九節木萃取物及其酸水解物對皮膚美白作用機轉之探討

酪胺酸酶為黑色素生成中最主要之關鍵酵素，近年來有許多研究從天然萃取物中尋找具美白之有效成分，進而應用於製造美白化妝品。本研究針對11種茜草科植物及其酸水解物以抑制圈平板試驗進行初步篩選，結果以九節木水解物之抑制效果最佳。接著進行九節木水解物對蘑菇酪胺酸酶抑制率試驗，再以小鼠黑色素瘤細胞檢測其對色素沉著機轉之探討，包括黑色素生成量、細胞內酪胺酸酶合成及直接阻斷酪胺酸酶活性之影響。此外亦進行兔子眼睛及皮膚刺激性試驗以評估其安全性。研究結果顯示，於抑制圈平板試驗篩選實驗中發現九節木酸水解物於相當於原植物含量57.6 mg/mL 時，即可達到比熊果素 (27.2 mg/mL) 較佳的抑制作用且具濃度依存性。於不同來源之酪胺酸酶 (蘑菇及小鼠) 活性抑制作用試驗結果，九節木酸水解物之抑制效果優於其萃取物。在抑制黑色素生成試驗，九節木酸水解物在5、10、30 μg/mL濃度下，其抑制率分別為11.8%、15.4% 及26.8%，具濃度依存性，且細胞數並無明顯改變。另九節木酸水解物對於細胞內酪胺酸酶並無作用，以西方墨點法試驗，顯示無抑制酪胺酸酶蛋白之表現。綜合以上結果發現九節木酸水解物對細胞外酪胺酸酶活性有抑制作用，雖對小鼠瘤細胞內之酪胺酸酶合成及蛋白質量無明顯作用，但對於細胞內黑色素生成量有降低之作用，仍具有開發成美白產品之價值，惟其作用機轉尚待進一步探討。

Tyrosinase is the key enzyme in the synthesis of melanin. In recent years, many researchers attempt to look for the active whitening ingredients from the plant extracts and use them to produce whitening cosmetics. In this investigation, the inhibition zone test of anti-tyrosinase was applied to preliminarily screen the eleven kinds of Rubiaceae plants extracts and their hydrolysates. Among the tests, Psychotria rubra (Lour.) Poir. hydrolysate (PRH) showed the strongest inhibition effect. Later, the PRH was tested by mushroom tyrosinase inhibition experiment. Then the effects of PRH on hyperpigmentation using mouse melanoma cells (B16-F0) were investigated. These tests included the melanin content, cell proliferation, tyrosinase synthesis, tyrosinase activity and tyrosinase expression. In addition, the Draize eye-irritation test and skin irritation test were applied to evaluate the safety of PRH.The results showed that while the PRH equaled to 57.6 mg/mL of the original weight of Psychotria rubra leaves, the inhibition effect was stronger than that of arbutin (27.2 mg/mL) and still kept the dose-dependent manner. In activity inhibition tests from the tyrosinase of mushrooms and mouse, the inhibition effects of the PRH were stronger than that those of its extract. In the experiment of the inhibition of melanin production, the inhibition rates of PRH at concentration of 5, 10, 30 μg/mL were 11.8%, 15.4% , 26.8% respectively. The PRH kept the dose-dependency and the cell numbers of B16 melanoma cells had no obvious change. The PRH showed no inhibition effect on tyrosinase in melanoma cells. In addition, using the western blot assay, it showed that PRH would not inhibit the protein expression of tyrosinase. In the irritation test, PRH showed mild irritation for eye-irritation and no irritation on the intact and abraded skin, respectively.In conclusion, the PRH showed inhibition effect on mushroom tyrosinase. Although, the PRH showed no inhibition effects on tyrosinase synthesis and the protein expression of tyrosinase, the PRH could inhibit the melanin production in melanoma cells. Through this investigation, the PRH might be developed as a safe and effective skin whitening product. The mechanisms on melanogenesis will be further investigated.