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 IC 50 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.
Once melanin is synthesized in melanocytes, it is incorporated into the melanosome, an organelle that is transported to adjacent keratinocytes, resulting in melanin distribution. Melanin, and in particular, eumelanin, protects human skin from ultraviolet radiation (UVR)-induced DNA and skin damage by absorbing UVR and scavenging UVR-induced reactive oxygen species (ROS). Hence, melanin is thought to serve as the primary photoprotective pigment that suppresses UVR-induced oxidative stress and damage. However, the unusual accumulation of melanin also causes dermatological disorders, including melasma, wrinkling, senile lentigines, and skin cancers. Hence, identification and characterization of anti-melanogenic compounds has attracted considerable attention.
Tyrosinase plays an important role in increasing melanin biosynthesis through hydroxylation of tyrosine into dihydroxyphenylalanine (DOPA), followed by further oxidation into dopaquinone, which is the precursor for melanin via cysteinyl-DOPA and dopachrome, respectively. Given that tyrosinase has been recognized as a major target molecule for the inhibition of melanin biosynthesis, many antagonists have been developed and applied clinically. Tyrosinase is a di-copper oxidase in which six histidine residues surround two copper ions in its catalytically active site. Goldfeder et al. reported that the main substrates of tyrosinase fit in the active site, whereas the presence of Zn 2+ ions forces out the Cu 2+ ions, effectively inhibiting the catalytic activity. In this way, many flavonoids competitively target the active site of tyrosinase, thereby inhibiting its activity. Accordingly, competitive inhibitors targeting tyrosinase may represent an excellent strategy for inhibiting melanin biosynthesis.
UVR increases the expression of α-melanocyte-stimulating hormone (α-MSH) in keratinocytes, which binds to the melanocortin 1 receptor (MC1R) in melanocytes and promotes melanin biosynthesis. Binding of α-MSH to MC1R primarily activates adenylyl cyclase (AC), which increases intracellular cyclic 3′,5′-cyclic adenosine monophosphate (cAMP) levels and consequently stimulates protein kinase A (PKA). Subsequently, cAMP-responsive element-binding protein (CREB) is phosphorylated, which, together with CBP/p300, enhances the expression of microphthalmia-related transcription factor (MITF), a main regulator of tyrosinase expression. Therefore, targeting the α-MSH-mediated signaling pathway may inhibit melanin biosynthesis by suppressing tyrosinase expression.
Pinostrobin is a natural flavonoid found in various plants, such as the leaves of Cajanus cajan (L.) Millsp and the rhizomes of Boesenbergia rotunda (L.). Pinostrobin possesses a broad spectrum of pharmacological activities, including anti-cancer, antioxidant, anti-inflammatory, and anti-virus properties. In fact, El-Nashar et al. recently reported that pinostrobin, isolated from Egyptian propolis, effectively reduces in vitro mushroom tyrosinase activity. However, there is currently a dearth of data regarding the anti-melanogenic effects of pinostrobin. Therefore, in this study, we investigated whether pinostrobin downregulates melanogenesis in B16F10 melanoma cells and zebrafish larvae by inhibiting the melanogenic signaling.
As tyrosinase is a rate-limiting enzyme in melanogenesis, we investigated whether pinostrobin negatively regulates mushroom tyrosinase activity in vitro. As expected, the tyrosinase inhibitors phenylthiourea (PTU), ascorbic acid (AA), and kojic acid (KA) significantly inhibited mushroom tyrosinase activity by 74.7% ± 0.6%, 63.8% ± 1.0%, and 67.4% ± 0.6%, respectively. Meanwhile, as the concentration of pinostrobin gradually increased, in vitro mushroom tyrosinase activity was inhibited, and 1000 μM pinostrobin exhibited the strongest inhibitory effect (58% ± 0.6%). In addition, the concentration required for 50% inhibition (IC 50) was confirmed to be approximately 700 μM. Collectively, these data suggest that pinostrobin directly inhibits tyrosinase activity in vitro at high concentrations.
Whether pinostrobin inhibits in vitro mushroom tyrosinase activity by competing with its substrate was analyzed using a protein–ligand docking simulation. Using SwissDock, 34 clusters in which pinostrobin binds to mushroom tyrosinase were identified. The major binding site was identified, to which approximately 50% of clusters (0, 1, 3, 4, 8, 13, 17, 20, 21, 22, 23, 24, 27, 28, 30, and 34) were bound. Meanwhile, clusters 2, 6, 9, 14, 18, 19, 31, and 33 were bound to alternative binding site, where the second highest binding force was observed. Additionally, four minor pinostrobin-binding sites were identified. The 3D conformation and ribbon structure also showed the major pinostrobin binding site and the active site of tyrosinase containing Cu 2+ ions. In particular, pinostrobin formed a hydrogen bond with TYR98 (HN) in the light chain (L, lectin-like fold protein) at a distance of 2.4769 Å. In addition to the conventional hydrogen bond with TYR98, the 2D interaction diagram showed the formation of carbon hydrogen bonds (THR324), alkyl or π-alkyl bonds (TYR78, ILE324, and PRO338), and many van der Waals interactions with the surrounding amino acids. These results indicate that pinostrobin does not compete with substrates at the active site of tyrosinase but rather primarily binds to heavy (H, polyphenol oxidase) and light chains.
To investigate whether pinostrobin is cytotoxic, B16F10 melanoma cells were treated with pinostrobin (0–1000 µM) for 72 h, and cytotoxicity was evaluated based on morphological changes and MTT assay. Observation under a phase-contrast microscope showed that pinostrobin treatment did not induce any morphological changes in the cells. However, the MTT assay showed that treatment with pinostrobin at concentrations ≥100 μM markedly decreased the relative cell viability after 24 h (77.3% ± 1.0% and 54.3% ± 0.5% at 100 and 200 μM, respectively); the inhibitory effect became stronger after 48 h (68.3% ± 1.1% and 49.2% ± 0.2% at 100 and 200 μM, respectively) and 72 h (39.6% ± 0.1% and 30.6% ± 0.2% at 100 and 200 μM, respectively).
