Institute of Nutrition, College of Health Care, China Medical University, Taichung, 406040, Taiwan.
Department of Cosmeceutics, College of Pharmacy, China Medical University, Taichung, 406040, Taiwan; Department of Health and Nutrition Biotechnology, Asia University, Taichung, 413005, Taiwan; Chinese Medicine Research Center, China Medical University, Taichung, 406040, Taiwan; Research Center of Chinese Herbal Medicine, China Medical University, Taichung, 406040, Taiwan.
Free Radic Biol Med. 2021 Sep;173:151-169. doi: 10.1016/j.freeradbiomed.2021.07.030. Epub 2021 Jul 24.
3-O-ethyl ascorbic acid (EAA) is an ether-derivative of ascorbic acid, known to inhibit tyrosinase activity, and is widely used in skincare formulations. Nevertheless, the molecular mechanisms underlying the EAA's effects are poorly understood. Here, the anti-melanogenic activity of EAA was demonstrated through Nrf2-mediated α-MSH inhibition in UVA-irradiated keratinocytes (HaCaT) and autophagy induction and inhibition of α-MSH-stimulated melanogenesis in melanocytes (B16F10). EAA pretreatment increased the HaCaT cell viability but suppressed ROS-mediated p53/POMC/α-MSH pathways in UVA-irradiated cells. Further, the conditioned medium from EAA-pretreated and UVA-irradiated HaCaT cells suppressed the MITF-CREB-tyrosinase pathways leading to the inhibition of melanin synthesis in B16F10 cells. EAA treatment increased nuclear Nrf2 translocation via the p38, PKC, and ROS pathways leading to HO-1, γ-GCLC, and NQO-1 antioxidant expression in HaCaT cells. However, Nrf2 silencing reduced the EAA-mediated anti-melanogenic activity, evidenced by impaired antioxidant gene expression and uncontrolled ROS (H0) generation following UVA irradiation. In B16F10 cells, EAA-induced autophagy was shown by enhanced LC3-II levels, AVO formation, Beclin-1 upregulation, and activation of p62/SQSTM1. Further, EAA-induced anti-melanogenic activity was substantially decreased in autophagy inhibitor (3-MA) pretreated or LC3 knockdown B16F10 cells. Notably, transmission electron microscopy data showed increased melanosome-engulfing autophagosomes in EAA-treated B16F10 cells. Moreover, EAA also down-regulated MC1R, TRP-1/-2, tyrosinase expressions, and melanin synthesis by suppressing the cAMP-CREB-mediated MITF expression in B16F10 cells stimulated with α-MSH. In vivo studies on the zebrafish model further confirmed that EAA inhibited tyrosinase expression/activity and endogenous pigmentation. In conclusion, 3-O-ethyl ascorbic acid is an effective skin-whitening agent and could be used as a topical agent for cosmetic purposes.
3-O-乙基抗坏血酸（EAA）是抗坏血酸的醚衍生物，已知其能抑制酪氨酸酶的活性，广泛应用于护肤品配方中。然而，EAA 作用的分子机制尚不清楚。在这里，通过 Nrf2 介导的 UVA 照射角质细胞（HaCaT）中 α-MSH 抑制和自噬诱导以及抑制黑色素细胞（B16F10）中 α-MSH 刺激的黑色素生成，证明了 EAA 的抗黑色素生成活性。EAA 预处理增加了 HaCaT 细胞的活力，但抑制了 UVA 照射细胞中 ROS 介导的 p53/POMC/α-MSH 途径。此外，来自 EAA 预处理和 UVA 照射的 HaCaT 细胞的条件培养基抑制了 MITF-CREB-酪氨酸酶途径，导致 B16F10 细胞中黑色素合成的抑制。EAA 处理通过 p38、PKC 和 ROS 途径增加核 Nrf2 易位，导致 HaCaT 细胞中 HO-1、γ-GCLC 和 NQO-1 抗氧化剂的表达。然而，Nrf2 沉默减少了 EAA 介导的抗黑色素生成活性，这表现为 UVA 照射后抗氧化基因表达受损和不可控的 ROS（H0）生成。在 B16F10 细胞中，通过增强 LC3-II 水平、AVO 形成、Beclin-1 上调和 p62/SQSTM1 的激活来显示 EAA 诱导的自噬。此外，在自噬抑制剂（3-MA）预处理或 LC3 敲低的 B16F10 细胞中，EAA 诱导的抗黑色素生成活性显著降低。值得注意的是，透射电子显微镜数据显示，在用 EAA 处理的 B16F10 细胞中，黑色素体吞噬自噬体增加。此外，EAA 通过抑制 α-MSH 刺激的 B16F10 细胞中 cAMP-CREB 介导的 MITF 表达，还下调了 MC1R、TRP-1/-2、酪氨酸酶的表达和黑色素的合成。斑马鱼模型的体内研究进一步证实，EAA 抑制了酪氨酸酶的表达/活性和内源性色素沉着。总之，3-O-乙基抗坏血酸是一种有效的皮肤美白剂，可作为化妆品的外用制剂。
