Protocatechuic Aldehyde Inhibits α-MSH-Induced Melanogenesis in B16F10 Melanoma Cells via PKA/CREB-Associated MITF Downregulation

Protocatechuic aldehyde (PA) is a naturally occurring phenolic compound that is a potent inhibitor of mushroom tyrosinase. However, the molecular mechanisms of the anti-melanogenesis activity of PA have not yet been reported. The aim of the current study was to clarify the melanogenesis inhibitory effects of PA and its molecular mechanisms in murine melanoma cells (B16F10). We first predicted the 3D structure of tyrosinase and used a molecular docking algorithm to simulate binding between tyrosinase and PA. These molecular modeling studies calculated a binding energy of −527.42 kcal/mol and indicated that PA interacts with Cu400 and 401, Val283, and His263. Furthermore, PA significantly decreased α-MSH-induced intracellular tyrosinase activity and melanin content in a dose-dependent manner. PA also inhibited key melanogenic proteins such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2 in α-MSH-stimulated B16F10 cells. In addition, PA decreased MITF expression levels by inhibiting phosphorylation of cAMP response element-binding protein (CREB) and cAMP-dependent protein kinase A (PKA). These results demonstrate that PA can effectively suppress melanin synthesis in melanoma cells. Taken together, our results show that PA could serve as a potential inhibitor of melanogenesis, and hence could be explored as a possible skin-lightening agent.     

Tyrosol and Its Analogues Inhibit Alpha-Melanocyte-Stimulating Hormone Induced Melanogenesis

Melanin is responsible for skin color and plays a major role in defending against harmful external factors such as ultraviolet (UV) irradiation. Tyrosinase is responsible for the critical steps of melanogenesis, including the rate-limiting step of tyrosine hydroxylation. The mechanisms of action of skin hypopigmenting agents are thought to be based on the ability of a given agent to inhibit the activity of tyrosinase and, hence, down regulate melanin synthesis. Tyrosol and its glycoside, salidroside, are active components of Rhodiola rosea, and in our preliminary study we found that Rhodiola rosea extract inhibited melanogenesis. In this study, we examined the effects of tyrosol and its analogues on melanin synthesis. We found that treatment of B16F0 cells to tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), 2-hydroxyphenylacetic acid (7), or salidroside (11) resulted in a reduction in melanin content and inhibition of tyrosinase activity as well as its expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5) and 2-hydroxyphenylacetic acid (7) suppressed MC1R expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) inhibited α-MSH induced TRP-1 expression, but salidroside (11) did not. All the compounds did not affect MITF and TRP-2 expression. Furthermore, we found that the cell viability of tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) at concentrations below 4 mM and salidroside (11) at concentrations below 0.5 mM were higher than 90%. The compounds exhibited metal-coordinating interactions with copper ion in molecular docking with tyrosinase. Our results suggest that tyrosol, 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, and salidroside are potential hypopigmenting agents.     

Inhibition of melanogenesis by Erigeron canadensis via down-regulating melanogenic enzymes in B16F10 melanoma cells

The effects of Erigeron canadensis extract on melanogenesis and cell toxicity in cultured B16F10 mouse melanoma cells were investigated. E. canadensis extract down regulated melanin synthesis effectively at a non-toxic concentration. Its extract was fractionated by using a recycling HPLC with GS310 column (21.5×500 mm, 10–15 μM) into five fractions. The fraction 1 showed melanin inhibition by 48.0% at 100 mg/ml which was 2.5 times more efficient than the depigmenting effect of commercial arbutin (17.5%) and also did not show cell toxicity. To elucidate the depigmenting mechanism of fraction 1, in vitro and cellular tyrosinase activity, antioxidant activity, and protein level of the main melanogenic enzymes, such as tyrosinase, TRP-1 and TRP-2 were evaluated. Fraction 1 inhibited melanin synthesis in B16F10 melanoma cells by decreasing protein levels of melanogenic enzymes, especially tyrosinase. In conclusion, we suggest that this fraction may be a safe and effective depigmentation agent.     

棕榈酰三肽-5皮肤美白功效及机制研究

采用MTT法检测细胞存活率,利用α-促黑素(α-MSH)诱导的B16小鼠黑色素瘤细胞模型和UVB照射诱导豚鼠皮肤色素沉着模型对棕榈酰三肽-5的美白功效进行评价。研究了其对细胞内酪氨酸酶活性、小眼畸形相关转录因子(MITF)和酪氨酸酶m RNA水平的影响。结果表明,棕榈酰三肽-5能够显著降低上清及细胞内的黑色素水平且无细胞毒性。进一步研究发现,棕榈酰三肽-5通过抑制酪氨酸酶活性,下调MITF和酪氨酸酶的mRNA水平,从而抑制黑色素的生成。在动物模型中,棕榈酰三肽-5能够显著降低UVB照射诱导的豚鼠皮肤色素沉着和黑色素分布区域灰度值(P0.05)。     

Synergistic Effect of DIBOA and Verbascoside from Acanthus mollis Leaf on Tyrosinase Inhibition

Overexpression of melanin contributes to darkening of plant and fruit tissues and skin hyperpigmentation, leading to melasma or age spots. Although melanin biosynthesis is complex and involves several steps, a single enzyme known as tyrosinase is key to regulating this process. The melanogenesis pathway is initiated by oxidation of the starting material l-tyrosine (or l-DOPA) to dopaquinone by tyrosinase; the resulting quinone then serves as a substrate for subsequent steps that eventually lead to production of melanin. Medicinal plants are considered a good source of tyrosinase inhibitors. This study investigated the tyrosinase inhibitory activity of A. mollis leaf extracts and their phytochemicals. Significant activity was verified in the ethanol extract –EEt (IC₅₀ = 1.21 µg/mL). Additionally, a kinetic study showed that this tyrosinase inhibition occurs by DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) and verbascoside contribution through a non-competitive reaction mechanism. A synergistic effect on tyrosinase inhibition was observed in the binary combination of the compounds. In conclusion, both EEt and a mixture of two of its phytochemicals can be effective tyrosinase inhibitors and can be used as a bleaching agent for cosmetic formulations in the future.     

Pinostrobin Suppresses the α-Melanocyte-Stimulating Hormone-Induced Melanogenic Signaling Pathway

Pinostrobin is a dietary flavonoid found in several plants that possesses pharmacological properties, such as anti-cancer, anti-virus, antioxidant, anti-ulcer, and anti-aromatase effects. However, it is unclear if pinostrobin exerts anti-melanogenic properties and, if so, what the underlying molecular mechanisms comprise. Therefore, we, in this study, investigated whether pinostrobin inhibits melanin biosynthesis in vitro and in vivo, as well as the potential associated mechanism. Pinostrobin reduced mushroom tyrosinase activity in vitro in a concentration-dependent manner, with an IC₅₀ of 700 μM. Molecular docking simulations further revealed that pinostrobin forms a hydrogen bond, as well as other non-covalent interactions, between the C-type lectin-like fold and polyphenol oxidase chain, rather than the previously known copper-containing catalytic center. Additionally, pinostrobin significantly decreased α-melanocyte-stimulating hormone (α-MSH)-induced extracellular and intracellular melanin production, as well as tyrosinase activity, in B16F10 melanoma cells. More specifically, pinostrobin inhibited the α-MSH-induced melanin biosynthesis signaling pathway by suppressing the cAMP–CREB–MITF axis. In fact, pinostrobin also attenuated pigmentation in α-MSH-stimulated zebrafish larvae without causing cardiotoxicity. The findings suggest that pinostrobin effectively inhibits melanogenesis in vitro and in vivo via regulation of the cAMP–CREB–MITF axis.