Bee pollen peptides as potent tyrosinase inhibitors with anti-melanogenesis effects in murine b16f10 melanoma cells and zebrafish embryos

Enrichment of bee pollen peptides treated with different proteases

TIPs are now widely prepared via enzymatic hydrolysis, due to the ease of controlling the process and the absence of extreme reaction conditions. There are many commercially produced enzymes which are available to support TIP production, among which are trypsin, chymotrypsin, flavourzyme, alkaline protease, neutral protease, papain, and so forth. It is understood that different enzymes can differ in their hydrolytic impact upon a given material because the binding specificity between substrate and enzymes will vary. However, in the case of the enzymolytic effect of an enzyme, the same enzyme will produce different results on different materials¹². In this study, the enzymes under investigation included neutrase, alcalase, flavourzyme, papain, and pepsin-pancresin. Hydrolysis of the bee pollen powder was conducted with various proteases at three specific concentrations before comparisons were drawn to consider the TYR inhibition of the bee pollen hydrolysates, with findings shown in Table 1. The lowest IC₅₀ values for the mono-phenolase activity (50.01 ± 1.15 µg/mL) and di-phenolase activity (39.93 ± 0.60 µg/mL) were recorded for the protein hydrolysate which was prepared using 5% (w/v) neutrase. The hydrolysates comprise a number of peptides, and thus differ from kojic acid, which is a single molecule and a very potent standard TYR inhibitor. For this reason, the true concentration of the active compound is not as high as that stated for the IC₅₀ value. This study therefore places emphasis upon those hydrolysates which offer the greatest enzyme inhibition⁷. A similar example can be found in the work of Zhao et al.²⁵. whose hydrolysis of the “Fengdan” peony (Paeonia ostii) seed meal protein with neutrase (at 1 mg/mL) revealed hydrolysates achieving 59.7% TYR inhibition. Before carrying out the process of enzymatic hydrolysis it is essential to select the appropriate proteases and raw materials, because the protein amino acid compositions vary and this leads to different sized peptides being released, which offer different levels of bioactivity.

Those proteins offering strong TYR inhibition typically contain high levels of hydrophobic amino acids, such as Trp, Phe, Gly, Val, Leu, Ile, Ala, Pro, and Met, and also aromatic amino acids including Tyr, Trp, and Phe. Neutrase, which can be described as a type of neutral protease has a tendency to hydrolyze proteins to generate peptides which have hydrophobic amino acids including Tyr, Trp, or Phe as C-terminals²⁶. Accordingly, neutrase could serve as an appropriate protease for the preparation of high-activity TIPs. Reactions take place between hydrogen donor hydrophobic amino acids and the various residues, free radicals, or metal ions, while the aromatic amino acids possess conjugated planar rings, which are able to absorb ultraviolet rays while also engaging in π-π interactions with the copper ions of TYR. The oxidative activity of TYR tends to be disrupted by the conjugation with Cu²⁺, which in turn limits melanin synthesis¹². To summarize, the current approach to obtain TIPs involves the screening of enzyme species followed by optimization of the enzymolytic process along with efficacious purification. This study employed neutrase in sequence since this was the approach which provided the highest levels of TYR inhibition. These findings were then taken forward to guide the ultrafiltration (UF) stage.

UF of the prepared BPPH

Having obtained the enzymatic hydrolysates from protein fractions and peptides, it is necessary to perform further filtration in order to separate these peptides prior to carrying out additional experimentation. The peptides must be purified isolated from other substances before it is possible to determine their abilities in melanogenesis inhibition in cell culture or to assess their anti-TYR properties through in vitro assay. This is because impurities could affect the outcomes of these analyses. Most research into bioactive peptides makes use of membrane fractionation techniques to commence the analysis. The purification stage begins by filtering the protein hydrolysates and peptides using membranes which are able to separate the fractions by molecular size. The food sector normally requires UF in which the sub-fractions fall within a range of 1 to 100 kDa for molecular size²⁷. The initial screening for bioactivity can then be carried out by comparing the activity levels of the different sub-fractions. The most promising protein hydrolysate can be separated by membranes to form different peptide sizes, typically of MW > 10 kDa, MW 5–10 kDa, MW 3–5 kDa, MW 3-0.65, and MW < 0.65 kDa. Assays can then be conducted for in vitro TYR inhibition to determine the capacity to serve as TYR inhibitors in mono-phenolase and di-phenolase contexts, as explained in the section describing the experimental procedures. Table 2 presents the findings. The lowest IC₅₀ values for both mono-phenolase activity (1.08 ± 0.50 µg/mL) and di-phenolase activity (1.82 ± 0.80 µg/mL) were obtained for protein hydrolysates which were prepared with 5% (w/v) of neutrase at a molecular weight < 0.65 kDa. Meanwhile, Prakot et al.¹⁹. reported the highest level of TYR inhibition from peptides derived from the protein hydrolysates of the spotted babylon snail, for which the molecular weight was lower than 3,000 Da. Deng et al.²⁸. reported similar findings, noting that greater TYR inhibition was exhibited by the smaller molecular weight fractions of the protein hydrolysates from Chinese quince seeds following UF. In line with this trend, the 0–3 kDa fraction was chosen to undergo additional purification. Pongkai et al.¹⁸. confirmed the benefits of using the UF membrane to enhance the activity of a peptide fraction in terms of TYR inhibition, since the inhibitory activity of a peptide is closely related to its molecular weight. Accordingly, the protein hydrolysates prepared using 5% (w/v) neutrase which has a molecular weight < 0.65 kDa were shown to offer strong TYR inhibition, since they were able to exert their inhibitory influence at low concentrations, thus exhibiting strong dependence upon molecular weight. The differences observed might be attributable to the susceptible bonds in the amino acid sequences, which might have been dependent on the mechanism for catalysis of the immobilized enzyme, the breaking point preference in the protein sequence, and protein isolates composition. The fraction with molecular weight < 0.65 kDa which offered strong mono-phenolase and di-phenolase inhibition was selected to be purified further using size exclusion chromatography (SEC) and reversed-phase high-performance liquid chromatography (RP-HPLC).

TIP purification through SEC and RP-HPLC

Figure 1a serves to explain that higher levels of TYR inhibitory activity may result from the fraction with molecular weight < 0.65 kDa at a half-maximal inhibitory concentration (IC₅₀) because of that low molecular weight. Further fractionation of the MW < 0.65 kDa ultrafiltrated fraction can be carried out by SEC using a Superdex 30 Increase column with a separation range of 100-7,000 Da. It is then possible to monitor the various peptide bonds, proteins, peptides, or amino acids with aromatic rings through the use of A₂₈₀, which revealed two individual peaks (F₁ and F₂), confirming that aromatic peptide or amino acid side chains were present. F₁ presented TYR inhibition for mono-phenolase with an IC₅₀ value of 46.10 ± 1.25 µg/mL, and for di-phenolase with an IC₅₀ value of 37.39 ± 1.26 µg/mL. However, it was not possible to calculate such values for F₂. On the basis of these outcomes, the F₁ fraction was chosen for subsequent analysis. This lyophilized active F₁ fraction underwent additional separation using RP-HPLC to create the five sub-fractions indicated as F_1–1, F_1–2, F_1–3, F_1–4 and F_1–5 (Fig. 1b). The inhibition of TYR varied among the various sub-fractions, with sub-fraction F_1–2 exhibiting the highest levels of TYR activity in both mono-phenolase (IC₅₀ = 17.28 ± 0.02 µg/mL) and di-phenolase (IC₅₀ = 25.69 ± 0.30 µg/mL). Furthermore, the F_1–1 and F_1–5 sub-fractions featured very low protein concentration levels so it was not possible to calculate an IC₅₀ value for the TYR activity (S1 Table). In the work of Kubglomsong et al.²⁹. papain hydrolyzed rice bran albumin hydrolysates were reported to offer greater TYR inhibition, and were then separated into 11 fractions via RP-HPLC. The greatest level of TYR inhibition was demonstrated by Fraction 1 (IC₅₀ = 1.31 mg/mL), which comprised 13 peptides with molecular weights in the range from 1.3 kDa to 4.8 KDa. One particular peptide displayed the amino acid sequence SSEYYGGEGSSSEQGYYGEG, and was noted for its residues and other features associated with TYR inhibition and chelating copper ions. It has been confirmed by a number of studies that TYR inhibition is linked to peptides’ amino acid composition and molecular weight distribution. Accordingly, it was necessary to investigate the amino acid sequence of the F_1–2 fraction, which showed significant TYR activity, via liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry (LC-Q-TOF-MS/MS).

(a) SEC of the F₁ and F₂ fractions from MW < 0.65 kDa neutrase hydrolysate. (b) RP-HPLC profile of the active fraction (F₁) obtained from BPPH.

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Identification and sequencing of isolated peptides and bioinformatic predictions of peptide properties

The selected F_1–2 fraction underwent LC-Q-TOF-MS/MS and de novo amino acid sequencing in order to evaluate its various characteristics, which could then be reassessed via the NCBI database in order to establish the identity in the Mimosa genus, as shown in Fig. 2. The VY-9 peptide, discovered through de novo sequencing, shows partial similarity to apyrase proteins from Mimosa pudica, suggesting a possible functional relationship while indicating novelty within the Mimosa genus. The F_1–2 peptide (VDGYPAAGY; Val-Asp-Gly-Tyr-Pro-Ala- Ala-Gly-Tyr; named VY-9) matches to the peptide of nine amino acids with the molecular weight of 911.95 Da. Protein BLAST allowed the location to be established in the homologous region. Accordingly, the VY-9 peptide showed full similarity to the apyrase protein which can be obtained from Mimosa pudica (S2 Table). Apyrases are enzymes that serve to eliminate other enzymes which would extract the terminal phosphate from NTPs and NDPs, while not doing so in the case of nucleotide monophosphates. Their role in plant and animal physiology is highly varied³⁰. The preparation of functional peptides requires a thorough comprehension of peptide properties and stability. One key property is the solubility of a peptide in water, since this will affect drug release, bioavailability, and absorption³¹. Water solubility is thus a critical property when considering the use of a particular peptide in the medical, cosmetic, or nutritional fields. The VY-9 peptide has poor water solubility according to the Innovagen server (Table 3), yet our own findings indicated that at concentration below 5.0 mg/mL the water solubility was not as poor as might be anticipated. However, as the concentration increased, the solubility did tend to become worse. Meanwhile, peptide or protein toxicity can be estimated in silico by ToxinPred, which is a very useful tool in the area of drug discovery³². In this case, ToxinPred was used to assess the toxicity of the peptide VY-9, with the outcome suggesting a lack of toxicity, and thus acceptability for applications in the food, medical, or cosmetic sectors. The findings can be seen in Table 3.

Mass fragmentation spectrum and analysis of the F_1–2 fraction (VY-9) amino acid sequence via LC-Q-TOF-MS/MS.

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The peptide VY-9 comprises 44.44% hydrophobic amino acid residues. It is normally the case that low molecular weight protein hydrolysates exhibit greater hydrophobicity as well as superior dispersion which leads to stronger overall bioactivity. Where hydrophobic amino acids appear at each end of the peptide chain, this increases the interactivity with the copper active sites of TYR. Furthermore, it was reported by Wang et al.³³. that a low-molecular-weight (700-1,700 Da) gelatin hydrolysate derived from the sea cucumber wall offered 55.7% hydrophobic amino acids. While the exterior environment of TYR is hydrophilic, the inner core forms a hydrophobic cavity such that those amino acids which contain a hydrophobic side chain will tend to bind more readily to the hydrophobic pocket close to the active site of TYR. Aromatic amino acids have benzene rings which can be buried at the active site as they bind to TYR, and since they are hydrophobic, this stabilizes the binding, increasing the inhibitory effects against TYR. According to Ge et al.³⁴. three new peptides offering notable inhibitory activity against TYR include ILFTLL (IC₅₀ = 9.25 mg/mL), TIPPPT (IC₅₀ = 7.59 mg/mL), and IIPFIF (IC₅₀ = 6.16 mg/mL). However, the peptide IIPFIF stands out through its aromatic amino acid located at the C-terminal enabling entry to the active pocket from the C-terminal, whereas in contrast there is no aromatic amino acid at either end of the peptide chain in the case of TIPPPT or ILFTLL. Instead, the IIPFIF peptide comprises solely hydrophobic amino acids and its larger portion enters the active pocket, whereas TIPPPT and ILFTLL only have one or two hydrophilic amino acids and the smaller portion enters the active pocket.

Validation of synthetic peptide VY-9 activity

In order to check our own hypothesis concerning the ability of the peptide VY-9 to inhibit TYR, we created a synthetic peptide with a matching sequence to test the inhibition in the case of both mono-phenolase and di-phenolase forms. Our synthetic version of the peptide VY-9 demonstrated good TYR inhibition, for which the IC₅₀ values of 0.55 ± 0.03, and 2.54 ± 0.06 µM were recorded in the case of the respective mono-phenolase and di-phenolase activities. The results indicated that the peptide VY-9 was more effective in the inhibition of mono-phenolase than di-phenolase, possibly because the inhibitory concentration threshold for the di-phenolase reaction could exceed that of the mono-phenolase reaction. In earlier research the protein from rice bran underwent enzymatic digestion with the simultaneous use of chymotrypsin and trypsin to create peptides. From the rice bran protein hydrolysates, six bioactive peptides were isolated and determined to exhibit TYR inhibitory activity. These peptides were classified as CT-1-6, and the amino acid sequence analysis determined that three of these peptides possessed tyrosine residue at the C-terminus side of the peptides (CT-1: HGGEGGRPY, CT-2: LQPSHY, and CT-3: HPTSEVY). These three peptides produced notable TYR inhibition in the mono-phenolase tyrosine substrate reaction, but importantly did not do so in the di-phenolase L-DOPA substrate reaction. Moreover, melanogenesis in melanoma cells was shown to be inhibited by the peptide CT-2 (> 50% at 500 µM) while no cytotoxic effects were recorded³⁵. On the basis of these data, it could be argued that protein hydrolysates from rice bran might serve as an excellent source of TYR inhibitory bioactive peptides. The inhibitory activity was shown to be lower for the di-phenolase when compared to the mono-phenolase in a majority of the substances examined³⁶. Meanwhile, Li et al.³⁷. reported significant differences in the measured IC₅₀ values between mono-phenolase and di-phenolase inhibitory activity towards mushroom TYR. Where such differences occur between the mono-phenolase and di-phenolase activity for a particular compound, it can most commonly be attributed to differences in the catalytic cycle mechanisms of monophenol (L-tyrosine) and diphenol (L-DOPA) oxidation by the mushroom TYR. It is not, however, possible to rule out the significant inhibition of mono-phenolase steady-state activity with no extension of the lag time. For TYR activity in the context of L-tyrosine as a monophenol substrate it is necessary to implement a lag-phase in order to achieve the transformation to diphenol, which acts as a substrate to di-phenolase.

VY-9 has C-terminal tyrosine residue which supports the TYR inhibition, possibly as a consequence of the aromatic ring structures in the amino acid Tyr residues. Ge et al.³⁴. noting that hydrophobic interactions with hydrophobic side chains located at the active sites of TYR could be increased by inhibitors which have aromatic ring structures. Furthermore, a number of researchers have suggested that the tyrosine residue makes an important contribution to TYR inhibition. For instance, Schurink et al.³⁸. performed the screening of seven different peptides offering TYR inhibition from protein-based peptide libraries via SPOT synthesis, with the results indicating that peptides which contained tyrosine were able to activate TYR since it was possible for them to undergo conversion by TYR. In addition, Ochiai et al.³⁵. examined TH10 (MRSRERSSWY), synthesizing it following a search of the rice DNA database. The peptide TH10 was found to have an IC₅₀ value of 102 µM. This peptide can be considered homologous to peptide P4 (YRSRKYSSWY) from an earlier study which achieved an IC₅₀ value of 123 µM where tyrosine was a substrate of TYR. These peptides both have sequences containing tyrosine on the C-terminal, while peptide P4 has an additional two tyrosine residues, located in the middle of the peptide and at the N-terminal. Simulations were performed to assess the sequence-shuffled variants of peptide P4 and the molecular docking of variants of peptide TH10, revealing that the peptide P4 inhibitory activity was not linked to the presence of tyrosine residues at the center of the peptide sequence and in the N-terminal position, although the tyrosine residue at the C-terminal in both of the peptides is important in TYR inhibition because tyrosine residue on both peptides plays very important role in the inhibition activity of TYR by acting as a substrate analogue at the TYR active site and coordinating with the copper ions of the TYR enzyme. Most peptides offering TYR inhibition have Tyr residue at the C-terminal.

Inhibition type and constant determination for the VY-9 peptide

In order to understand the VY-9 peptide mechanism for TYR inhibition, the inhibition mode was investigated through the analysis of Lineweaver-Burk plots. The plots can be seen in Fig. 3a-d, where four lines of differing gradients meet at the same intersection point for the vertical coordinate at varying concentrations of the VY-9 peptide. The results show that the Km values rose as the concentration of the inhibitor rose, whereas the Vmax values remained unchanged suggesting that the peptide VY-9 acted as a competitive inhibitor in the case of tyrosine mono-phenolase and also for di-phenolase. In this way, the peptide VY-9 was capable only of binding to free enzymes, but could not form bonds with enzyme-substrate complexes. The inhibition constant Ki had a value of 0.6 ± 0.09 mM for tyrosine mono-phenolase and 0.82 ± 0.07 mM for di-phenolase. Feng et al.³⁹. conducted similar investigations, determining that the inhibitory activity of the peptide FPY, a competitive inhibitor obtained from de-fatted walnut, had a Ki value of 22.04 ± 0.09 mM for mono-phenolase and 4.82 ± 0.07 mM for di-phenolase. Meanwhile, the Phage Display Library revealed the peptide IQSPHFF to act as a competitive inhibitor for di-phenolase, with the Ki value reported to be 0.765 mM⁴⁰. In other studies, Yap and Gan reported that GYSLGNWVCAAK, obtained from egg white protein, could competitively inhibit TYR¹³.

The Lineweaver-Burk plot of the peptide VY-9 when inhibiting (a) mono-phenolase activity and (b) di-phenolase activity. Reactions were carried out at varying inhibitor concentrations: No inhibitor (filled black circle), 0.25 mM (filled red circles), 0.5 mM (filled green circle), and 1.0 mM of VY-9 (filled blue circle). Lineweaver-Burk secondary plots revealed the determination of the inhibitor constant (Ki) in inhibiting (c) mono-phenolase activity and (d) di-phenolase activity by the peptide VY-9.

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Molecular docking

Studies of molecular docking via computer simulation have recently become increasingly widely used. The technique allows the mechanisms by which inhibitors and enzymes interact to be investigated⁴¹. In this study, it was necessary to better understand the peptide inhibition mechanism in the context of TYR, so molecular docking was employed to examine the peptide VY-9 interacting with TYR. The results can be observed in Fig. 4a,b. When comparing the findings, it was evident that the VY-9 formed effective bonds to the peripheral residues of TYR via hydrogen bonds, hydrophobic interactions, van der Waals forces, and π-σ bonds. In another study, Ismaya examined the crystal structure of the mushroom TYR (abTYR) obtained from Agaricus bisporus, revealing that abTYR has a crystal structure which is an H2L2 tetramer comprising two H subunits and two L subunits. There are 150 subunits in the L subunit which is the result of an unrelated gene which plays an undetermined role. In contrast, the H subunit may derive from the TYR ppo3 gene since it comprises 391 amino acid residues which perfectly match the residues 2-392 of ppo3. The H subunit has an active binding site for abTYR which has two copper atoms (Cu400 and Cu401) along with six histidines (His). CuA will bind to the His61, His85, and His94 ligands, while CuB will bind to the His259, His263, and His296 ligands⁴². As a consequence of these histidine residues undergoing ligation with copper ions, the rotational freedom is restricted. The integrity of the binding site, Phe90, located between His94, His259, and His296, and Phe292, located between His61, His263, and His296, is maintained through constraining the histidine side chain conformation. Accordingly, it is clear that His61, His85, His94, His259, His263, His296, Phe90, and Phe292 are vital amino acid residues which affect TYR activity.

In order to predict the potential protein–peptide binding interactions, AutoDock Vina molecular docking analysis was first conducted. Then to predict the protein–peptide binding affinity, the LigPlot⁺ 2.0 server was employed. Further study of molecular docking was then performed to determine how the peptide VY-9 interacts with the TYR activity pocket. The interaction model deemed to be most probably for the peptide VY-9 binding with TYR displayed a binding energy of − 8.3 kcal mol^− 1. In its central activity region, the peptide VY-9 could use hydrogen bonds at the residuals Tyr65, Asn81, His85, His244, Asn260, Ser282, and Val283 to interact with TYR, along with hydrophobic interactions with Phe264, His263, H259, Pro277, Phe192, Pro284, Leu 63, and Glu322. Direct chelation with the copper ions is observed for the residue His85, and when the tyrosine residue oxygen atom on the peptide VY-9 bonds with the His85 hydrogen atom, this will influence enzyme activity, placing the tyrosine residue near to the copper ions, indicating that the peptide VY-9 binds to TYR as a substrate analogue, preventing melanin production.

The VY-9 peptide contains tyrosine which has a benzene ring that performs powerful hydrophobic interactions with Pro277 and Phe192, potentially blocking the TYR hydrophobic pocket, and lowering its hydrophobicity. In addition, the docking simulation reveals that the tyrosine residues of CRY and RCY interact with the Trp227 and Met257 side chains via hydrophobic interactions which serve to cover the TYR active pocket. While the exterior environment of TYR is hydrophilic, the inner core forms a hydrophobic cavity such that those amino acids which contain a hydrophobic side chain will tend to bind more readily to the hydrophobic pocket close to the active site of TYR. Furthermore, aromatic amino acids have benzene rings which can be buried at the active site as they bind to TYR, and since they are hydrophobic, this stabilizes the binding, increasing the inhibitory effects against TYR⁴³. When comparing the binding of arbutin and the VY-9 peptide with TYR, the VY-9 peptide exhibited the lowest binding energy at -8.3 kcal/mol, while arbutin showed a binding energy of -6.5 kcal/mol (S2 Figure, and S3 Table). Furthermore, the VY-9 peptide engages with seven binding residues: Tyr65, Asn81, His85, His244, Asn260, Ser282, and Val283. In contrast, Arbutin interacts with only four residues: Gln144, Glu173, Asn174, and His178. This result suggests that the VY-9 peptide binds to TYR more strongly than arbutin. In previous research there was a proposal for the inhibition mechanism for “tyrosine-type” TYR inhibitory peptides, including those similar to TH10. Furthermore, docking simulation has indicated that these peptides serve as substrate analogues which block access to the TYR active site for L-tyrosine. A simulation was also carried out using the mushroom TYR structure to determine the peptide CT-2 binding mode. These investigations revealed that the C-terminal tyrosine residue of CT-2 is bound to TYR active site with its copper through a mechanism similar to that of TH10, which would indicate that there is a strong affinity for CT-2 to the TYR active site, while CT-2 also serves as a substrate analogue. It is not, however, clear exactly why CT-2 proved more effective than the other isolated peptides in terms of inhibitory activity. Previous reports have suggested that while the tyrosine residue at the C-terminal plays a major inhibitory role in “tyrosine-type” TYR inhibitory peptides, there may also be a significant contribution made by the nearby amino acid residues³⁶.

The molecular docking model for the peptide VY-9 and TYR. (a) The 3D map of the peptide VY-9 docking at the TYR activity center. (b) The 2D projection of the peptide VY-9 and TYR docking model. This image was produced using LigPlot.

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The influence of the peptide VY-9 on B16F10 melanoma cells

In producing cosmetic products and functional foods, safety is of the highest priority during the formulation process. For instance, while hydroquinone is widely used in the treatment of hyperpigmentation and serves as a common skin-whitening treatment, it is known to induce a number of undesirable side effects such as contact dermatitis, irritation of the skin, and exogenous ochronosis in individuals of a darker skin tone. For these reasons, hydroquinone cannot be used as a cosmetic component in the European Union, and is strictly supervised by the Food and Drug Administration in the USA⁴⁴. Accordingly, this study commenced by assessing the cell viability of the peptide VY-9 in order to estimate the safety. The cells investigated in vitro included B16F10 melanoma cells from mice. Many studies focusing on pigmentation make use of the B16F10 melanoma cell line, since TYRs in humans and mice tend to be homologous with 86% identity at the protein level and 84% at the cDNA level. Moreover, these cell lines regulate the mechanisms of melanin synthesis in a similar manner to that of human melanocytes. Also, the cells are able to become attached to the culture flask and will create homogenous cell populations. It can then be feasible to observe the cellular differentiation from the perspectives of both morphology and melanogenesis^45,46. The cytotoxicity of the various concentrations of the peptide VY-9 was evaluated using the MTT colorimetric assay, and comparisons were drawn with arbutin (0.2 mM). The influence of the peptide VY-9 peptide with arbutin was examined in the context of B16F10 cell viability, with the outcome shown in Fig. 5a. As the peptide concentration rose, there was a decline in the survival rate of the cells. However, inside the concentration range used for testing, from 0 to 1.6 µM, there were no clear signs of cytotoxicity (viability > 50%). The ISO 10993-5:2009 guidelines suggest that any treatment which leads to a reduction in cell viability of more than 30% can be classified as toxic⁴⁷.

For additional investigation of the TYR activity which takes place in B16F10 melanoma cells after treatment by the peptide VY-9, different concentrations of the peptide VY-9 were used along with arbutin (0.2 mM). Figure 5b presents the outcomes. We are not aware of any previous studies examining mono-phenolase activity which make use of TYR derived from cell models. The lack of research in this area may be due to the need to initially accumulate L-DOPA via di-phenolase activity in order to stabilize TYR so that its catalytic capabilities can be fully activated. TYR can be found in three different oxidation forms, which in turn present different capabilities in phenol catalysis or the catalysis of catechol substrates. Meanwhile, mono-phenolase activity kinetics can be complex and are not east to observe⁴⁸. As a consequence, we did not examine the mono-phenolase reaction in this work. As the concentration of the peptide VY-9 was increased, the TYR activity began a steady decline, suggesting that TYR inhibition was increasing. This rate of inhibition was measured at 44.88% using 1.6 µM of VY-9, exceeding the performance of arbutin which offered 50.45% inhibition at 0.2 mM. Although molecular docking predicted that VY-9 interacts with the tyrosinase catalytic site, further experimental validation is required to confirm the peptide’s ability to penetrate cell membranes and reach intracellular tyrosinase. Future studies will involve permeability assays, such as Franz diffusion or cellular uptake studies⁴⁹, to verify the peptide’s intracellular delivery.

In skin, melanin content determines the level of whiteness. It is understood that melanin is produced when tyrosine is oxidized to form DOPA due to the action of TYR, whereupon DOPA undergoes further oxidization to DOPA-quinone, which in turn undergoes a number of reactions which create eumelanin. The TYR enzyme is therefore crucial in producing melanin. Figure 5c shows the steady decline in melanin content as peptide VY-9 increased in concentration, revealing a relationship strongly dependent on concentration levels and confirming the ability of VY-9 to restrict melanin production. The inhibition level was shown to reach a 54.34% reduction in melanin content when using arbutin (0.2 mM) to treat the cells, while the peptide VY-9 achieved a 52.31% reduction at 1.6 µM. While the arbutin rate was better, melanin synthesis is influenced by a number of factors in addition to TYR, and it would appear that arbutin is able to regulate the transcription of TYR pathway genes, bleaching the melanin which is produced⁵⁰.

The influence of the peptide VY-9 on B16F10 melanoma cell cytotoxicity (a), tyrosinase activity (b), and melanin content (c). The positive control used was arbutin (0.2 mM). The findings are presented in the form of percentage of the control, while the values represent the mean ± SD from three independent replications. For Duncan’s test, the different letters are used to denote a significant statistical difference between groups when p < 0.05.

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Abu Ubeid et al.⁵¹. conducted the screening of an internal library to identify the active oligopeptides capable of inhibiting TYR in mushrooms. Notably, the oligopeptides YRSRKYSSWY (IC₅₀ = 40 µM) and RADSRADC (IC₅₀ = 123 µM), showed greater activity than hydroquinone (IC₅₀ = 680 µM). However, some peptides, such as KFEKKFEK (IC₅₀ = 3.6 mM) and SFLLRN (IC₅₀ = 8 mM) showed lower levels of activity. Meanwhile, human TYR was also inhibited by the peptides YRSRKYSSWY and RADSRADC to a greater extent than was the case with hydroquinone. When human melanocytes underwent treatment for seven days with YRSRKYSSWY and RADSRADC at 100 µM there was a reduction in melanin content by 43% for the former and 27% for the latter. In addition, a docking study based on a library of short sequence oligopeptides in the context of the mushroom TYR crystal structure allowed the identification of several oligopeptides which displayed favorable binding free energies and interacted directly with the catalytic enzyme pocket. In the work of Shen et al.⁵². the TYR inhibition of the peptide ECGYF was reported, with the peptide comprising a short sequence of the protein midasin (MDN1) which is obtained from Vigna seeds. The reported TYR inhibition capabilities of ECGYF (IC₅₀ = 0.46 mM) exceeded those of arbutin and glutathione and the peptide (0.5-1 mM) was also able to lower the melanin levels in cultured A375 melanoma cells more effectively than arbutin or glutathione in the absence of cytotoxicity.

Effect of the peptide VY-9 on gene and protein expression of the melanogenesis pathway in B16F10 cells

MITF (microphthalmia-associated transcription factor) was a basic helix-loop-helix leucine zipper transcription factor relative to line age specific pathway regulation of certain cell models including osteoclasts, melanocytes, and mast cells. Importantly, MITF was the transcription factor of TYR, TRP-1, and TRP-2 in the melanogenesis pathway. Furthermore, in melanin production, TYR was an oxidase serving as the rate-limiting enzyme which controlled the production of melanin, while TRP-1 was a melanocyte-specific gene associated with melanin synthesis. In addition to this role, TRP-1 also served to stabilize the TYR protein, governing its catalytic abilities⁵³. The expression of TRP-1 and TRP-2 was altered by the MITF, and the influence of the peptide in inhibiting the expression of melanogenesis was examined using qRT-PCR. The qRT-PCR data presented in Fig. 6 revealed that once the B16F10 cells had undergone treatment with the peptide, at a concentration of 1.6 µM, peptide VY-9 significantly reduced the expression levels of all measured genes relative to the control. For Mitf and Trp-2, expression decreases were observed starting from the lowest concentration of 0.2 µM. In contrast, Tyr expression began to decrease at 0.4 µM, while Trp-1 showed a reduction only at the highest concentration of 1.6 µM. Notably, at a peptide concentration of 1.6 µM, the expression of Mitf and Trp-2 significantly decreased compared to arbutin, which served as a positive control. The protein expression results of the four target proteins measured by western blot were consistent with the gene expression results, as can be observed in Fig. 7a, b, and Fig. S1. At a concentration of peptide VY-9 at 1.6 µM, the expression of all proteins decreased compared to the control. However, compared to arbutin, only MITF showed a lower expression level. Starting from a concentration of 0.2 µM, the protein expression of TRP-1 and TRP-2 gradually decreased with increasing concentration. In contrast, the protein expression of MITF and TYR began to decrease at a concentration of 0.4 µM onwards.

The mRNA expression levels of Mitf, Tyr, Trp-1, and Trp-2 in B16F10 cells treated with different concentrations (0.2, 0.4, 0.8, and 1.6 µM) of the peptide VY-9. Results are presented as a percentage of the control, with values representing the mean ± SD of three independent replicates. All data are shown as mean ± SD. Post hoc analysis was conducted using Dunnett’s test. Significant differences compared to the control group are indicated as follows: *p < 0.05 and **p  <  0.01. Arbutin (0.2 mM) was used as the positive control.

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Western blot analysis was conducted for the comparison of melanin-related protein expressions in B16F10 cells in both the presence and absence of the peptide VY-9 for 48 h at concentrations of 0.2, 0.4, 0.8, and 1.6 µM. (A) Western blotting; and (B) Relative intensity of the protein band quantified by ImageJ software, with the value normalized to match the corresponding loading control. All data are shown as mean ± SD. Post hoc analysis was conducted using Dunnett’s test. Significant differences compared to the control group are indicated as follows: *p < 0.05 and **p < 0.01. Arbutin (0.2 mM) served as the positive control.

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During the course of our experiments, the highest concentration used was 1.6 µM, which reduced cell toxicity and effectively restricted the production of melanin and gene expression. Treatment with 1.6 µM resulted in a reduction in the gene and protein expression levels of MITF, TYR, TRP-1, and TRP-2, aligning with the findings for melanin content at that same concentration, as shown in Fig. 5c. Notably, at the lowest peptide concentration (0.2 µM), TRP-2 mRNA and protein levels decreased slightly compared to the control. The inhibitory effect became stronger as the concentration increased, as illustrated in Figs. 6 and 7. That is, TRP-2 activity could be influenced by additional physiological factors as well as the peptide itself. The main role of TRP-2 lies in converting L-dopachrome to DHICA in the melanogenesis pathway. The peptide examined in this study was able to inhibit the upstream transcription factor of TRP-2, but when the concentration of the peptide was increased, there was a fall in TRP-2 expression. This might arise as a consequence of MITF inhibition, and it could significantly change both TYR and TRP-1. We believed this was because the peptide could inhibit TYR and TRP-1 transcription, while a further possibility was that declining MITF expression might prevent TRP-2 from expressing normally. However, it was not possible to establish the precise reason for TRP-2 alterations which might allow the VY-9 peptide mechanism to be determined. While the peptide was capable of promoting TRP-2 expression, TYR was still significant as the rate-determining step. Furthermore, melanin synthesis must also occur via TRP-1, which was inhibited. We therefore take the view that this peptide would be capable of inhibiting the production of melanin. The peptide has also shown strong inhibitory capability based upon the dosage used in the inhibition of mushroom TYR, so enzyme activity inhibition may potentially be another outcome. These findings suggest that the peptide VY-9 may support skin whitening through the inhibition of melanogenesis via the obstruction of the mRNA and protein expression pathway.

Effects of the peptide VY-9 on zebrafish embryos

This study has a number of different models to evaluate melanogenesis, and there are pros and cons to each of these models. For instance, the B16F10 model is inexpensive and allows the rapid assessment of de-pigmentation capabilities in an assay which permits high throughput, yet there are differences in the post-translational modification of TYRs when compared to the situation in humans, while the lack of metabolic activity which would typically be enriched in the epidermal layer can lead to inaccuracies in assessing the de-pigmentation efficacy. In such scenarios it would be necessary to carry out further investigation in human melanocytes and in models which more accurately represent the human metabolism. The zebrafish model was recently implemented for in vivo studies of melanogenesis^54,55. It offers several advantages such as high levels of drug penetration, ease of maintenance, and does not require the application of stringent ethical rules which limit cosmetics testing on animals. The zebrafish provides an accepted vertebrate model for testing in the fields of toxicology and pharmacology. Zebrafish can be externally fertilized, are highly fecund, and the embryos develop quickly while offering transparency. The genetics and physiology of zebrafish are relatively similar to those of humans and other mammals⁵⁶. Melanin can be synthesized in zebrafish, and it is possible to observe the pigment visually at around 28 h after fertilization. Accordingly, zebrafish embryos can readily serve as a model when testing melanin inhibition in various compounds^57,58,59. In this research, we used zebrafish embryos to investigate the peptide VY-9 for melanin inhibition and also safety. Empirical determination guided the choice of concentrations examined in this study (2–10 µM). In zebrafish embryos, the peptide VY-9 displayed a “no observed effect concentration” (NOEC) of 10 µM. However, at 20 µM, the development of the embryo was significantly retarded. In this case, we found no significant difference in the rates for survival or malformation in the zebrafish embryo sample groups undergoing treatment when compared to the control group. These results suggest that the concentrations used for the in vitro analysis were safe when used with zebrafish embryos. Furthermore, embryo malformation was not observed when using these concentrations. Embryo samples following exposure can be seen in Fig. 8a. Additionally, the quantitative evaluation of melanin content showed no significant reduction in melanin levels at any concentration of the peptide VY-9 compared to the control. Although the relative melanin content at 4 µM was the lowest among all treatment groups, it did not achieve statistical significance compared to the control group, as depicted in Fig. 8b.

It was also shown that in embryos treated with PTU, the melanin content differed significantly (p < 0.01) to the control. These conflicting outcomes might be a consequence of the complexity of the organism involved. Since the zebrafish are able to absorb compounds comprising small molecules within the embryo medium through the skin, it may be possible for the peptide VY-9 to be exposed to biomolecules, potentially disrupting the inhibition of melanin synthesis^60,61. This study has produced in vitro and in vivo findings which demonstrate the ability of the peptide VY-9 to inhibit melanin, although it may be more suitable for use in cosmetic products rather than nutraceuticals. That is, the inhibition of melanin synthesis might be more readily accomplished by the direct application of the peptide VY-9 to the skin, since this may offer greater efficacy than using the same peptide in the form of a supplement or drug. However, in a nutraceutical context, it is necessary to further examine the metabolism of the peptide VY-9 after entry to different organisms.

Influence of the peptide VY-9 upon melanin pigments in zebrafish embryos at 48 h after fertilization. (a) Representative zebrafish embryos under treatment with the peptide VY-9. Scale bar = 500 μm. (b) Quantitative analysis zebrafish embryo melanin content after treatment using the peptide VY-9. The findings are presented as percentage of the control. Data from three independent replications are shown in the form of mean ± SD.

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• Source: https://www.nature.com/articles/s41598-024-81495-8