The market for cosmetic products, particularly skin care items like lighteners and treatments for hyperpigmentation, is experiencing rapid growth. A significant aspect of this demand is for skin whitening, which requires restricting the body’s melanin production. Melanin synthesis is primarily controlled by enzymes, with tyrosinase (TYR) playing a key role as the initial, rate-limiting enzyme in this process. Inhibiting TYR is considered an effective way to achieve skin whitening or treat conditions like hyperpigmentation. While the pharmaceutical industry offers skin whiteners based on TYR inhibition, the main inhibitors currently used, such as kojic acid, hydroquinone, and arbutin, are associated with notable adverse consequences, including instability, irritation, potential DNA damage, and increased cancer risk. This highlights the need to discover natural whitening agents that offer high skin permeability, low irritation, and increased safety while effectively inhibiting TYR.
Recent studies have focused on TYR inhibitors based on peptides, particularly bioactive peptides derived from natural proteins or food sources. These peptides offer several advantages, including stable structure, excellent nutritional value, and safety, and they can be readily absorbed by the skin with high biological safety. They have potential applications in both food products and skincare treatments, including whitening and repair of photodamaged skin. Natural compounds, in general, are favored subjects of research in this field due to their perceived lower likelihood of causing adverse incidental consequences and their high effectiveness in TYR inhibition.
Zingiber cassumunar Roxb., a perennial plant belonging to the Zingiberaceae family, is known for its medicinally valuable rhizomes and has a long history of use in Southeast Asian traditional medicine for various ailments5. Phytochemical analyses have identified several bioactive compounds in Z. cassumunar5. Previous research on Z. cassumunar rhizome extracts demonstrated that derived peptides possess strong antioxidant activity and cytotoxic effects against certain cancer cells. Among these, a specific peptide, Asp-Gly-Ile-Phe-Val-Leu-Asn-Tyr (DGIFVLNY), referred to as DY-8, was identified for its potent free-radical scavenging activity in vitro. Notably, the DY-8 sequence contains a terminal tyrosine residue, a structural feature often found in peptides with TYR-inhibitory properties5. These tyrosine-containing peptides have shown efficacy in topical applications for skin depigmentation5. Based on these characteristics, it was hypothesized that DY-8 could serve as a promising natural inhibitor of TYR activity. This study aimed to investigate the mechanism and efficacy of DY-8 in tyrosinase inhibition and melanin suppression using B16F10 mouse melanoma cells and zebrafish embryos.
Methods
The study involved synthesizing the DY-8 peptide for testing. Molecular docking simulations were conducted to predict the interaction between DY-8 and mushroom TYR. In vitro assays evaluated TYR inhibition kinetics using L-tyrosine and L-DOPA substrates, determined the inhibition type via Lineweaver-Burk plots, and calculated IC50 and Ki values. The cytotoxicity of DY-8 on B16F10 murine melanoma cells was assessed using the MTT assay, and its effect on melanin content in these cells was measured spectrophotometrically. Furthermore, the influence of DY-8 on the expression of melanogenesis-related genes (Mitf, Tyr, Trp-1, Trp-2) and proteins (MITF, TYR, TRP-1, TRP-2) in B16F10 cells was investigated using qRT-PCR and Western blot analysis, respectively. Finally, the in vivo efficacy and toxicity of DY-8 were validated in zebrafish embryos by evaluating mortality, morphological abnormalities, and quantitative melanin content reduction. Statistical analysis was performed using one-way ANOVA.
Key Findings
•Molecular docking predicted that DY-8 binds to the active tyrosinase site through various interactions, including hydrogen bonds, hydrophobic interactions, π-π and π-σ bonds, and van der Waals forces. The binding energy was measured at -7.1 Kcal/mol, which was better than that of the positive control, arbutin (-6.5 Kcal/mol).
•DY-8 demonstrated effective inhibition of tyrosinase activity in vitro, with IC50 values of 0.18 ± 0.01 μg/mL for mono-phenolase activity and 0.81 ± 0.06 μg/mL for di-phenolase activity8…. These values were comparable to those obtained from kojic acid21.
•Lineweaver-Burk plots indicated that DY-8 is a competitive inhibitor for both mono-phenolase and di-phenolase activities, suggesting it binds directly to the active site of the free enzyme. The inhibition constant (Ki) values suggested a higher binding affinity for DY-8 to TYR when L-tyrosine was the substrate compared to L-DOPA.
•DY-8 showed no significant cytotoxicity on B16F10 cells at concentrations ranging from 0 to 100 μM, with cell viability exceeding 50%.
•In α-MSH-stimulated B16F10 cells, DY-8 significantly reduced melanin content in a dose-dependent manner compared to the control.
•DY-8 treatment led to the significant downregulation of mRNA levels for melanogenesis-related genes, including Mitf, Tyr, Trp-1, and Trp-2, in a dose-dependent manner. MITF regulates TYR, TRP-1, and TRP-2 expression, promoting melanin synthesis.
•Western blot analysis confirmed that DY-8 treatment also resulted in a decrease in protein levels of MITF, TYR, TRP-1, and TRP-2 in B16F10 cells.
•In zebrafish embryos, DY-8 demonstrated a non-toxic profile, with no significant effect on survival or normal development within the tested concentration range (40-200 µM).
•DY-8 significantly reduced melanin pigmentation in zebrafish embryos in a dose-dependent manner, with significant reductions observed starting from 80 µM and the most pronounced effect at 160 and 200 µM.
This research successfully isolated and identified DY-8 as a novel TYR inhibitory peptide derived from the rhizome extract of Zingiber cassumunar. The findings from molecular docking, in vitro enzymatic assays, cellular studies, and in vivo validation in zebrafish embryos collectively demonstrate that DY-8 is a promising, safe, and potent melanin inhibitor. The study elucidated that DY-8 likely functions through competitive inhibition of TYR activity and by downregulating the expression of key genes and proteins involved in the melanogenesis pathway (MITF, TYR, TRP-1, TRP-2). The demonstration of efficacy in both cellular and zebrafish models, coupled with its low toxicity, highlights the significant potential of DY-8.
The novelty lies in the identification of this specific peptide, DY-8, from Zingiber cassumunar, a plant previously overlooked for this application. This research contributes a natural and sustainable alternative to existing synthetic skin whitening agents which have known drawbacks. The future implications of this research are significant, suggesting that DY-8 holds strong potential for application as a safe and effective bio-based ingredient in the cosmetic and personal care industries, as well as potentially the food industry, for managing skin pigmentation disorders and hyperpigmentation. Further development of DY-8 based on these findings is warranted for therapeutic or cosmetic applications.
Link to the study: https://www.sciencedirect.com/science/article/pii/S0926669025006454
