| 摘要: | 近年來，消費者對於化粧保養品的認知已和以往有所不同，化粧品的使用已從容顏的修飾轉變為對皮膚功效性的訴求，其中以保濕、美白、抗老、抗皺等功效為目前的主流。現今市面上皮膚美白的成分眾多，在莽草酸循環之生合成化合物中，已有 ferulic acid、gallic acid、ellagic acid 被應用在美白及抗氧化上。因此，本研究的目的是希望從莽草酸生合成路徑上的天然物評估其美白及抗氧化活性的關聯性，瞭解莽草酸生合成路徑的衍生物之結構與活性上的差異。實驗設計以抑制酪胺酸酶活性測定與利用斑馬魚的動物模式來評估美白效果；以清除 DPPH 自由基能力、清除 ABTS＋．陽離子自由基能力與螯合亞鐵離子能力等評估其抗氧化效能。
研究結果顯示，化合物在濃度100 μg/ml下 syringic acid（4）、ferulic acid（8）、gallic acid（9） 皆有95%的以上清除 DPPH 自由基的能力；而在清除 ABTS＋． 陽離子自由基的測定結果，顯示 orcinol monohydrat（7）、ferulic acid（8）、gallic acid（9）具有95% 以上的清除能力；在亞鐵離子螫合能力的實驗結果， phenylpyruvic acid （10）表現出最佳的螫合效能（72.1 ± 3.2%）。在抑制酪胺酸酶活性部分：p-coumaric acid（2）、orcinol monohydrate（7）、gallic acid （9） 均有50%以上之抑制能力，其中以 orcinol monohydrate（7） （93.2 ± 3.4%）抑制酪胺酸酶的能力最為優異。
在斑馬魚體色之抑制實驗結果顯示，魚體體色總面積與控制組相較下， shikimic acid（6）、ferulic acid（8）、gallic acid（9） 對於斑馬魚體色面積皆有減少，所生成黑色素面積分別為魚體總面積之 79.1 ± 3.6 %，85.9 ± 2.7 %，79.3 ± 4.0 %，顯示這三種化合物是具有抑制體色生成的效能。在抑制小鼠黑色素瘤細胞之細胞酪胺酸酶活性的評估實驗，其結果顯示 benzoic acid（1）、shikimic acid（6）、 orcinol monohydrate（7）、ferulic acid（8）、gallic acid（9）皆有50 % 以上抑制 B16 細胞酪酸酶活性的能力，其中以 shikimic acid（6）（76.6 ± 2.9 %）抑制能力最為優異。
在細胞毒性試驗中，以纖維母細胞及黑色素細胞進行 24 小時及 48 小時的測試，纖維母細胞 24 小時的測試結果，於濃度為 200 μM 下之 p-coumaric acid（2）、quinic acid（5）、orcinol monohydrate（7） 與濃度於 500 μM 下之vanillic acid（3）、shikimic acid（6）、ferulic acid（8） 等化合物的細胞存活率均大於 80%以上；而黑色素細胞經過 48 之測試後，除化合物 quinic acid（5） 之細胞存活率為 76.6 ± 1.3%，其餘細胞存活率均大於 80%以上。另外化合物 orcinol monohydrate（7）、vanillic acid（3） 在 24 及 48 小時的測試中，對於纖維母細胞及黑色素細胞的細胞存活率皆大於控制組的細胞存活率，推測可能具有促進細胞存活的生理活性。
本研究所選用的十種莽草酸生合成路徑的化合物中，以 orcinol monohydrate（7）、ferulic acid（8）、gallic acid（9） 等三種化合物具有顯著的美白與抗氧化效能，且其細胞毒性甚低，因此這四種化合物是具美白潛力的成分，可廣泛應用在訴求皮膚美白的保養品中。
In the recent years, the perceptions of cosmetics have been different to customers. The purpose of using cosmetics have changed from modification of face to the demands of of the skin efficacy in which moisturizing, whitening, and anti-aging, anti-wrinkle efficacy becomes the current mainstream. There are numerous skin-whitening ingredients on the market, such as ferulic acid, gallic acid and ellagic acid. These compounds synthesized from shikimic acid cycle have been applied on skin whitening and anti-oxidant. Therefore, in this study, we hope to evaluate the correlation of skin whitening and anti-oxidant activities among natural compounds from shikimic acid biosynthesis pathway. The whitening efficacy was evaluated of experiment in the design of the experiment by the inhibition of tyrosinase and the animal model of zebrafish while then the anti-oxidant activity was evaluated by DPPH radical scavenging, ABTS+. free radical scavenging and chelating ferrous ion.
Experimental results showed that the DPPH free radical scavenging of syringic acid (4), ferulic acid and gallic acid was over 95% of at a concentration of 100 μg/ml .In the ABTS free radical scavenging test,it showed that orcinol monohydrat, ferulic acid and gallic acid were over 95%. The result of chelating ferrous ion showed that phenylpyruvic acid had the best chelation effect (72.1 ± 3.2% ). As for the tyrosinase activity, the inhibition rates of p-coumaric acid (2), orcinol monohydrate (7) and gallic acid were over 50%, especially orcinol monohydrate (7) had the best inhibitory activity of tyrosinase. (93.2 ± 3.4% ).
The results of melanin-inhibition assay of embryo zebrafish demonstrated that the total area of melanin had decreased after treating with shikimic acid (6), ferulic acid (8) and gallic acid (9) compared with control group. The melanin area were 79.1 ± 3.6%, 85.9 ± 2.7%, 79.3 ± 4.0% , respectively.In the experiment of the inhibition of tyrosinase activity of B16 melanoma cell , the results showed taht benzoic acid (1), orcinol monohydrate (7), shikimic acid (6), ferulic acid (8) and gallic acid (9) were over 50%, and shikimic acid (6) had the most outstanding inhibitory activity (76.6 ± 2.9 %).
In the cytotoxicity test, we used the fibroblast and melanocytes to experiment for 24 or 48 hours. The results of fibroblast cytotoxicity for 24 hours showed that the cell viability of p-coumaric acid (2), quinic acid (5) and orcinol monohydrate (7) at a concentration of 200 μM, and vanillic acid (3), shikimic acid (6) and ferulic acid (8) at a concentration of 500 μM were over 80% ; The cell viability of above compounds were also over 80% in melanocytes cytotoxicity for 48 hours, except quinic acid was 76.6 ± 1.3%. Furthermore, the cell viability of orcinol monohydrate(7) and vanillic acid (3) for fibroblast and melanocytes cytotoxicites were higher than control group after incubated for 24 or 48 hours, therefore they was spectulated that these two compounds would probably promote cell proliferation.
In this study, the ten compounds from shikimic acid biosynthesis pathway which are orcinol monohydrate, ferulic acid (8) and gallic acid (9) have the efficacy of skin whitening and anti-oxidant, and also have low cell toxicity.As a result, the compounds of orcinol monohydrate (7), ferulic acid (8) and gallic acid (9) were the ingredients of whitening performance the can widely be use in skin care whitening products. |
