Inhibition mechanism investigation of quercetagetin as a potential tyrosinase inhibitor

Tyrosinase is one important rate limiting enzyme in melanin synthesis, directly affecting the melanin synthesis. Quercetagetin is one active ingredient from marigold. Thence, the inhibition effects of quercetagetin against tyrosinase were investigated. The results showed quercetagetin could inhibit tyrosinase activity with IC50 value of 0.19 ± 0.01 mM and the inhibition type was a reversible mixed-type. Results of fluorescence quenching showed quercetagetin could quench tyrosinase fluorescence in static process. CD and 3D fluorescence results showed the interaction of quercetagetin to tyrosinase could change tyrosinase conformation to inhibit activity. Moreover, docking revealed details of quercetagetin’s interactions with tyrosinase.


Introduction
As we all know, various diseases affect people's health (Zhang et al., 2020;Zhang X. et al., 2021;Shi et al., 2023;Zhou S. et al., 2022).As a therapeutic target, tyrosinase is one important metal enzyme containing two copper, which widely presents in the organism (Min et al., 2023;Fan et al., 2017).Tyrosinase has been confirmed to be involved in the synthesis of melanin (Li et al., 2023;Hassan et al., 2023;Li J. et al., 2021).The first reaction process is monophenolase activity, in which L-tyrosine is hydroxylated into L-dopa and the second reaction process is diphenolase activity, in which L-dopa is subsequently oxidized into dopaquinone (Djafarou et al., 2023;Lee et al., 2023;Zargaham et al., 2023).In the organism, melanin acts crucial roles to protect the skin from UV radiation (Lu et al., 2023a;Znajafi et al., 2023).But, excessive elevated melanin content leads to lots of pigmentation disorders, including age spots and melanoma (Broulier et al., 2023;De Barros et al., 2023;Xue et al., 2023).Inhibition of tyrosinase activity would reduce melanin generation, thence, the finding on novel tyrosinase inhibitors is attracting more attention due to their potential application in medicine and cosmetics fields (Wang and Mu, 2021).Although kojic acid and arbutin (Figure 1) are applied as tyrosinase inhibitors in the medical and industrial fields, they still have been found to lots of adverse side effects (Wang et al., 2021;Esma et al., 2023).Now, to find novel tyrosinase inhibitors is essential for treatment of melanin synthesis (Romagnoli et al., 2022;Nasab et al., 2023).
Hence, in this study, we investigated the inhibition effects of quercetagetin against tyrosinase by the multispectral method, followed by the molecular docking.

Inhibitory activity
The tyrosinase inhibitory activity of quercetagetin was examined on mushroom tyrosinase.The tyrosinase activity was measured under different concentrations of quercetagetin (Figure 2).It could be observed that tyrosinase relative activity gradually reduced with quercetagetin concentration (0-0.64 mM), meaning that quercetagetin could inhibit the tyrosinase activity with quercetagetin concentration.Its IC 50 value was calculated to be 0.19 ± 0.01 mM, which was lower than that of kojic acid.This result showed that quercetagetin could be used as a natural tyrosinase inhibitor.

Kinetic study
There are two kinds of inhibitors, reversible and non-reversible inhibitors.For a reversible inhibitor, it can reduce the enzyme activity by the binding to enzyme, which can restore the enzyme activity through the remove of inhibitor.There are three reversible inhibitors, including competitive, non-competitive and mixed-type inhibitors.The inhibition type of quercetagetin on tyrosinase was subsequently investigated.With different concentration of quercetagetin and tyrosinase, the absorbance changes were measured (Figure 3A) and found that the lines of quercetagetin with different concentration passed origin and slopes reduced with quercetagetin concentration.The results suggested quercetagetin as a reversible inhibitor.With different concentration of quercetagetin and substrate, the absorbance changes were analyzed using Lineweaver-Burk plots (Figure 3B).The lines of quercetagetin intersected in the third quadrant and their slops increased with quercetagetin concentrations.The results indicated that quercetagetin inhibited tyrosinase in a mixed-type, meaning that quercetagetin bound to tyrosinase and tyrosinase-substrate complex to inhibit its activity.Similar phenomena were obtained in inhibition type of indole-carbohydrazides (Iraji et al., 2022).
Moreover, as shown in Figures 4A, B, the inhibition constants K i and K is were obtained from the secondary curves of inhibitory kinetics to be 0.24 and 0.12 mM.Smaller K is value than K i meant that binding force of quercetagetin with tyrosinase-substrate complex was stronger than with tyrosinase.That was to say that quercetagetin preferred to bind with tyrosinase-substrate complex.

Fluorescence quenching
The fluorescence quenching process of tyrosinase by quercetagetin was investigated.For fluorescence spectra of tyrosinase, they all showed characteristic peaks at 344 nm at 295, 298, and 305 K, respectively (Figures 5A-C).But, quercetagetin did not show effective fluorescence spectra.Moreover, when treated by quercetagetin, tyrosinase presented the gradually decreasing peak intensity (Figures 5A-C), which indicated that quercetagetin bound to tyrosinase.
The fluorescence quenching data at 295, 298, and 305 K were future analyzed by Stern-Volmer plots.As is shown in Figure 5D the Stern-Volmer plots of quercetagetin, lines with different temperature presented good linearity, meaning that there was only one quenching type of static or dynamic in quenching process.Then the quenching constant (K sv ) the bimolecular quench rate constant (K q ) were obtained (Table 1).The K sv results found that K sv values decreased with the temperature.And K q values at 295, 298, and 305 K were higher than 2 × 10 10 L mol −1 S −1 .Above results suggested the quenching process of quercetagetin against tyrosinase was a static process.This quenching type presented in the quenching of indole derivatives on α-glucosidase (Hu et al., 2024).The quenching process also was the process of thermodynamic changes.Thence, the thermodynamic parameters were calculated (Table 1).The negative ΔG value unlocked one spontaneous process of quercetagetin binding to tyrosinase.The positive ΔH and ΔS values suggested hydrophobic interactions as the important forces between quercetagetin and tyrosinase.

CD spectra
The effect of quercetagetin on conformation change of tyrosinase was investigated using CD spectra.Figure 6 showed CD spectra of tyrosinase, the characteristic peaks around 210-220 nm stood for the peptide chains of tyrosinase.When treated with quercetagetin, the CD spectra of tyrosinase appeared some changes, meaning its conformation change.Then its secondary structure contents were calculated (Table 2) and found that quercetagetin treatment (molar ratio: 2:1) caused reduction of  α-helix and random coils and increase of β-sheet and β-turn.These results indicated that quercetagetin treatment could lead to conformation changes of tyrosinase.

3D fluorescence spectra
3D fluorescence spectra were monitored to further investigate the effect on conformation of tyrosinase by quercetagetin.As shown in Figure 7A the 3D fluorescence spectra tyrosinase, two characteristic peaks appeared, including Peak A for Tyr and Trp residues and Peak B for peptide backbone.When treated with quercetagetin, Peak A and Peak B were reduced the intensity by 35.4% and 13.3%, respectively (Figure 7B), which suggested that quercetagetin treatment would cause the changes of Tyr and Trp residues, and peptide backbone.That was to say that quercetagetin treatment would cause the conformation change of tyrosinase.

Molecular docking
The interaction of quercetagetin to tyrosinase was simulated by molecular docking method.As shown in Figure 8A the docking results, quercetagetin bound to the active pocket of tyrosinase with trihydroxychromone section in the active catalytic zone of active pocket.From Figure 8B the docking results in detail, it could be observed that quercetagetin made one hydrogen bond with His259 and hydrophobic interaction with Val248, His263, Val283, and Ala286.The results indicated that the interactions

Copper-chelating activity
The copper-chelating activity of quercetagetin was finally assayed using copper sulfate.Figure 9A showed the quercetagetin-copper sulfate mixture.Quercetagetin showed its UV spectra with characteristic peak at 370 nm.While, after added copper sulfate, the UV characteristic peak of quercetagetin was gradually decreased.These results indicated that copper might complex with quercetagetin to change its UV characteristic peak.The peak intensity at different molar ratios of quercetagetin to copper sulfate was analyzed (Figure 9B) and found that the decreasing trend of quercetagetin peak intensity become equilibrium, meaning that the binding molar ratio of quercetagetin with copper sulfate was 1.The copper-chelating activity of quercetagetin also might be one reason of the reduction of tyrosinase activity.

Conclusion
Tyrosinase is one important rate limiting enzyme in melanin synthesis, directly affecting the melanin synthesis.Quercetagetin is one active ingredient from marigold.In this study, the inhibition effects of quercetagetin against tyrosinase were investigated.The results showed that quercetagetin could inhibit the tyrosinase activity with IC 50 value of 0.19 ± 0.01 mM and the inhibition type was a reversible mixed-type.Results of fluorescence quenching showed that quercetagetin could quench tyrosinase fluorescence in static process.CD and 3D fluorescence results showed interaction of quercetagetin to tyrosinase could change tyrosinase conformation to inhibit activity.Moreover, docking revealed details of quercetagetin's interactions with tyrosinase.

Tyrosinase activity assay
Tyrosinase inhibitory activity of quercetagetin was measured (Lu et al., 2023b).10 μL of quercetagetin in DMSO solutions was added into 140 μL of tyrosinase in PBS solution and incubated for CD spectra of tyrosinase with quercetagetin.  10 min.50 μL of L-dopa (in PBS) was added as the substrates.Then, the absorbance was measured at 475 nm on a microplate reader.Kojic acid was selected as the positive control.All experiments were tested in triplicate.Inhibition rate (%) = [(OD 1 -OD 0 )/OD 0 ]×100%.OD 0 and OD 1 were the absorbance of tyrosinase and tyrosinasequercetagetin mixture.

Inhibitory kinetics
The reversibility of quercetagetin against tyrosinase was analyzed at different concentration of quercetagetin with tyrosinase or L-dopa.And the inhibitory kinetics of quercetagetin against tyrosinase was analyzed at different concentration of quercetagetin with tyrosinase or L-dopa.The inhibition constants K i and K is were obtained from the secondary curves of inhibitory kinetics (Xu et al., 2020;Deng et al., 2022;Lin et al., 2023).

Fluorescence quenching
To the 3.0 mL of tyrosinase solution, 1 μL of quercetagetin was added by titration method (Li et al., 2024a;Min et al., 2024).The mixture was measured fluorescence spectra at excitation of 280 nm.The experiments were conducted at temperatures of 295, 298, and 305 K, respectively.The Stern-Volmer equation and the vanʼt Hoff equation were employed to obtained parameters.

CD spectra
To the 100 μL of tyrosinase solution, 1 μL of quercetagetin was added.CD spectra were measured at room temperature (Xiao et al., 2023;Li et al., 2024b).Tyrosinase solution without quercetagetin was also measured its CD spectra.

Copper-chelating activity
To the 2 mL of quercetagetin solution, 5 μL of copper sulfate solution was gradually added.UV absorption spectra were detected.The molar ratios of quercetagetin to copper sulfate ranged from 10: 0 to 10: 14.

Molecular docking
Docking of quercetagetin with tyrosinase was simulated using the SYBYL software (Zhang et al., 2022;Feng et al., 2024).The tyrosinase crystal structure (PDB: 2Y9X) was optimized by removing water, adding hydrogen, and generation of active pocket.Quercetagetin structure was performed energy minimization.Thence, docking procedure was run in the default format.

FIGURE 4 (
FIGURE 4 (A) K i assay of quercetagetin; (B) K is assay of quercetagetin.

FIGURE 8 (
FIGURE 8 (A,B) The molecular docking of quercetagetin to tyrosinase.

FIGURE 9 (
FIGURE 9(A) UV spectra of quercetagetin-copper sulfate mixture; (B) UV peak of quercetagetin versus molar ratios of quercetagetin to copper sulfate.

TABLE 1
Parameters of quercetagetin with tyrosinase.

TABLE 2
The secondary structure contents of tyrosinase with quercetagetin.