Purification and Characterization of RNA Allied Extracellular Tyrosinase from Aspergillus Species

Production of l-DOPA, an anti-Parkinson’s drug, using biological sources is widely studied in which tyrosinase is known to play a vital role. Tyrosinase is an omnipresent type 3 copper enzyme participating in many essential biological functions. Understanding properties of tyrosinase is essential for developing useful tyrosinase-based applications. Hence, extracellular tyrosinase from Aspergillus flavus UWFP 570 was purified using ammonium sulphate precipitation and DEAE ion exchange chromatography up to 8.3-fold. Purified protein was a riboprotein in nature containing significant amount of RNA which was confirmed colorimetrically and by electrophoresis. Removal of RNA reduced the activity and altered the conformation of tyrosinase as suggested by spectroflurometric results. Optimum pH and temperature of this 140 kDa protein were 7 and 40 °C, respectively. Copper sulphate and magnesium chloride enhanced the activity whereas in contrast FeCl₃ inhibited the activity completely. Purified tyrosinase transformed l-tyrosine (5 mM) to l-DOPA within 5 h.     

An ultrafiltration high-performance liquid chromatography coupled with diode array detector and mass spectrometry approach for screening and characterising tyrosinase inhibitors from mulberry leaves

Tyrosinase is a key enzyme in melanin synthesis. Its inhibitor may be used to efficiently treat hyperpigmentation and widely applied in cosmetic products and food supplements. In the present study, a new assay based on ultrafiltration high-performance liquid chromatography coupled with diode array detector and mass spectrometry (HPLC–DAD–MS) was developed for the rapid screening and identification of ligands for tyrosinase. Experiments were carried out to select the optimal binding conditions, tyrosinase concentration, and incubation time. Non-specific binding to the denatured tyrosinase was also investigated. Twelve compounds with tyrosinase binding activity were found in mulberry leaf extracts. The identities of these compounds were characterised by HPLC–DAD–MSⁿ. Particularly, two compounds, namely, quercetin-3-O-(6-O-malonyl)-β-d-glucopyranoside and kaempferol-3-O-(6-O-malonyl)-β-d-glucopyranoside, were identified as new tyrosinase inhibitors. The screening results were verified by tyrosinase inhibition assays. Experimental results proved that the proposed method could rapidly screen tyrosinase inhibitors in complex mixtures.     

Identification of Mushroom and Murine Tyrosinase Inhibitors from Achillea biebersteinii Afan. Extract

Growing scientific evidence indicates that Achillea biebersteinii is a valuable source of active ingredients with potential cosmetic applications. However, the data on its composition and pharmacological properties are still insufficient. This study aims to optimize the extraction procedure of the plant material, evaluate its phytochemical composition, and compare anti-tyrosinase potential of A. biebersteinii extracts obtained by various methods. In order to identify compounds responsible for the tyrosinase inhibitory activity of A. biebersteinii, the most active anti-tyrosinase extract was fractionated by column chromatography. The fractions were examined for their skin lightening potential by mushroom and murine tyrosinase inhibitory assays and melanin release assay. HPLC-ESI-Q-TOF-MS/MS analysis of the total extract revealed the presence of several phenolic acids, flavonoids, flavonoid glucosides, and carboxylic acid. Among them, fraxetin-8-O-glucoside, quercetin-O-glucopyranose, schaftoside/isoschaftoside, gmelinin B, 1,3-dicaffeoylquinic acid (1,3-DCQA), and ferulic acid were found in the fractions with the highest skin lightening potential. Based on obtained qualitative and quantitative analysis of the fractions, it was assumed that the caffeoylquinic acid derivatives and dicaffeoylquinic acid derivatives are more likely responsible for mushroom tyrosinase inhibitory activity of A. biebersteinii extracts and fractions. Ferulic acid was proposed as the most active murine tyrosinase inhibitor, responsible also for the reduced melanin release from B16F10 murine melanoma cells.     

Methoxy-Substituted Tyramine Derivatives Synthesis,Computational Studies and Tyrosinase Inhibitory Kinetics

Targeting tyrosinase for melanogenesis disorders is an established strategy. Hydroxyl-substituted benzoic and cinnamic acid scaffolds were incorporated into new chemotypes that displayed in vitro inhibitory effects against mushroom and human tyrosinase for the purpose of identifying anti-melanogenic ingredients. The most active compound 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2,4-dihydroxyphenyl) acrylate (Ph9), inhibited mushroom tyrosinase with an IC₅₀ of 0.059 nM, while 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) had an IC₅₀ of 2.1 nM compared to the positive control, kojic acid IC₅₀ 16700 nM. Results of human tyrosinase inhibitory activity in A375 human melanoma cells showed that compound (Ph9) and Ph6 exhibited 94.6% and 92.2% inhibitory activity respectively while the positive control kojic acid showed 72.9% inhibition. Enzyme kinetics reflected a mixed type of inhibition for inhibitor Ph9 (K_i 0.093 nM) and non-competitive inhibition for Ph6 (K_i 2.3 nM) revealed from Lineweaver–Burk plots. In silico docking studies with mushroom tyrosinase (PDB ID:2Y9X) predicted possible binding modes in the catalytic site for these active compounds. Ph9 displayed no PAINS (pan-assay interference compounds) alerts. Our results showed that compound Ph9 is a potential candidate for further development of tyrosinase inhibitors.     

A Novel Heptapeptide with Tyrosinase Inhibitory Activity Identified from a Phage Display Library

Peptidic inhibition of the enzyme tyrosinase, responsible for skin pigmentation and food browning, would be extremely useful for the food, cosmetics, and pharmaceutical industries. In order to identify novel inhibitory peptides, a library of short sequence oligopeptides was screened to reveal direct interaction with the tyrosinase. A phage displaying heptapeptide (IQSPHFF) was found to bind most strongly to tyrosinase. The inhibitory activity of the heptapeptide was evaluated using mushroom tyrosinase. The results showed that the peptide inhibited both the monophenolase and diphenolase activities of mushroom tyrosinase with IC₅₀ values of 1.7 and 4.0 mM, respectively. The heptapeptide is thought to be a reversible competitive inhibitor of diphenolase with the inhibition constants (Ki) of 0.765 mM. To further investigate how the heptapeptide exerts its inhibitory effect, a docking study between tyrosinase and heptapeptide was performed. The simulation showed that the heptapeptide binds in the active site of the enzyme near the catalytically active Cu ions and forms hydrogen bonds with five histidine residues on the active site. Phage display technology is thus a useful approach for the screening of potential tyrosinase inhibitors and could be widely applicable to a much wider range of enzymes.     

Tyrosinase immobilized magnetic nanobeads for the amperometric assay of enzyme inhibitors: application to the skin whitening agents

The immobilization of tyrosinase onto glutaraldehyde activated streptavidine magnetic particles and subsequent retention onto a magnetized carbon paste electrode for the amperometric assay of tyrosinase inhibitors is described. Tyrosine was used as substrate as it is the first substrate in the melanogenesis process. The sensing mode is based on monitoring the decrease of the amperometric signal corresponding to the electrochemical reduction of dopaquinone enzymatically generated. This current decrease is due to the presence of inhibitors acting directly on the enzyme or inhibitors acting on the product of the enzymatic reaction, i.e. dopaquinone. The methodology is designed for the evaluation of the inhibitory potency of the most frequently used active substances in cosmetic marketed products against hyperpigmentation such as kojic acid, azelaic acid and benzoic acid. These compounds bind to the tyrosinase active center. Ascorbic acid is also investigated as it interrupts the synthesis pathway of melanin by reducing the melanin intermediate dopaquinone back to l-dopa. By comparing the obtained IC₅₀, under the same experimental conditions, the order of their inhibitory potency was: kojic acid (IC₅₀ = 3.7 × 10⁻⁶ M, K_i = 8.6 × 10⁻⁷ M), ascorbic acid (IC₅₀ = 1.2 × 10⁻⁵ M), benzoic acid (IC₅₀ = 7.2 × 10⁻⁵ M, K_i = 2.0 × 10⁻⁵ M) and azelaic acid (IC₅₀ = 1.3 × 10⁻⁴ M, K_i = 4.2 × 10⁻⁵ M) in close agreement with literature spectrophotometric inhibition data using the soluble tyrosinase.     

The Hypopigmentation Mechanism of Tyrosinase Inhibitory Peptides Derived from Food Proteins: An Overview

Skin hyperpigmentation resulting from excessive tyrosinase expression has long been a problem for beauty lovers, which has not yet been completely solved. Although researchers are working on finding effective tyrosinase inhibitors, most of them are restricted, due to cell mutation and cytotoxicity. Therefore, functional foods are developing rapidly for their good biocompatibility. Food-derived peptides have been proven to display excellent anti-tyrosinase activity, and the mechanisms involved mainly include inhibition of oxidation, occupation of tyrosinase’s bioactive site and regulation of related gene expression. For anti-oxidation, peptides can interrupt the oxidative reactions catalyzed by tyrosinase or activate an enzyme system, including SOD, CAT, and GSH-Px to scavenge free radicals that stimulate tyrosinase. In addition, researchers predict that peptides probably occupy the site of the substrate by chelating with copper ions or combining with surrounding amino acid residues, ultimately inhibiting the catalytic activity of tyrosinase. More importantly, peptides reduce the tyrosinase expression content, primarily through the cAMP/PKA/CREB pathway, with PI3K/AKT/GSK3β, MEK/ERK/MITF and p38 MAPK/CREB/MITF as side pathways. The objective of this overview is to recap three main mechanisms for peptides to inhibit tyrosinase and the emerging bioinformatic technologies used in developing new inhibitors.     

Kinetics of Ergothioneine Inhibition of Mushroom Tyrosinase

The native amino acid ergothioneine, a thiourea derivative of histidine, inhibits mushroom tyrosinase activity in a dose-dependent manner, with an IC₅₀ value of 1.025 mg/ml (4.47 mM). By contrast, histidine exhibited no inhibitory effect on mushroom tyrosinase activity. We characterized ergothioneine as a noncompetitive tyrosinase inhibitor using a Lineweaver–Burk plot of experimental kinetic data. The IC₅₀ value for ergothioneine scavenging of 2,2-diphenyl-1-picrylhydrazyl was 6.110 ± 0.305 mg/ml, much higher than the IC₅₀ for inhibition of tyrosinase activity which indicating ergothioneine on tyrosinase shows a weak correlation to its antioxidative activity. The results demonstrated that ergothioneine has a potent inhibition effect on tyrosinase enzyme activity, resulting from the presence of the sulfur substituted imidazole ring in ergothioneine.     

K-Nearest Neighbor and Random Forest-Based Prediction of Putative Tyrosinase Inhibitory Peptides of Abalone Haliotis diversicolor

Skin pigment disorders are common cosmetic and medical problems. Many known compounds inhibit the key melanin-producing enzyme, tyrosinase, but their use is limited due to side effects. Natural-derived peptides also display tyrosinase inhibition. Abalone is a good source of peptides, and the abalone proteins have been used widely in pharmaceutical and cosmetic products, but not for melanin inhibition. This study aimed to predict putative tyrosinase inhibitory peptides (TIPs) from abalone, Haliotis diversicolor, using k-nearest neighbor (kNN) and random forest (RF) algorithms. The kNN and RF predictors were trained and tested against 133 peptides with known anti-tyrosinase properties with 97% and 99% accuracy. The kNN predictor suggested 1075 putative TIPs and six TIPs from the RF predictor. Two helical peptides were predicted by both methods and showed possible interaction with the predicted structure of mushroom tyrosinase, similar to those of the known TIPs. These two peptides had arginine and aromatic amino acids, which were common to the known TIPs, suggesting non-competitive inhibition on the tyrosinase. Therefore, the first version of the TIP predictors could suggest a reasonable number of the TIP candidates for further experiments. More experimental data will be important for improving the performance of these predictors, and they can be extended to discover more TIPs from other organisms. The confirmation of TIPs in abalone will be a new commercial opportunity for abalone farmers and industry.     

Kinetic Evaluation of Aminoethylisothiourea on Mushroom Tyrosinase Activity

This study demonstrates that aminoethylisothiourea (AET), a potent inhibitor of inducible nitric oxide synthase, is an irreversible competitive inhibitor of mushroom tyrosinase by chelation to the active site of tyrosinase when l-3,4-dihydroxyphenylalanine was assayed spectrophotometrically. The spectrophotometric recordings of the inhibition of tyrosinase by AET were characterized by the presence of a lag period prior to the attainment of an inhibited steady-state rate. The lag period corresponded to the time in which AET was reacting with the enzymatically generated o-quinone. Increasing AET concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady-state rate were dependent on AET, substrate, and tyrosinase concentrations. The inhibition of diphenolase activity of tyrosinase by AET showed positive kinetic cooperativity which arose from the protection of both substrate and o-quinone against inhibition by AET. The UV-visible spectrum of a mixture of tyrosinase and AET exhibited a characteristic shoulder peak ascribed to the chelation of AET to the active site of tyrosinase. Moreover, the presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition when CuSO₄ was added to the assay medium on tyrosinase activity.     

Tyrosinase inhibitors from Rhododendron collettianum and their structure-activity relationship (SAR) studies

A new coumarinolignoid 8'-epi-cleomiscosin A (1) together with the new glycoside 8-O-beta-D-glucopyranosyl-6-hydroxy-2-methyl-4H-1-benzopyrane-4-one (2) have been isolated from the aerial parts of Rhododendron collettianum and their structures determined on the basis of spectroscopic evidences. Tyrosinase inhibition study of these compounds and their structure-activity relationship (SAR) were also investigated. The compounds exhibited potent to mild inhibition activity against the enzyme. Especially, the compound 1 showed strong inhibition (IC50=1.33 microM) against the enzyme tyrosinase, as compared to the standard tyrosinase inhibitors kojic acid (IC50=16.67 microM) and L-mimosine (IC50=3.68 microM), indicating its potential used for the treatment of hyperpigmentation associated with the high production of melanocytes.     

Analysis of Kojic Acid Derivatives as Competitive Inhibitors of Tyrosinase: A Molecular Modeling Approach

Tyrosinases belong to the functional copper-containing proteins family, and their structure contains two copper atoms, in the active site, which are coordinated by three histidine residues. The biosynthesis of melanin in melanocytes has two stages depending on the actions of the natural substrates L-DOPA and L-tyrosine. The dysregulation of tyrosinase is involved in skin cancer initiation. In the present study, using molecular modeling tools, we analyzed the inhibition activity of tyrosinase activity using kojic acid (KA) derivatives designed from aromatic aldehydes and malononitrile. All derivatives showed conformational affinity to the enzyme active site, and a favorable distance to chelate the copper ion, which is essential for enzyme function. Molecular dynamics simulations revealed that the derivatives formed promising complexes, presenting stable conformations with deviations between 0.2 and 0.35 Å. In addition, the investigated KA derivatives showed favorable binding free energies. The most stable KA derivatives showed the following binding free energies: −17.65 kcal mol⁻¹ (D6), −18.07 kcal mol⁻¹ (D2), −18.13 (D5) kcal mol⁻¹, and −10.31 kcal mol⁻¹ (D4). Our results suggest that these derivatives could be potent competitive inhibitors of the natural substrates of L-DOPA (−12.84 kcal mol⁻¹) and L-tyrosine (−9.04 kcal mol⁻¹) in melanogenesis.     

Partial Purification and Characterization of a Novel Extracellular Tyrosinase from Auricularia auricula

Extracellular tyrosinase from Auricularia auricula RF201 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 12.6 kDa on SDS-PAGE. The optimum pH for tyrosinase activity was 7, and the enzyme was stable between pH 6 and 9. Tyrosinase has optimal activity at 40 °C and retained most of its activity between 4 and 50 °C. A. auricula tyrosinase could oxidize l-tyrosine, l-DOPA, catechol, and caffeic acid and displayed dark brown or peach color. However, the enzyme was unable to catalyze l-phenylalanine and ferulic acid. In comparison with other substrates, l-tyrosine displayed the highest affinity (K _m of 0.11 mM) and the maximal reaction velocity (V _max of 102.58 μmol/min). Tyrosinase activity was reduced in the presence of numerous tested compounds. Particularly SDS, it significantly inhibited enzyme activity. CuSO₄ and NaCl showed an activation effect on enzyme activity, with the maximum activation found in the presence of CuSO₄.     

Alternative Approach for Specific Tyrosinase Inhibitor Screening: Uncompetitive Inhibition of Tyrosinase by Moringa oleifera

Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC₅₀ values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC₅₀ values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of K_ii 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.     

Insights on the Inhibitory Power of Flavonoids on Tyrosinase Activity: A Survey from 2016 to 2021

Tyrosinase is a multifunctional copper-containing oxidase enzyme that initiates melanin synthesis in humans. Excessive accumulation of melanin pigments or the overexpression of tyrosinase may result in skin-related disorders such as aging spots, wrinkles, melasma, freckles, lentigo, ephelides, nevus, browning and melanoma. Nature expresses itself through the plants as a source of phytochemicals with diverse biological properties. Among these bioactive compounds, flavonoids represent a huge natural class with different categories such as flavones, flavonols, isoflavones, flavan-3-ols, flavanones and chalcones that display antioxidant and tyrosinase inhibitor activities with a diversity of mechanistic approaches. In this review, we explore the role of novel or known flavonoids isolated from different plant species and their participation as tyrosinase inhibitors reported in the last five years from 2016 to 2021. We also discuss the mechanistic approaches through the different studies carried out on these compounds, including in vitro, in vivo and in silico computational research. Information was obtained from Google Scholar, PubMed, and Science Direct. We hope that the updated comprehensive data presented in this review will help researchers to develop new safe, efficacious, and effective drug or skin care products for the prevention of and/or protection against skin-aging disorders.     

Development of nanoformulation for hyperpigmentation disorders: Experimental evaluations,in vitro efficacy and in silico molecular docking studies

Hyperpigmentation is a crucial dermatological disorder. This study aims to formulate a nanoemulsion formulation containing chlorogenic acid (CA) for hyperpigmentation treatment, to carry out characterization studies, and to investigate its efficacy and safety in vitro and in silico analysis.In line with this purpose, CA nanoemulsions (CA-NEs) were developed using the ultrasonic homogenization method. Accelerated stability tests were performed to examine the kinetic and thermodynamic stability of the CA-NEs to ascertain the presence of any stability issues. After the heating–cooling test, appropriate CA-NEs were stored for 60 days in three different stability environments to examine the physicochemical stability and determine the finalized formulation. The toxicity of the finalized CA-NE formulation was evaluated by genotoxicity/mutagenicity and cytotoxicity tests. The tyrosinase and melanogenesis activities of the finalized CA-NE formulation were determined on the Melanoma B16F0 cell line. Finally, the molecular docking method was used to reveal interactions of CA that play an essential role in tyrosinase inhibition. Additionally, the mushroom and human tyrosinase enzymes were used to determine the activity of CA. In addition, the comparison study with the molecular docking method was carried out using kojic acid as a reference molecule.In conclusion, the molecular docking study, pharmacokinetic analyses, and in vitro studies showed that F4P1 coded CA-NE formulation might hold promise as an innovative formulation in cosmetic applications such as skin-lightening effects with its high efficacy and safety profile.     

Inhibitory Effects of p-Phenylene-bis and Phenyl Dithiocarbamate on Mushroom Tyrosinase

The binding properties and structural changes of mushroom tyrosinase enzyme, MT, due to its interactions with phenyl dithiocarbamate (I) and p-phenylene-bis dithiocarbamate (II), were investigated at 27 and 37 °C in phosphate buffer (10?mmol?L^?1) at pH = 6.8 by isothermal titration calorimetric (ITC). The extended solvation model was used to calculate the solvation parameters, which were attributed to the stability of this enzyme. Thermodynamic analysis indicated that the predominant mode of interaction was hydrophobic in the binding of compound I to MT, while the binding of II to MT essentially depends on electrostatic interactions. It seems that II is a more potent MT inhibitor due to its two charged head groups that are able to chelate copper ions in the active enzyme site. It was concluded that MT has two distinct sites for p-phenylene-bis and phenyl dithiocarbamate.     

Electrochemical Detection of Tyrosinase in Cell Lysates at Functionalized Nanochannels via Amplifying of Ionic Current Response

Tyrosinase is an important enzyme in the metabolism of melanin and catecholamine, as well as an important antigen in autoimmunity of vitiligo. However, it is still a challenge to develop a selective and sensitive sensing approach for monitoring of tyrosinase in cell environment. In this work, a sensing strategy based on ion current rectification induced by nanochannels was established to electrochemically detect inactive tyrosinase. Functionalized alumina nanochannels were decorated by tyramine as an electrochemical platform to selectively determine tyrosinase via chemically specific reaction between phenolic hydroxyl groups of tyramine and tyrosinase. On the other hand, Fe²⁺ was further bonded with dihydroxyl groups generated by tyrosinase in nanochannels to amplify charge changes, resulting in the enhancement on the detection sensitivity for tyrosinase. Based on this principle, the developed electrochemical sensor showed a good linearity towards tyrosinase from 2 to 50 U/mL with a detection limit down to 0.83 U/mL, which can fulfil the requirement for detection of tyrosinase in cells. Eventually, the present electrochemical sensor was successfully employed in the selective and sensitive determination of tyrosinase in cell lysates. The results suggested that the activity of tyrosinase in B16 cells was 1.7-fold higher than that of Hela cells.     

Tyrosinase inhibition organic phase biosensor for triazinic and benzotriazinic pesticide analysis (part two)

Several triazine pesticides, such as atrazine, are much more soluble in several organic solvents, such as chloroform, than in water. Our recent research was aimed at analyzing this class of pesticides using tyrosinase OPEE (organic phase enzyme electrodes), exploiting their inhibiting action on the tyrosinase enzyme when operating in water-saturated chloroform medium. In this work we studied the response of a tyrosinase inhibition enzyme sensor to several triazinic (simazine, propazine, terbuthylazine) and benzotriazinic (azinphos-ethyl and azinphos-methyl) pesticides (LOD=0.5×10⁻⁹ mol l⁻¹). Recovery trials were also performed in vegetal matrixes (corn, barley, lentils). Lastly, the effect of the solvent (chloroform or water) on the inhibition process was investigated via Hill’s equation and the diffusion of analyte from the solvent to the enzyme membrane.     

Novel Anti-Melanogenic Compounds, (Z)-5-(Substituted Benzylidene)-4-thioxothiazolidin-2-one Derivatives: In Vitro and In Silico Insights

To confirm that the β-phenyl-α,β-unsaturated thiocarbonyl (PUSTC) scaffold, similar to the β-phenyl-α,β-unsaturated carbonyl (PUSC) scaffold, acts as a core inhibitory structure for tyrosinase, twelve (Z)-5-(substituted benzylidene)-4-thioxothiazolidin-2-one ((Z)-BTTZ) derivatives were designed and synthesized. Seven of the twelve derivatives showed stronger inhibitory activity than kojic acid against mushroom tyrosinase. Compound 2b (IC₅₀ = 0.47 ± 0.97 µM) exerted a 141-fold higher inhibitory potency than kojic acid. Kinetic studies’ results confirmed that compounds 2b and 2f are competitive tyrosinase inhibitors, which was supported by high binding affinities with the active site of tyrosinase by docking simulation. Docking results using a human tyrosinase homology model indicated that 2b and 2f might potently inhibit human tyrosinase. In vitro assays of 2b and 2f were conducted using B16F10 melanoma cells. Compounds 2b and 2f significantly and concentration-dependently inhibited intracellular melanin contents, and the anti-melanogenic effects of 2b at 10 µM and 2f at 25 µM were considerably greater than the inhibitory effect of kojic acid at 25 µM. Compounds 2b and 2f similarly inhibited cellular tyrosinase activity and melanin contents, indicating that the anti-melanogenic effects of both were due to tyrosinase inhibition. A strong binding affinity with the active site of tyrosinase and potent inhibitions of mushroom tyrosinase, cellular tyrosinase activity, and melanin generation in B16F10 cells indicates the PUSTC scaffold offers an attractive platform for the development of novel tyrosinase inhibitors.