Bacterioruberin和鞣花酸代謝物urolithin A、urolithin B抑制B16F0黑色素形成之研究

Abstract

本研究係探討bacterioruberin以及鞣花酸代謝物urolithin A、urolithin B作為美白化妝品應用新素材之可行性。以小鼠黑色素瘤細胞株B16F0做為試驗目標物，將上述材料分別與其共培養後，得到細胞存活率達80%以上之最高濃度，並定此濃度為最高測試濃度，再以此濃度處理B16F0，探討其對黑色素生成量、對B16F0酪胺酸酶的活性之抑制、以及對B16F0酪胺酸酶mRNA之表現等的影響情形，作為判斷urolithin A和urolithin B以及bacterioruberin是否具有作為美白素材的潛力。試驗結果顯示，最高測試濃度分別為urolithin A 10 μM、urolithin B 10 μM與bacterioruberin 3.75 nM。以上述之濃度分別處理B16F0（培養基為含測試樣品之5 ％胎牛血清的DMEM，持續培養72小時），可減少黑色素生成達23%以上。Urolithin A和urolithin B以及bacterioruberin可有效抑制B16F0酪胺酸酶與蕈酪胺酸酶活性。B16F0分別與urolithin A和urolithin B以及bacterioruberin共培養72小時後，不會影響B16F0之酪胺酸酶mRNA的表現量，顯示這些測試樣品對B16F0黑色素生成能力的抑制係直接來自於測試樣品對酪胺酸酶活性之抑制作用。進一步探討上述測試樣品與酪胺酸酶之作用機制，結果顯示urolithin A和urolithin B以及bacterioruberin抑制B16F0酪胺酸酶與基質L-DOPA之模式為競爭型之抑制反應。

This study was to find out the potential of urolithin A, urolithin B and bacterioruberin in whitening application of cosmetics. B16F0 cells were employed as the tested cells for evaluating the potent use of above three samples. B16F0 cells were treated by various concentrations of urolithin A, urolithin B and bacterioruberin. The concentrations of three samples, made the viability of B16F0 cells higher than 80% , were set as the highest concentration for further tests. The experimental works including the inhibition of melanin formation and tyrosinase activity of B16F0 cells as well as the corresponding mRNA expression of B16F0 tyrosinase were conducted in this study. The results revealed that the highest concentrations of three samples, without significant cytotoxic effect, were respective 10 μM for urolithin A, 10 μM for urolithin B and 3.75 nM for bacterioruberin, and the above concentrations of three samples incubated with B16F0 for 72 h could effectively reduce the melanin formation more than 23% in B16F0 cells. Urolithin A, urolithin B and bacterioruberin could not only inhibit the activity of B16F0 tyrosinase, but also inhibit the activity of mushroom tyrosinase. In addition, the results of RT-PCR revealed that there were no significant influences on the mRNA expression of B16F0 tyrosinase. Therefore, we suggested that the melanin formation of B16F0 cells reduced by urolithin A, urolithin B and bacterioruberin could be attributed their capabillity to act directly with the tyrosinase of cells. Furthermore, the results from the enzyme kinetics studies of tyrosinase inhibited by urolithin A, urolithin B and bacterioruberin revealed that all tested samples were the same mode of competitive inhibition toward the L-DOPA substrate of B16F0 tyrosinase.