protein expressions in B16F10 cells. PPSW and PPLW extracts could down regulate the related protein expressions of MITF, MC1R, TRP-2, TRP-1 and tyrosinase.Besides, PPSW and PPLW extracts 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 flow cytometer. Animal experiment confirmed that the PPSW and PPLW extracts inhibited inflammatory factors (p38, IL-6, IL-1β and TNF-α) release in SKH-1 mice skin tissue by UVB stimulation and they also decreased the level of inflammatory-related protein expression as determined by immunohistochemistry (IHC) assay.Additionally, the DNA repair mechanism of PPSW and PPLW extracts were studied. These results showed that PPSW and PPLW extracts could promote the HaCaT cells to repair after UVB-irradiated and they increased significantly of protein expression on NER (nucleotide excision repair) system. The results demonstrated that PPSW and PPLW had antioxidant, antimelanogenesis and DNA repair properties. Thus, the nature products can be used as whitening and repair skincare cosmetics additive |
