Bioassay-Guided and DeepSAT-Driven Precise Mining of Monoterpenoid Coumarin Derivatives with Antifeedant Effects from the Leaves of Ailanthus altissima.

Demand for the exploration of botanical pesticides continues to increase due to the detrimental effects of synthetic chemicals on human health and the environment and the development of resistance by pests. Under the guidance of a bioactivity-guided approach and HSQC-based DeepSAT, 16 coumarin derivatives were discovered from the leaves of Ailanthus altissima (Mill.) Swingle, including seven undescribed monoterpenoid coumarins, three undescribed monoterpenoid phenylpropanoids, and two new coumarin derivatives. The structure and configurations of these compounds were established and validated via extensive spectroscopic analysis, acetonide analysis, and quantum chemical calculations. Biologically, 5 exhibited significant antifeedant activity toward the Plutella xylostella. Moreover, tyrosinase being closely related to the growth and development of larva, the inhibitory potentials of 5 against tyrosinase was evaluated in vitro and in silico. The bioactivity evaluation results highlight the prospect of 5 as a novel category of botanical insecticide.

The Effect of α-Arbutin on UVB-Induced Damage and Its Underlying Mechanism.

Ultraviolet radiation can heighten tyrosinase activity, stimulate melanocyte production, impede the metabolism of numerous melanocytes, and result in the accumulation of plaques on the skin surface. α-Arbutin, a bioactive substance extracted from the arbutin plant, has been widely used for skin whitening. In this study, the whitening effect of α-arbutin by inhibiting tyrosinase activity and alleviating the photoaging effect induced by UVB are investigated. The results indicate that α-arbutin can inhibit skin inflammation, and its effectiveness is positively correlated with concentration. Moreover, α-arbutin can reduce the skin epidermal thickness, decrease the number of inflammatory cells, and down-regulate the expression levels of IL-1ß, IL-6 and TNF-α, which are inflammatory factors. It also promotes the expression of COL-1 collagen, thus playing an important role in anti-inflammatory action. Network pharmacology, metabolomics and transcriptomics further confirm that α-arbutin is related to the L-tyrosine metabolic pathway and may interfere with various signaling pathways related to melanin and other photoaging by regulating metabolic changes. Therefore, α-arbutin has a potential inhibitory effect on UVB-induced photoaging and possesses a whitening effect as a cosmetic compound.

Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins - A case study for Mefp-3 from Mytilus edulis.

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

Development of an Innovative Biosensor Based on Graphene/PEDOT/Tyrosinase for the Detection of Phenolic Compounds in River Waters.

Phenolic compounds, originating from industrial, agricultural, and urban sources, can leach into flowing waters, adversely affecting aquatic life, biodiversity, and compromising the quality of drinking water, posing potential health hazards to humans. Thus, monitoring and mitigating the presence of phenolic compounds in flowing waters are essential for preserving ecosystem integrity and safeguarding public health. This study explores the development and performance of an innovative sensor based on screen-printed electrode (SPE) modified with graphene (GPH), poly(3,4-ethylenedioxythiophene) (PEDOT), and tyrosinase (Ty), designed for water analysis, focusing on the manufacturing process and the obtained electroanalytical results. The proposed biosensor (SPE/GPH/PEDOT/Ty) was designed to achieve a high level of precision and sensitivity, as well as to allow efficient analytical recoveries. Special attention was given to the manufacturing process and optimization of the modifying elements' composition. This study highlights the potential of the biosensor as an efficient and reliable solution for water analysis. Modification with graphene, the synthesis and electropolymerization deposition of the PEDOT polymer, and tyrosinase immobilization contributed to obtaining a high-performance and robust biosensor, presenting promising perspectives in monitoring the quality of the aquatic environment. Regarding the electroanalytical experimental results, the detection limits (LODs) obtained with this biosensor are extremely low for all phenolic compounds (8.63 × 10-10 M for catechol, 7.72 × 10-10 M for 3-methoxycatechol, and 9.56 × 10-10 M for 4-methylcatechol), emphasizing its ability to accurately measure even subtle variations in the trace compound parameters. The enhanced sensitivity of the biosensor facilitates detection and quantification in river water samples. Analytical recovery is also an essential aspect, and the biosensor presents consistent and reproducible results. This feature significantly improves the reliability and usefulness of the biosensor in practical applications, making it suitable for monitoring industrial or river water.

Artificial Intelligence-Assisted Optimization of Antipigmentation Tyrosinase Inhibitors: De Novo Molecular Generation Based on a Low Activity Lead Compound.

Artificial intelligence (AI) de novo molecular generation is a highly promising strategy in the drug discovery, with deep reinforcement learning (RL) models emerging as powerful tools. This study introduces a fragment-by-fragment growth RL forward molecular generation and optimization strategy based on a low activity lead compound. This process integrates fragment growth-based reaction templates, while target docking and drug-likeness prediction were simultaneously performed. This comprehensive approach considers molecular similarity, internal diversity, synthesizability, and effectiveness, thereby enhancing the quality and efficiency of molecular generation. Finally, a series of tyrosinase inhibitors were generated and synthesized. Most compounds exhibited more improved activity than lead, with an optimal candidate compound surpassing the effects of kojic acid and demonstrating significant antipigmentation activity in a zebrafish model. Furthermore, metabolic stability studies indicated susceptibility to hepatic metabolism. The proposed AI structural optimization strategies will play a promising role in accelerating the drug discovery and improving traditional efficiency.

Considerations about the inhibition of monophenolase and diphenolase activities of tyrosinase. Characterization of the inhibitor concentration which generates 50 % of inhibition, type and inhibition constants. A review.

Tyrosinase is a copper oxidase enzyme which catalyzes the first two steps in the melanogenesis pathway, L-tyrosine to L-dopa conversion and, then, to o-dopaquinone and dopachrome. Hypopigmentation and, above all, hyperpigmentation issues can be originated depending on their activity. This enzyme also promotes the browning of fruits and vegetables. Therefore, control of their activity by regulators is research topic of great relevance. In this work, we consider the use of inhibitors of monophenolase and diphenolase activities of the enzyme in order to accomplish such control. An experimental design and data analysis which allow the accurate calculation of the degree of inhibition of monophenolase activity (iM) and diphenolase activity (iD) are proposed. The IC50 values (amount of inhibitor that causes 50 % inhibition at a fixed substrate concentration) can be calculated for the two activities and from the values of IC50M (monophenolase) and IC50D(diphenolase). Additionally, the strength and type of inhibition can be deduced from these values. The data analysis from these IC50D values allows to obtain the values of [Formula: see text] or [Formula: see text] , or and [Formula: see text] from the values of IC50M. In all cases, the values of the different must satisfy their relationship with IC50M and IC50D.

Isolation, Purification and Tyrosinase Inhibitory Activity of Anthocyanins and Their Novel Degradation Compounds from Solanum tuberosum L.

To explore the composition of anthocyanins and expand their biological activities, anthocyanins were systematically isolated and purified from tubers of Solanum tuberosum L., and their tyrosinase inhibitory activity was investigated. In this study, two new anthocyanin degradation compounds, norpetanin (9) and 4-O-(p-coumaryl) rhamnose (10), along with 17 known anthocyanins and their derivatives, were isolated and purified from an acid-ethanolic extract of fresh purple potato tubers. Their structures were elucidated via 1D and 2D NMR and HR-ESI-MS and compared with those reported in the literature. The extracts were evaluated for anthocyanins and their derivatives using a tyrosinase inhibitor screening kit and molecular docking technology, and the results showed that petanin, norpetanin, 4-O-(p-coumaryl) rhamnose, and lyciruthephenylpropanoid D/E possessed tyrosinase inhibitory activity, with 50% inhibiting concentration (IC50) values of 122.37 ± 8.03, 115.53 ± 7.51, 335.03 ± 12.99, and 156.27 ± 11.22 µM (Mean ± SEM, n = 3), respectively. Furthermore, petanin was validated against melanogenesis in zebrafish; it was found that it could significantly inhibit melanin pigmentation (p < 0.001), and the inhibition rate of melanin was 17% compared with the normal group. This finding may provide potential treatments for diseases with abnormal melanin production, and high-quality raw materials for whitening cosmetics.

Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

Probiotics from kefir: Evaluating their immunostimulant and antioxidant potential in the carpet shell clam (Ruditapesdecussatus).

This study aimed to investigate the impact of incorporating kefir into the diet on biometric parameters, as well as the immune and antioxidant responses of the carpet shell clam (Ruditapes decussatus) after an experimental infection by Vibrio alginolyticus. Clams were divided into a control group and a treated group. The control group was fed on spirulina (Arthrospira platensis) alone. While, the treated group was fed on spirulina supplemented with 10% dried kefir. After 21 days, clams were immersed in a suspension of V. alginolyticus 5 × 105 CFU mL -1 for 30 min. Seven days after experimental infection, survival was 100% in both groups. The obtained results showed a slight increase in weight and condition index in clams fed with kefir-supplemented diet for 21 days compared to control clams. Regarding antioxidant responses, the treated group showed higher superoxide dismutase activity compared to the control group. However, the malondialdehyde level was lower in the treated clams than in the control. In terms of immune parameters, the treated group showed slightly elevated activities of phenoloxidase, lysozyme and alkaline phosphatase, whereas a decreased lectin activity was observed compared to the control group. The obtained results suggest that kefir enhanced both the antioxidant and immune response of infected clams.

SERS-Based Microneedle Biosensor for In Situ and Sensitive Detection of Tyrosinase.

Tyrosinase (TYR) emerges as a key enzyme that exerts a regulatory influence on the synthesis of melanin, thereby assuming the role of a critical biomarker for the detection of melanoma. Detecting the authentic concentration of TYR in the skin remains a primary challenge. Distinguished from ex vivo detection methods, this study introduces a novel sensor platform that integrates a microneedle (MN) biosensor with surface-enhanced Raman spectroscopy (SERS) technology for the in situ detection of TYR in human skin. The platform utilized dopamine (DA)-functionalized gold nanoparticles (Au NPs) as the capturing substrate and 4-mercaptophenylboronic acid (4-MPBA)-modified silver nanoparticles (Ag NPs) acting as the SERS probe. Here, the Au NPs were functionalized with mercaptosuccinic acid (MSA) for DA capture. In the presence of TYR, DA immobilized on the MN is preferentially oxidized to dopamine quinone (DQ), a process that results in a decreased density of SERS probes on the platform. TYR concentration was detected through variations in the signal intensity emitted by the phenylboronic acid. The detection system was able to evaluate TYR concentrations within a linear range of 0.05 U/mL to 200 U/mL and showed robust anti-interference capabilities. The proposed platform, integrating MN-based in situ sensing, SERS technology, and TYR responsiveness, holds significant importance for diagnosing cutaneous melanoma.

Melanin Inhibitory Effect of Tuber himalayense Isolated in Incheon, Korea.

There has been a growing interest in skin beauty and antimelanogenic products. Melanogenesis is the process of melanin synthesis whereby melanocytes are activated by UV light or hormone stimulation to produce melanin. Melanogenesis is mediated by several enzymes, such as tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), and TRP-2. In this study, we investigated the effect of Tuber himalayense extract on melanin synthesis in α-melanocyte-stimulating hormone (α-MSH)-treated B16F10 melanoma cells. We confirmed that T. himalayense extract was not toxic to α-MSH-treated B16F10 melanoma cells and exhibited a significant inhibitory effect on melanin synthesis at concentrations of 25, 50, and 100 µg/ml. Additionally, the T. himalayense extract inhibited melanin, TRP-1, TRP-2, tyrosinase, and MITF, which are enzymes involved in melanin synthesis, in a concentration-dependent manner. Furthermore, T. himalayense extract inhibited the mitogen-activated protein kinase (MAPK) pathways, such as extracellular signal-regulated kinase-1/2 (ERK), c-Jun N-terminal kinase (JNK), and p38. Therefore, we hypothesized that various components of T. himalayense extract affect multiple factors involved in melanogenesis in B16F10 cells. Our results indicate that T. himalayense extract could potentially be used as a new material for preparing whitening cosmetics.

In vitro Evaluation of Skin Related Enzyme Inhibitory Effect and Emulgel Formulation Development Studies of Onobrychis Argyrea subsp. Argyrea with Phytochemical Analysis.

Species of Onobrychis have been used to treat skin disorders such as wounds and cuts in folk medicine and Onobrychis argyrea subsp. argyrea (OA) commonly known as 'silvery sainfoin', is a member of this genus. In this study, it was aimed to investigate the skin-related biological activities and phytochemical characterization of OA. Moreover, an emulgel formulation was developed from the main methanolic extract of the plant (OAM). Initially, to identifiy of the active fractions, aerial parts of the plant material was extracted with methanol and fractionated by n-hexane, chloroform, ethyl acetate and n-butanol, respectively. Antioxidant activity was determined by CUPRAC, TOAC, FRAP and DPPH assays. Thereafter, the inhibition potential of OAM, novel formulation and all fractions was measured against elastase, collagenase, tyrosinase and hyaluronidase enzymes. OAM was analyzed and characterized by LC/MS-MS. The major bioactive flavonoids which are rutin and isoquercetin were measured and compared as qualitative and quantitative via high performance thin layer chromatography (HPTLC) analysis in OAM and fractions. The results showed that extracts of OA can be a potential cosmeceutical agent for skin related problems.

Chemical Composition of Artemisia Scoparia and Their Bioactivities.

Three undescribed sesquiterpenes (1-3), two enantiomeric pairs of monoterpenes (4a/4b-5a/5b), one alkyne (6), two known alkynes (7-8) and eight known coumarins (9-16) were isolated from the aerial parts extracts of Artemisia scoparia. The structures of these compounds were fully elucidated by their 1D and 2D NMR, HRESIMS spectral data analyses, and comparison with literature. The absolute configurations of compounds were determined by single-crystal X-ray crystallography (1), a comparison of experimental and calculated electronic circular dichroism (ECD) data (2-6). 15 showed moderate inhibitory activity with the NO release in LPS-induced RAW264.7 cells. 9-16 showed varying degrees of promoting melanogenesis and tyrosinase activity in B16 cells.

Synthesis of eco-friendly layered double hydroxide and nanoemulsion for jasmine and peppermint oils and their larvicidal activities against Culex pipiens Linnaeus.

Mosquito-borne diseases represent a growing health challenge over time. Numerous potential phytochemicals are target-specific, biodegradable, and eco-friendly. The larvicidal activity of essential oils, a jasmine blend consisting of Jasmine oil and Azores jasmine (AJ) (Jasminum sambac and Jasminum azoricum) and peppermint (PP) Mentha arvensis and their nanoformulations against 2nd and 4th instar larvae of Culex pipiens, was evaluated after subjecting to different concentrations (62.5, 125, 250, 500, 1000, and 2000 ppm). Two forms of phase-different nanodelivery systems of layered double hydroxide LDH and oil/water nanoemulsions were formulated. The synthesized nanoemulsions showed particle sizes of 199 and 333 nm for AJ-NE and PP-NE, with a polydispersity index of 0.249 and 0.198, respectively. Chemical and physiochemical analysis of TEM, SEM, XRD, zeta potential, drug loading capacity, and drug release measurements were done to confirm the synthesis and loading efficiencies of essential oils' active ingredients. At high concentrations of AJ and PP nanoemulsions (2000 ppm), O/W nanoemulsions showed higher larval mortality than both LDH conjugates and crude oils. The mortality rate reached 100% for 2nd and 4th instar larvae. The relative toxicities revealed that PP nanoemulsion (MA-NE) was the most effective larvicide, followed by AJ nanoemulsion (AJ-NE). There was a significant increase in defensive enzymes, phenoloxidase, and α and ß-esterase enzymes in the treated groups. After treatment of L4 with AJ, AJ-NE, PP, and PP-NE, the levels of phenoloxidase were 545.67, 731.00, 700.00, and 799.67 u/mg, respectively, compared with control 669.67 u/mg. The activity levels of α-esterase were 9.71, 10.32, 8.91, and 10.55 mg α-naphthol/min/mg protein, respectively. It could be concluded that the AJ-NE and PP-NE nanoformulations have promising larvicidal activity and could act as safe and effective alternatives to chemical insecticides.

Molecular Modeling of the Multiple-Substrate Activity of the Human Recombinant Intra-Melanosomal Domain of Tyrosinase and Its OCA1B-Related Mutant Variant P406L.

Tyrosinase serves as the key enzyme in melanin biosynthesis, catalyzing the initial steps of the pathway, the hydroxylation of the amino acid L-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA), followed by the subsequent oxidation of L-DOPA into dopaquinone (DQ), and it facilitates the conversion of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into 5,6-indolequinone-2-carboxylic acid (IQCA) and 5,6-dihydroxy indole (DHI) into indolequinone (IQ). Despite its versatile substrate capabilities, the precise mechanism underlying tyrosinase's multi-substrate activity remains unclear. Previously, we expressed, purified, and characterized the recombinant intra-melanosomal domain of human tyrosinase (rTyr). Here, we demonstrate that rTyr mimics native human tyrosinase's catalytic activities in vitro and in silico. Molecular docking and molecular dynamics (MD) simulations, based on rTyr's homology model, reveal variable durability and binding preferences among tyrosinase substrates and products. Analysis of root mean square deviation (RMSD) highlights the significance of conserved residues (E203, K334, F347, and V377), which exhibit flexibility during the ligands' binding. Additionally, in silico analysis demonstrated that the OCA1B-related P406L mutation in tyrosinase substantially influences substrate binding, as evidenced by the decreased number of stable ligand conformations. This correlation underscores the mutation's impact on substrate docking, which aligns with the observed reduction in rTyr activity. Our study highlights how rTyr dynamically adjusts its structure to accommodate diverse substrates and suggests a way to modulate rTyr ligand plasticity.

Tyrosinase-Modified UHMW SELP Polymers as Wet and Underwater Adhesives to Achieve Multi-interface Adhesion.

The presence of a hydration layer in humid and underwater environments challenges adhesive-substrate interactions and prevents effective bonding, which has become a significant obstacle to the development of adhesives in the industrial and biomedical fields. In this study, ultrahigh-molecular-weight (UHMW) silk-elastin-like proteins (SELP) with 3,4-dihydroxyphenylalanine (DOPA) converted from tyrosine residues by tyrosinase exhibited excellent adhesive properties on different interfaces, such as glass, aluminum, wood, polypropylene sheets, and pigskin, under both dry and wet conditions. Additionally, by incorporating trace amounts of cross-linking agents like Fe3+, NaIO4, and tris(hydroxymethyl) phosphine (THP), the mussel-inspired adhesives maintained a stable and excellent adhesion, broadening the conditions of application. Notably, the UHMW SELP adhesive exhibited remarkable underwater adhesion properties with a shear strength of 0.83 ± 0.17 MPa on glass. It also demonstrated good adhesion to biological tissues including the kidney, liver, heart, and lungs. In vitro cytocompatibility testing using L929 cells showed minimal toxicity, highlighting its potential application in the biomedical field. The sustainable, cytocompatible, cost-effective, and highly efficient adhesive provides valuable insights for the design and development of a new protein-based underwater adhesive for medical application.

Tyrosinase-Based Proximity Labeling in Living Cells and In Vivo.

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries.

The biotic effects of lignite on humic acid components conversion during chicken manure composting.

Targeted regulation of composting to convert organic matter into humic acid (HA) holds significant importance in compost quality. Owing to its low carbon content, chicken manure compost often requires carbon supplements to promote the humification progress. The addition of lignite can increase HA content through biotic pathways, however, its structure was not explored. The Parallel factor analysis revealed that lignite can significantly increase the complexity of highly humified components. The lignite addition improved phenol oxidase activity, particularly laccase, during the thermophilic and cooling phases. The abundance and transformation functions of core bacteria also indicated that lignite addition can influence the activity of microbial transformation of HA components. The structural equation model further confirmed that lignite addition had a direct and indirect impact on enhancing the complexity of HA components through core bacteria and phenol oxidase. Therefore, lignite addition can improve HA structure complexity during composting through biotic pathways.

A turn-on fluorescence probe for imaging tyrosinase at the wound site in broken tail of zebrafish.

Tyrosinase (TYR) is a copper-containing oxidase that affects the synthesis of melanin in the human body, which is regulate to the pigmentation of the skin. Nevertheless, abnormal expression of TYR can lead to albinism, vitiligo and other skin diseases. Excessive accumulation of TYR is a marker of melanoma cancer and an important factor leading to pigmentation during wound healing, freckles and browning of fruits and vegetables. Efficient tracking of TYR is of significance for studying its pathophysiological mechanism. Herein, we synthesized a benzindole-based fluorescent probe Pro-OH to detect TYR in living cells and zebrafish. The probe displayed a high selectivity and sensitivity in distinguishing TYR from other analytes with the low detection limit of 1.024 U/mL. Importantly, Pro-OH was successfully used to imagine TYR at the wound site of broken tail of zebrafish.

Molecular mechanisms underlying Tao-Hong-Si-Wu decoction treating hyperpigmentation based on network pharmacology, Mendelian randomization analysis, and experimental verification.

CONTEXT: Hyperpigmentation, a common skin condition marked by excessive melanin production, currently has limited effective treatment options. OBJECTIVE: This study explores the effects of Tao-Hong-Si-Wu decoction (THSWD) on hyperpigmentation and to elucidate the underlying mechanisms. MATERIALS AND METHODS: We employed network pharmacology, Mendelian randomization, and molecular docking to identify THSWD's hub targets and mechanisms against hyperpigmentation. The Cell Counting Kit-8 (CCK-8) assay determined suitable THSWD treatment concentrations for PIG1 cells. These cells were exposed to graded concentrations of THSWD-containing serum (2.5%, 5%, 10%, 15%, 20%, 30%, 40%, and 50%) and treated with α-MSH (100 nM) to induce an in vitro hyperpigmentation model. Assessments included melanin content, tyrosinase activity, and Western blotting. RESULTS: ALB, IL6, and MAPK3 emerged as primary targets, while quercetin, apigenin, and luteolin were the core active ingredients. The CCK-8 assay indicated that concentrations between 2.5% and 20% were suitable for PIG1 cells, with a 50% cytotoxicity concentration (CC50) of 32.14%. THSWD treatment significantly reduced melanin content and tyrosinase activity in α-MSH-induced PIG1 cells, along with downregulating MC1R and MITF expression. THSWD increased ALB and p-MAPK3/MAPK3 levels and decreased IL6 expression in the model cells. DISCUSSION AND CONCLUSION: THSWD mitigates hyperpigmentation by targeting ALB, IL6, and MAPK3. This study paves the way for clinical applications of THSWD as a novel treatment for hyperpigmentation and offers new targeted therapeutic strategies.