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Indian Journal of Pharmaceutical Education and ResearchAn Official Publication of Association of Pharmaceutical Teachers of India (APTI)
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Analysis on the Active Components and Mechanisms of Action of Ecliptae Herba Intervention on Melanin Synthesis Applying UHPLC-Q-Orbitrap HRMS and Network Pharmacology in Combination with Molecular Docking Screening

1, 1,2★★ Corresponding author

  1. 1School of Chinese Materia Medica, Guangdong Pharmaceutical University, Guangzhou, CHINA.
  2. 2Key Laboratory of Digital Quality Evaluation of Chinese Materia Medica of SATCM (YGZXKT2024313), Guangzhou, CHINA.

CORRESPONDENCE

Shuyu Guan

School of Chinese Materia Medica, Guangdong Pharmaceutical University, Guangzhou, CHINA.

gdpugsy@126.com

Received: 17-04-2026; Revised: 09-06-2026; Accepted: 26-08-2026.

Volume 61, Issue 1 · pp. 399-410 · 2026 · DOI: 10.5530/ijper.20271427

ABSTRACT

Background: White hair, a prevalent condition associated with melanin metabolism, has become increasingly common among the modern young population. Ecliptae Herba has been used since ancient time for white hair treatment, but its active components and underlying mechanisms of action remain to be explored.Materials and Methods: The chemical compositions of Ecliptae Herb were analyzed using liquid chromatography-mass spectrometry and UHPLC-Q-Orbitrap HRMS. To evaluate its effects on melanin synthesis, a zebrafish model was employed and treated by different concentrations of Ecliptae Herba extract. The TCMSP and PharmMapper platform were utilized to identify the chemical compounds of Ecliptae Herba and its potential targets. Additionally, melanin metabolism-related targets were screened using the GeneCards and OMIM databases and integrated to construct a drug-target-disease network. Finally, the structural data of the target proteins were obtained from Uniprot and PDB databases and pre-processed by PyMOL 2.6.0 software for molecular docking using Autodock Tool.Results: UHPLC-Q-Orbitrap HRMS screened a total of 34 components of Ecliptae Herb. Network pharmacology and molecular docking analysis showed that linarin, pratensin, demethylwedelolactone, echinocystic acid, Ecliptasaponin A, and quercetin were the active compounds. These components affected the drug metabolism, cytochrome P450, EGFR tyrosine kinase inhibitor activity, PPAR signaling pathway, and other signaling pathways by regulating the targets, such as GSTP1, PPARA, IGF1, PAH, NR1I2, and MAPK1, suggesting a potential mechanism underlying the effect of Ecliptae Herba on melanin metabolism.Conclusion: This study elucidated the multi-component, multi-target, and multi-pathway mechanisms by which Ecliptae Herba regulated melanin metabolism, contributing to future research on developing novel products for growing black hair.

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INTRODUCTION

The occurrence of white hair, characterized by the abnormal whitening of partial or complete hair strands, is closely related to human health.1 Melanin is a high molecular weight polymer with an uncertain molecular structure. Synthesis of melanin is realized through a series of reactions in the melanosome of melanocytes. Firstly, tyrosine is catalyzed by Tyrosinase (TYR) to produce L-DOPA, which is further oxidized to obtain DOPAquinone.2 Eumelanin and zeaxanthin are two types of melanin generated by dopaoquinone through different pathways, and the colors of these two types of melanin are also different. Tyrosinase-Related Protein-1 (TRP1) and TRP2 play important role in the formation of melanin.3–5 In recent years, the increasing incidence of white hair among younger population has emerged as a significant dermatological prolem,6 especially among young and middle-age individuals. According to research, the effects of traditional Chinese and Western medical products for black hair on the market are not satisfactory.7,8 Current therapeutic products for white hair, exemplified by minoxidil in Western medicine, are limited by its low efficacy and inconvenience in daily usage. Though hair dyeing has become a solution for white hair, this approach may potentially compromise hair quality and scalp health.9 Therefore, it is urgent to discover and produce safe drugs that control white hair problems through oral or external use.

According to traditional Chinese Medicine (TCM), the pathogenesis of melanin deficiency is attributed to the factors such as qi and blood blockage, liver qi stagnation, kidney yin deficiency, and spleen dysfunction due to overexertion. TCM with blood nourishing, blood activating, and qi-regulating effects is used to restore the formation of melanin by nourishing the blood, promoting blood circulation, and relieving liver qi stagnation.10,11 Ecliptae Herba, the dry aerial part of the Asteraceae plant Eclipta prolata L., is mainly used to treat liver and kidney yin deficiency, loose teeth, premature graying of the beard, dizziness and tinnitus, soreness and weakness of the waist and knees, yin deficiency with blood heat, vomiting of blood, bleeding, hematuria, blood dysentery, collapse and bleeding, and external bleeding.12 Modern pharmacological studies have shown that Ecliptae Herba has liver protective, anti-inflammatory, melanin-promoting, antioxidant and some other effects. Clinical studies also validated that Ecliptae Herba can be used to treat symptoms such as bleeding, white hair, vitiligo, etc.1314,15 Previous study investigated the effects of ethanol extract from Ecliptae Herba on seven cancer cell lines, and discovered that the growth and movement of breast cancer cells were significantly restricted.16 Nevertheless, the specific mechanism by which Ecliptae Herba influences human melanin metabolism remains unclear, necessitating further investigation into the mechanism underlying the effects of Ecliptae Herba on the prevention and treatment of white hair.

To elucidate the material basis and molecular mechanism underlying the effect of Ecliptae Herba on enhancing melanin production, this study used UHPLC-Q-Orbitrap HRMS to identify the components of Ecliptae Herba and employed a zebrafish juvenile model for verification. The underlying mechanism was explored by applying network pharmacology and molecular docking methods. As the first study to investigate the effects of Ecliptae Herba on melanin metabolism, the current research provided valuable insights into the therapeutic potential of Ecliptae Herba for treating white hair, laying a solid foundation for further investigation on Ecliptae Herba.

MATERIALS AND METHODS

Experimental reagents and instruments

Ethanol was provided by Xilong Science Co., Ltd., (Shantou, China, lot No. 24030201). Acetonitrile was provided by Anaqua Anke Chemical (Cleveland, America, lot No. Y24AC0110K). Formic acid was provided by Tianjin Kemio Chemical Reagent Co., Ltd., (Tianjin, China, lot No. 20220710). Tricaine (lot No. C12590064) and 1-phenyl-2-thiourea (PTU, lot No. N816213) were provided by Shanghai McLean Biochemical Technology Co., Ltd., (Shanghai, China). Dimethyl Sulfoxide (DMSO) was provided by Tianjin Zhiyuan Chemical Reagent Co., Ltd., (Tianjin, China, lot No. 20190111). Melanocyte Stimulating Hormone (α-MSH) was provided by GloBio Technology (Shanghai, China, lot No. GC14374). UHHPLC-Q-Orbitrap HRMS was purchased from Thermo Scientific (Waltham, MA, USA).

Medicinal plant materials

The dried aerial part of Eclipta prostrata L. was purchased from Anhui Tonghuatang Traditional Chinese Medicine Decoction Technology Co., Ltd., in 2023, with batch number No. 20230901.

Preparation of Ink Lotus Samples

Sample 1: Approximately 1 g of the powder of Ecliptae Herba (sieved through the third sieve) was accurately weighted, and placed in a conical flask with a stopper. After adding 50 mL of 70% ethanol, the total volume was weighted. The mixture was subjected to heat reflux for 1 hr and allowed to cool down. After weighing, weight loss was compensated with 70% ethanol. Next, the mixture was well shaken, filtered, and the filtrate was collected as the final product.

Sample 2: Precisely 5 g of Ecliptae Herba powder (sieved through a 65-mesh sieve) was soaked in 50 mL of cold pure water for 60 min, then boiled for 60 min, then cooled down and filtered. The medicine residue was added again with 50 mL of pure water and boiled for 30 min. The filtrate was combined, heated and concentrated, and then diluted to 100 mL using a 100 mL volumetric flask. The solution was to be sociated for 30 min to prepare a mother liquor at a concentration of 50g/L,17 followed by sterilization in a high-temperature sterilization pot and storage in a refrigerator for later use. For the experiments, the mother liquor diluted with 0.1% DMSO embryo culture water to the desired concentration was used as the treatment group of drought lotus, while the control group contained an equal volume of 0.1% DMSO embryo culture water solution.

Testing conditions

Chromatographic conditions: Using a Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 μm) with a flow rate of 0.3 mL/min and a column temperature of 30ºC, gradient elution was performed using acetonitrile (A) -0.1% formic acid (B) as the mobile phase, with an injection volume of 1 μL. The specific elution gradient program was as follows: 0-7 min, 5% A -13% A; 7-25 min, 13% A-30% A; 25-30 min, 30% A-45% A; 3040 min, 45% A-95% A; 40-45 min, 95% A-95% A; 45-50 min, 95% A-5% A.

Mass spectrometry conditions: Analysis was conducted using a Heated Electrospray Ionization (H-ESI) in both positive and negative ion modes. The parameters of the instrument were as follows: nitrogen as the carrier gas is, with a sheath gas pressure and auxiliary air pressure at of 3 5 MPa and 1 0 MPa, respectively; spray voltage set at 3.80 kV(+), 2.50 kV(-); capillary temperature and auxiliary heating temperature set at 350ºC and 350ºC, respectively; resolution set at 120000 and Normalized Collision Energy (NCE) set at 20, 40, 60 eV. The scanning range was between m/z 100 and 1500.

Drug administration and sample collection

Four days post-fertilization (dpf), zebrafish fries were obtained through normal fertilization and exposed to 0.03 g/L PTU solution for 24 hr. The fries were divided into six groups: control group, model group (PTU group), positive control group (α-MSH group), and three treatment groups (treated by 0.5 g/L, 1 g/L, and 2 g/L PTU solution). Each group of 20 juvenile fishes were placed in a 6-well plate, with three treatment groups administered with PTU solution at the concentrations of 0.5 g/L, 1 g/L, and 2 g/L. In addition, a positive control group was processed with 0.01 g/L α-MSH (positive drug), according to literature. The model group was treated by PTU after 24 hr and continuously processed until the end of the experiment,18 which was set at 7dpf (zebrafish fry can no longer absorb drugs through the skin and gills starting from 7dpf). At the end of the experiment, 5 fries from each group were anesthetized with 0.01% lidocaine solution and observed using a stereomicroscope. The images were then processed using ImageJ-win64 software.

Target collection

The chemical constituents of Ecliptae Herba were searched in The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) using the keyword "Ecliptae Herba" under the initial screening criteria of Oral Bioavailability (OB) ≥ 30% and Drug-Like index (DL) ≥0.18. Potential molecular targets of the identified chemical constituents were then predicted using PharmMapper (http://www.lilab-ecust.cn/pharmmapper/) database. Subsequently, the resulting targets were processed by UniProt database (https://www.uniprot.org/) to standardize the gene names and establish a database of monomeric active ingredients and target genes.

Targets related to melanin metabolism, including melanin, melanogenesis, melanin biosynthesis, melanin metabolism, and melanin pigmentation were collected from the GeneCards (http://www.genecards.org/) and OMIM (https://omim.org/) databases. After removing duplicate data, a dataset of targets linked to melanin metabolism was combined for subsequent analysis.

Systems Pharmacology Database

Protein-Protein Interaction (PPI) network

The active component targets and disease targets were processed using Venny (https://bioinfogp.cnb.csic.es/tools/venny/index. html) tool to obtain the intersection targets. The common targets were imported into STRING (http://string-db.org) platform for PPI network construction, under the species of Homo sapiens. Cytoscape 3.9.1 software was employed to develop a network of the target genes in the active components of Ecliptae Herba.19,20

Enrichment analysis

Using the Metascape tool (https://metascape.org/gp/index.html#/main/step1), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted on the 48 common targets between the chemical components of the herb and disease-related targets. Enrichment entries for the three GO terms were obtained under a significance threshold of p<0.05 as the screening criterion.21,22

Molecular docking

Based on the degree value, the top-ranked key targets and key components were selected from both the PPI network and the "Ecliptae Herba Single Ingredient-Active Ingredient-Action Target" network diagram for molecular docking. The mol2 format structure of the active ingredients were downloaded from the TCMSP database, and the pdb format structure of the target protein was obtained from the Uniprot and PDB database. Using PyMOL2.6.0 and Autodock Tool-v1.5.7 software,23 dehydration, hydrogenation, ligand removal, and charge removal on the target protein and target molecules were performed. Finally, Autodock Tool-v1.5.7 was employed to dock the processed active molecules with the target protein. Binding energy is a parameter that reflects the interaction strength between pharmacological molecules and their target proteins. Specifically, binding energy<-4.25kcal/ mol indicates that the ligand may bind to the receptor, binding energy<-5.0kcal/mol indicates a relatively high binding activity, and binding energy<-7.0kcal/mol indicates an excellent binding activity.24,25

Statistical analysis

The images were processed using Image J-win64 software, and the data were analyzed using GraphPad Prism 8.0.1 and Excel 2019 software. The experimental data were expressed as ± s, and one-way analysis of variance was used to compare differences between groups. p<0.05 indicated a significant difference.

RESULTS

Identification of the components of Ecliptae Herba

Analysis of the main molecular chromatogram of Eclipta Herba in positive and negative ion modes identified 34 compounds (Figure 1). In positive ion mode, 6 compounds, namely, wedelolactone, Demethylwedelolactone, chlorogenic acid, isoquercetin, D,L-glutamine, L-glutamate, were identified (Table S1). In negative ion mode, 32 compounds were identified, such as wedelolactone, echinocystic acid, quercetin, luteolin, Ecliptasaponin A/D, Demethylwedelolactone, pratensein, chlorogenic acid, isoquercetin, apigenin, quinic acid, caffeic acid, Glucose, Shikimic acid, etc. (Table S2).

Potential effects of Ecliptae Herba on melanin metabolism

We used zebrafish fry to further explore the potential effects of Ecliptae Herba on melanin metabolism. As shown in Figures 2A-2D, 0.5 g/L of Ecliptae Herba significantly reduced gray values of eye and eye end to neck when compared to the treatment with α-MSH. These data revealed that Ecliptae Herba may possess a potential capacity to promote melanin formation and modulate pigmentation in specific body regions.

Construction of component-target network diagram and PPI network diagram

Using Venny 2.1.0 software, a total of 51 common targets in the intersection between 437 chemical targets of Ecliptae Herba and 652 targets related to melanin metabolism were obtained (Figure 3A). These 51 common targets were imported into Cytoscape 3.9.1 software to generate a component-target network diagram for Ecliptae Herba and melanin metabolism (Figure 3B). The network contained 63 nodes and 487 edges. According to the degree value, the top five chemical components in the network diagram were Pratensein, Wedelolactone, butin, acacetin, and Linarin, and the top five targets were GSTP1, PAH, NR1I2, MAPK1, and ADH1B. After loading the 51 common targets into the STRING platform, the interaction relationships between the 51 potential targets were obtained based on the condition of confidence>0.4 (Figure 3C).

Enrichment analysis

GO enrichment analysis showed that the therapeutic targets of Ecliptae Herba on melanin metabolism mainly exerted their effects through Biological Process (BP) such as hormone response, hormone level regulation, and MARK cascade regulation. The Cellular Component (CC) were mainly related to acrosome vesicles, receptor complexes, and mitochondrial matrix. The activity of nuclear receptors, monooxygenase activity, insulin-like growth factor receptor binding, protein serine/ threonine/tyrosine kinase activity, etc. were the main Molecular Function (MF) involved. These results were shown in Figure 4A and Table S3. KEGG pathway enrichment analysis showed that the signaling pathways significantly affected by Ecliptae Herba on melanin metabolism were insulin resistance pathway, EGFR tyrosine kinase inhibitor activity, etc. (Figure 4B, Table S4).

Molecular docking

The heatmap of the docking result was shown in Figure 4C. Specifically, Linarin showed a high binding affinity with GSTP1, PPARA, and IGF1; pratensin showed a high binding affinity with PPARA; Demethylwedelolactone showed a high binding affinity with GSTP1; Echinocystic acid showed a high binding affinity with GSTP1, PAH, NR1I2, MAPK1, IGF1; Ecliptaaponin A showed a high binding affinity with PPARA and IGF1; quercetin showed a high binding affinity with GSTP1.

Figure 1 Ion diagram of Ecliptae Herba using UHPLC-Q-Orbitrap HRMS. (A) Total ion current diagram of Ecliptae Herba in positive ion mode. (B) Total ion current diagram of Ecliptae Herba in negative ion mode.

DISCUSSION

The Chinese Pharmacopoeia currently considers pyrethroid as the indicator component of Ecliptae Herba, but a single indicator cannot fully represent the quality of traditional Chinese medicine. This study investigated the effects of Ecliptae Herba on melanin synthesis using UHPLC-Q-Orbitrap HRMS, network pharmacology, and molecular docking. Employing a zebrafish model, we identified key components such as linarin, pratensein, and quercetin, which targeted pathways such as drug metabolism.

The present results indicated that Ecliptae Herba enhanced melanin production through multiple components and pathways, providing a foundation for developing novel treatments for gray hair.

GSTP1, PAH, NR1I2, MAPK1, and ADH1B were identified as the key targets that played significant roles in melanin formation. GSTP1, a glutathione S-transferase, is involved in detoxification and oxidative stress regulation, which are critical for melanocyte function and melanin synthesis.26 PAH is essential for the conversion of phenylalanine to tyrosine, a precursor for melanin production, highlighting its importance in melanogenesis.27 Nuclear receptor subfamily 1 group I member 2 (NR1I2), also known as PXR, regulates drug metabolism and detoxification pathways and may indirectly influence melanin synthesis.5 Mitogen-Activated Protein Kinase 1 (MAPK1) is a key gene in the MAPK signaling pathway, which regulates melanocyte proliferation and differentiation.3 Alcohol Dehydrogenase 1B (ADH1B) is implicated in retinol metabolism and is linked to melanocyte function and skin pigmentation.6 According to the existing research, Scutellaria baicalensis lactone has been identified as an effective tyrosinase inhibitor that can inhibit the enzyme in a reversible and competitive manner.28 Red clover extract inhibits the pharmacological activity of melanin and also has anti-inflammatory and antioxidant effects.29 Prickling acid can not only inhibit the expression of pro-inflammatory cytokines IL-1β and TNF-α, but can also suppress the activation of NF-Kβ and MAPKs, thereby improving inflammation.30 Butin has a therapeutic effect on vitiligo. Studies have shown that butin treats vitiligo by elevating the expressions of TYR and TRP-1 proteins, reducing the activity of malondialdehyde and cholinesterase.31 Meanwhile, coumarin, isodemethylated coumarin, luteolin, and apigenin have been found to have strong anti-hepatotoxic activity.32 Linarin could inhibit changes in the inflammatory system by reducing NF-Kβ p65/ Inhibitor of Nuclear Factor Kappa-B Kinase Subunit Beta (IKKβ), IL-6, and TNF-α in animal bones, confirming that Linarin can suppress oxidative stress and inflammation and regulate the RANK/RANKL/OPG pathway.33 In recent years, studies reported that inflammatory response plays an important role in the regulation of melanin production.34 In addition, the active components of Ecliptae Herba regulate melanogenesis by binding to key proteins involved in melanogenesis and oxidative stress. Molecular docking results showed that Linarin, Pratensein, Demethylwedelolactone, Echinocystic acid, Ecliptasaponin A, and Quercetin had a high affinity with multiple targets, suggesting that they may act synergistically through multiple targets. For instance, Leis et al. found that the extracts of French maritime pine bark not only downregulates tyrosinase, but also reduces pigmentation-related mediators (including PPARα), which in turn inhibits melanin production in vitro and the pathways associated with hyperpigmentation of the skin.35 Moreover, Hu et al. showed that administering IGF1 at a concentration of 10 ng/ mL significantly influences melanogenesis in alpaca melanocytes via the cAMP pathway. 36 The binding of the active components of Ecliptae Herba to these targets indicates that they may enhance melanin formation through multifaceted interactions, highlighting the potential of Ecliptae Herba as a therapeutic agent for treating melanin-related diseases.

However, there were also some limitations in our study. Firstly, although the zebrafishes were an effective model for pigmentation studies, its melanogenesis mechanism cannot fully mimic the melanin physiological processes in mammals and humans. Future studies should include mammalian systems such as mouse pigmentation model or human melanocyte culture to enhance the clinical applicability of the current findings. Secondly, due to the synergistic action of multiple components in Ecliptae Herba, analysis on the specific function of a single component may be less reliable. Therefore, these components should be isolated and purified, and their individual effects are required to be verified by melanocyte models or enzyme activity assay systems. Finally, although three dose gradients were analyzed in this study, a systematic and complete dose-effect relationship has not been established, making it difficult to clarify the optimal effective dose and safety range of Ecliptae Herba. Therefore, further studies are encouraged to analyze the concentration of the gradients to clarify the effective dose range for the development of medicinal or functional products of Ecliptae Herba for white hair treatment.

Table S1 Identification of Chemical Components of Ecliptae Herba under LC-MS Positive Ion Mode.
Sl. No.tR/ minIonic modeMolecular formulaRelative molecular weighterrorchemical compoundSecondary mass spectrometry fragments(m/z)
1.21.33+C16H10O7315.0496-0.6wedelolactone297.03961,287.05524, 271.06055,269.04453, 243.06551
2.14.68+C15H8O7301.0341-0.5Demethylwedelolactone283.02371,273.03918
3.5.90+C16H18O9355.1022-0.5chlorogenic acid163.03903,135.04422
4.10.74+C21H20O12465.1027-0.0isoquercetin303.04990
5.0.84+C5H10O3N2147.076490.5D,L-glutamine130.02884,102.09171, 84.04498
6.0.84+C5H9O4N148.060490.4L-glutamate130.02884,102.09171, 84.04498
Table S2 Identification of Chemical Components of Ecliptae Herba by LC-MS Negative Ion Mode.
Sl. No.tR/minIonic modeMolecular formulaRelative molecular weighterrorchemical compoundSecondary mass spectrometry fragments(m/z)
1.21.35-C H O 16 10 7313.03564.0wedelolactone298.01205,270.01691,242.02057
2.36.13-C H O 30 48 4471.34843.1echinocystic acid471.34824,407.33243,453.33719
3.20.36-C H O 15 10 7301.03574.7quercetin273.04175,151.00270, 121.02834,107.01261
4.20.37-C H O 15 10 6285.04074.6Luteolin175.03954,151.00276, 133.02829
5.31.78-C H O 36 58 9633.40142.6Ecliptasaponin A/D587.39362,161.04463, 113.02319
6.9.08-C HO 15 8 7299.02004.6Demethylwedelolactone299.01965,271.02518, 227.03479,199.04007, 183.04451
7.24.29-C H O 16 12 6299.05644.7pratensein284.03293
8.5.90-C H O 16 18 9353.08793.2chlorogenic acid191.05544,173.04439, 161.02347
9.10.75-C H O 21 20 12463.08893.8isoquercetin463.08469,301.03522, 300.02789,255.02884
10.19.92-C H O 15 10 5269.04543.5apigenin269.04572,159.04472, 151.00327
11.0.88-CH O 7 12 6191.05531.6Quinic acid191.05537,173.04453, 127.03889
12.6.65-CHO 9 8 4179.03411.1caffeic acid179.03427,135.04401
13.0.83-CH O 6 12 6179.05531.3Glucose161.04459,117.01807, 89.02309,71.01247
14.4.87-CH O 7 10 5173.04471.2Shikimic acid155.03409,136.86159, 129.31128,111.04359
15.0.97-CHO 4 6 5133.0131-0.7Malic acid115.00243,71.01249
16.1.01-CHO 6 8 7191.01891.6Citric acid173.00734,154.99715, 129.01807,103.03882
17.1.62-CHO 7 6 5169.01331.0Gallic acid125.02319,107.01245, 97.02811,69.03322
18.3.41-C H O 13 18 8301.09314.4Tachioside139.00240,123.86333
19.11.82-C H O 13 16 10331.0658-0.6Gallic acid 3-O-β-D-glucopyranoside169.01337,125.02319
20.3.14-CHO 7 6 4153.01830.1Protocatechuic acid109.02820,91.01732, 81.03316
21.6.32-CHO 9 8 3163.03910.7P-Coumaric acid119.04897
22.4.65-C H O 15 20 10359.09884.1Glucosyringic acid197.04495,153.05473, 121.02814
23.15.48-C H O 21 20 10431.09883.6Cosmosiin269.04413,151.00262
24.9.41-C H O 14 18 9329.08834.8Phloracetophene 4’-O-glucoside167.03410,123.04431
25.24.29-C H O 16 12 6299.05644.73’-Hydroxybiochanin A284.03293,256.03690, 255.02956,239.03397, 227.03429
26.37.53-C H O 30 46 4469.33262.93-Oxo-16α-hydroxyolean-12 -en-28-oic acid423.33286,407.33228
27.10.27-C H O 21 20 13479.08343.3Quercetagitin-7-O-glucoside317.03040,271.02435
28.19.50-C H O 15 12 6287.05655.3Isookain151.00266,135.04401
29.12.52-C H O 21 20 12463.08904.0Quercetin-7-O-β-D glucopyranoside301.03552,151.00272
30.16.59-C H O 25 24 12515.119872.93,5-O-dicaffeoylquinic acid191.05542,173.04477, 161.02350,135.04402
31.13.11-C H O 21 20 11447.09373.3Astragalin285.04044,284.03296
32.1.27-C H ON 10 13 5 5282.08454.4Guanosine150.04547,133.06430
Table S3 GO enrichment analysis results.
Sl. No.CategoryNameGene target
1.BPresponse to hormoneAKT1|AKT2|BCHE|CTNNA1|ACE|GBA|GCK| NR3C1|GSK3B|HADH|INSR|KIT|NR3C2|NOS3 |PPARA|PPARD|PPARG|PTPN11|REN|NR1H3
2.BPregulation of protein transportAKT2|GCK|GSK3B|HADH|HMGCR|HRAS|IGF 1|PPARD|PPARG|PRKACA|PTPN11|NR1H2|N R1H4|NR1H3
3.BPreproductive structure developmentAKT1|CTNNA1|CYP19A1|ACE|INSR|KIT|NOS 3|PPARD|PPARG|MAPK1|PTPN11|REN|VDR
4.BPregulation of hormone levelsADH1C|BCHE|CYP2C8|CYP2C9|CYP19A1|A CE|GCK|HADH|PPARD|PPARG|PTPN11|REN |NR1H4
5.BPmonocarboxylic acid metabolic processADH1B|ADH1C|CYP2C8|CYP2C9|GCK|GSTM 1|GSTP1|HADH|IGF1|PPARA|PPARD|PPARG| NR1H4
6.BPorganic hydroxy compound metabolic processADH1C|ALDH2|CYP2C8|CYP2C9|CYP19A1|G BA|HMGCR|IGF1|PAH|PPARD|SPR|NR1H4
7.BPregulation of system processACE|GBA|IGF1|KIT|NOS3|PPARA|PPARG|PRK ACA|PTPN11|REN|NR1H2|NR1H3
8.BPregulation of MAPK cascadeBRAF|GBA|GSTP1|HMGCR|HRAS|IGF1|INSR| KIT|PPARG|MAPK1|PTPN11|REN
9.BPBehaviorAKT1|BCHE|ACE|GBA|HMGCR|INSR|KIT|PP ARA|MAPK1|REN
10.BPresponse to xenobiotic stimulusBCHE|CYP2C8|CYP2C9|ACE|GSTM1|GSTP1|H ADH|PNP|REN|NR1I2
11.BParomatic compound biosynthetic processATIC|FECH|IMPDH1|MTHFD1|PNP|PAH|PPA RG|SPR|VDR|NR1H3|NMNAT1
12.BPnegative regulation of lipid transportAKT1|AKT2|PTPN11|NR1H2|NR1H3
13.BPsmall molecule catabolic processALDH2|CBS|GCK|HADH|NOS3|PNP|PAH|PP ARD
14.CCplasma membrane raftCTNNA1|INSR|NOS3|PRKACA|MAPK1
15.CCvacuolar lumenARSA|GBA|IMPDH1|MAPK1|PSAP
16.CCacrosomal vesicleCTNNA1|KIT|PRKACA
17.CCreceptor complexINSR|KIT|NR3C2|PPARG|VDR|NR1H4|NR1H3
18.CCcell leading edgeAKT1|AKT2|CTNNA1|INSR
19.CCmitochondrial matrixALDH2|FECH|NR3C1|HADH
20.CCcell projection membraneAKT2|ACE|INSR
21.CCspindleAKT1|NR3C1|MAPK1
22.MFnuclear receptor activityNR3C1|NR3C2|PPARA|PPARD|PPARG|NR1H2 |VDR|NR1I2|NR1H4|NR1H3
23.MFoxidoreductase activityADH1C|ALDH2|CBS|CYP2C8|CYP2C9|CYP19 A1|GSTP1|HADH|HMGCR|HSD11B1|IMPDH1 |MTHFD1|NOS3|PAH|SPR
24.MFmonooxygenase activityCYP2C8|CYP2C9|CYP19A1|NOS3|PAH
25.MFinsulin-like growth factor receptor bindingIGF1|INSR|REN
26.MFprotein serine/threonine/tyrosine kinase activityAKT1|AKT2|BRAF|GSK3B|INSR|KIT|PRKACA |MAPK1
27.MFprotein homodimerization activityAKT1|ATIC|CBS|FECH|GSTM1|HSD11B1|KIT
28.MFscaffold protein bindingBRAF|NOS3|PSAP
29.MFamide bindingBCHE|GSTM1|INSR|PPARG|PSAP
30.MFcell adhesion molecule bindingATIC|CTNNA1|IGF1|PTPN11
31.MFhydrolase activity, acting on ester bondsALDH2|ARSA|BCHE|PTPN11
32.MFprotease bindingGSK3B|KIT|PSAP
33.MFsulfur compound bindingCBS|GSTM1|GSTP1
Table S4 KEGG enrichment analysis results.
Sl. No.KEGG IDKEGG PathwayGene target
1.hsa04931Insulin resistanceAKT1|AKT2|GSK3B|INSR|NOS3|PPARA|PTPN11|NR1H2|NR1H3
2.hsa05200Pathways in cancerAKT1|AKT2|BRAF|CTNNA1|GSK3B|GSTM1|GSTP1|HRAS|IGF1|KIT|PPARD|P PARG|MAPK1
3.hsa00982Drug metabolism - cytochrome P450ADH1B|ADH1C|CYP2C8|CYP2C9|GSTM1|GSTP1
4.hsa01524Platinum drug resistanceAKT1|AKT2|GSTM1|GSTP1|MAPK1
5.hsa04960Aldosterone-regulated sodium reabsorptionIGF1|INSR|NR3C2|MAPK1
6.hsa01240Biosynthesis of cofactorsALDH2|FECH|MTHFD1|SPR|NMNAT1
7.hsa03320PPAR signaling pathwayPPARA|PPARD|PPARG|NR1H3
8.hsa04936Alcoholic liver diseaseADH1B|ADH1C|AKT1|AKT2|ALDH2|GSK3B|PPARA
9.hsa00230Purine metabolismATIC|IMPDH1|PNP
10.hsa00600Sphingolipid metabolismARSA|GBA|PSAP
11.hsa04924Renin secretionACE|PRKACA|REN
12.hsa04976Bile secretionHMGCR|PRKACA|NR1H4
13.hsa04310Wnt signaling pathwayGSK3B|PPARD|PRKACA
14.hsa01521EGFR tyrosine kinase inhibitor resistanceAKT1|AKT2|BRAF|GSK3B|HRAS|IGF1|MAPK1
Figure 2 Effects of Ecliptae Herba on different parts of the body of zebrafish fry, including whole body (A), eyes (B), eye end to neck (C), and neck to tail (D).
Figure 3 Multidimensional network analysis diagram of Ecliptae Herba based on the association of targets and components. (A) Venn diagram showing the screening of 51 target genes from Ecliptae Herba drug targets and disease targets. (B) Network diagram of single herb-active ingredient-target of Ecliptae Herba. Blue represents the target, purple represents the active ingredient of Ecliptae Herba, and green represents the single herb of Ecliptae Herba. (C) PPI network diagram of active component target genes in Ecliptae Herba.
Figure 4 Multifaceted exploration of Ecliptae Herba’s regulatory mechanisms on melanin-related targets. (A) GO enrichment analysis (including BP, CC, and MF terms) of common targets between the active components of Ecliptae Herba and melanin-related genes. (B) KEGG enrichment analysis of common targets between the active components of Ecliptae Herba and melanin-related genes. (C) Molecular docking results of key pharmacological components in Ecliptae Herba with target proteins.

CONCLUSION

In this study, UHPLC-Q-Orbitrap HRMS identified 34 active components of Ecliptae Herba, and network pharmacology combined with molecular docking revealed key targets and pathways involved in melanogenesis. Functional enrichment analysis showed that Ecliptae Herba regulated melanogenesis through oxidative stress modulation, hormone response, and PPAR signaling pathways. Importantly, the experiments using the zebrafish model demonstrated that Ecliptae Herba water extract significantly reduced the gray values in specific body regions of the model, indicating a potential role of Ecliptae Herba in promoting melanin formation. In summary, this study offered valuable insights into the mechanisms by which Ecliptae Herba promoted melanin production, establishing a solid foundation for future investigations in this field.

SUMMARY

This study explored the active components and mechanisms of Ecliptae Herba in regulating melanin synthesis. UHPLC-Q-Orbitrap HRMS identified 34 compounds, while network pharmacology and molecular docking revealed key targets such as GSTP1, PPARA, and IGF1 involved in oxidative stress and PPAR signaling pathways. Zebrafish experiments showed that the Ecliptae Herba extract can reduced gray values in specific regions. These results suggest that Ecliptae Herba exerts multi-component, multi-target effects on melanin regulation, offering a scientific basis for its development in treating pigmentary disorders.

Abbreviations

UHPLC-Q-Orbitrap HRMS: Ultra-High Performance Liquid Chromatography-Quadrupole-Orbitrap High-Resolution Mass Spectrometry; TCMSP: The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform; OMIM: Online Mendelian Inheritance in Man; PDB: the Protein Data Bank; TRP1: Tyrosinase-related protein-1; NP: network pharmacology; DMSO: Dimethyl Sulfoxide; OB: oral bioavailability; DL: drug-like index; PPI: Protein-protein interaction; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Conflict of Interest

The authors declare that there is no conflict of interest.

Author Contributions

All authors contributed to this present work: MYS and SYG designed the experiment, conducted the experiment, and analyzed the data, MYS and SYG designed the study, MYS and SYG acquired the data. MYS and SYG drafted the manuscript, MYS and SYG revised the manuscript. All authors have been involved in revising it critically for important intellectual content. All authors read and approved the final manuscript.

REFERENCES

As published

Showing references and in-text citations exactly as published.

  1. 1.Gude, D., Bansal, D. P., Ambegaonkar, R., Prajapati, J. Paraphenylenediamine: blackening more than just hair. J Res Med Sci Off J Isfahan Univ Med Sci 2012;17(6):584-6.GOOGLE SCHOLAR
  2. 2.Liu, Y. Q., Xu, C. Y., Liang, F. Y., Jin, P. C., Qian, Z. Y., Luo, Z. S. Selecting and Characterizing tyrosinase Inhibitors from Atractylodis macrocephalae rhizoma Based on Spectrum-Activity Relationship and Molecular Docking. J Anal Methods Chem 2021;2021:5596463. https://doi.org/10.1155/2021/5596463CrossrefGOOGLE SCHOLAR
  3. 3.Molagoda, I. M., Lee, K. T., Athapaththu, A. M., Choi, Y. H., Hwang, J., Sim, S. J. Flavonoid glycosides from Ziziphus jujuba var. inermis (Bunge) Rehder Seeds Inhibit α-Melanocyte-Stimulating Hormone-Mediated Melanogenesis. Int J Mol Sci 2021;22(14):7701. https://doi.org/10.3390/ijms22147701CrossrefGOOGLE SCHOLAR
  4. 4.Vachtenheim, J., Borovanský, J. Transcription physiology" of pigment formation in melanocytes: central role of MITF. Exp Dermatol 2010;19(7):617-27. https://doi.org/10.1111/j.1600-0625.2009.01053.xCrossrefGOOGLE SCHOLAR
  5. 5.D’Mello, S. A., Finlay, G. J., Baguley, B. C., Askarian-Amiri, M. E. Signaling pathways in melanogenesis. Int J Mol Sci 2016;17(7):1144. https://doi.org/10.3390/ijms17071144CrossrefGOOGLE SCHOLAR
  6. 6.Lee, C. Y., Wei, C. C., Yu, M. C., Lin, C. C., Sheu, S. J., Yang, J. H. Hair growth effect of traditional Chinese medicine BeauTop on androgenetic alopecia patients: A randomized double-blind placebo-controlled clinical trial. Exp Ther Med 2017;13(1):194-202. https://doi.org/10.3892/etm.2016.3935CrossrefGOOGLE SCHOLAR
  7. 7.Sarkar, C., Singh, S. K., Mandal, S. K., Saha, B., Bera, R., Ratha, J. Human placental protein/ peptides stimulate melanin synthesis by enhancing tyrosinase gene expression. Mol Cell Biochem 2006;285(1-2):133-42. https://doi.org/10.1007/s11010-005-9069-3CrossrefGOOGLE SCHOLAR
  8. 8.Sarkar, D., Leung, E. Y., Baguley, B. C., Finlay, G. J., Askarian-Amiri, M. E. Epigenetic regulation in human melanoma: past and future. Epigenetics 2015;10(2):103-21. https://doi.org/10.1080/15592294.2014.1003746CrossrefGOOGLE SCHOLAR
  9. 9.He, L., Michailidou, F., Gahlon, H. L., Zeng, W. Hair dye ingredients and potential health risks from exposure to hair dyeing. Chem Res Toxicol 2022;35(6):901-15. https://doi.org/10.1021/acs.chemrestox.1c00427CrossrefGOOGLE SCHOLAR
  10. 10.Liu, S. C., Sheu, M. L., Tsai, Y. C., Lin, Y. C., Chang, C. W., Lai, D. W. Attenuation of in vitro and in vivo melanin synthesis using a Chinese herbal medicine through the inhibition of tyrosinase activity. Phytomedicine 2022;95:153876. https://doi.org/10.1016/j.phymed.2021.153876CrossrefGOOGLE SCHOLAR
  11. 11.Tian, S., Li, Y. L., Wang, J., Dong, R. C., Wei, J., Ma, Y. Chinese Ecliptae herba (Eclipta prostrata L.) extract and its component wedelolactone enhances osteoblastogenesis of bone marrow mesenchymal stem cells via targeting METTL3-mediated m6A RNA methylation. J Ethnopharmacol 2023;312:116433. https://doi.org/10.1016/j.jep.2023.116433CrossrefGOOGLE SCHOLAR
  12. 12.Wu, J., Gou, W., Wang, Z., Chang, H., Li, D., Hou, W. Discovery of the radio-protecting effect of Ecliptae Herba, its constituents and targeting p53-mediated apoptosis in vitro and in vivo. Acta Pharm Sin B 2023;13(3):1216-30. https://doi.org/10.1016/j.apsb.2022.09.003CrossrefGOOGLE SCHOLAR
  13. 13.Tao, R., Liu, E., Zhao, X., Han, L., Yu, B., Mao, H. Combination of Ligustri Lucidi Fructus with Ecliptae Herba and their phytoestrogen or phytoandrogen like active pharmaceutical ingredients alleviate oestrogen/testosterone-induced benign prostatic hyperplasia through regulating steroid 5-α-reductase. Phytomedicine 2022;102:154169. https://doi.org/10.1016/j.phymed.2022.154169CrossrefGOOGLE SCHOLAR
  14. 14.Liu, Y. Q., Zhan, L. B., Liu, T., Cheng, M. C., Liu, X. Y., Xiao, H. B. Inhibitory effect of Ecliptae herba extract and its component wedelolactone on pre-osteoclastic proliferation and differentiation. J Ethnopharmacol 2014;157:206-11. https://doi.org/10.1016/j.jep.2014.09.033CrossrefGOOGLE SCHOLAR
  15. 15.Yadav, N. K., Arya, R. K., Dev, K., Sharma, C., Hossain, Z., Meena, S. Alcoholic extract of Eclipta alba Shows in vitro antioxidant and anticancer activity without exhibiting toxicological effects. Oxid Med Cell Longev 2017;2017:9094641. https://doi.org/10.1155/2017/9094641CrossrefGOOGLE SCHOLAR
  16. 16.Lai, Y., Feng, Q., Zhang, R., Shang, J., Zhong, H. The Great Capacity on Promoting Melanogenesis of Three Compatible Components in Vernonia anthelmintica (L.) Willd. Int J Mol Sci 2021;22(8):4073. https://doi.org/10.3390/ijms22084073CrossrefGOOGLE SCHOLAR
  17. 17.Wu, C. S., Cioanca, A. V., Gelmi, M. C., Wen, L., Di Girolamo, N., Zhu, L. The multifunctional human ocular melanocortin system. Prog Retin Eye Res 2023;95:101187. https://doi.org/10.1016/j.preteyeres.2023.101187CrossrefGOOGLE SCHOLAR
  18. 18.Chen, D. J., Lai, C. H. Exploring the mechanisms of magnolol in the treatment of periodontitis by integrating network pharmacology and molecular docking. Biocell 2023;47(6):1317-27. https://doi.org/10.32604/biocell.2023.028883CrossrefGOOGLE SCHOLAR
  19. 19.Qian, F., Ren, P., Zhao, L. I., Zheng, D., He, W., Jin, J. Study of molecular mechanisms underlying the medicinal plant Tripterygium wilfordii-derived compound celastrol in treating diabetic nephropathy based on network pharmacology and molecular docking. Biocell 2023;47(8):1853-67. https://doi.org/10.32604/biocell.2023.029353CrossrefGOOGLE SCHOLAR
  20. 20.Song, Z., Yu, J., Wang, M., Shen, W., Wang, C., Lu, T. CHDTEPDB: transcriptome expression profile database and interactive analysis platform for congenital heart disease. Congenit Heart Dis 2023;18(6):693-701. https://doi.org/10.32604/chd.2024.048081CrossrefGOOGLE SCHOLAR
  21. 21.Zhang, W., Tian, W., Wang, Y., Jin, X., Guo, H., Wang, Y. Explore the mechanism and substance basis of Mahuang FuziXixin Decoction for the treatment of lung cancer based on network pharmacology and molecular docking. Comput Biol Med 2022;151(A):106293. https://doi.org/10.1016/j.compbiomed.2022.106293CrossrefGOOGLE SCHOLAR
  22. 22.Seeliger, D., de Groot, B. L. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput-Aid Mol Des 2010;24(5):417-22. https://doi.org/10.1007/s10822-010-9352-6CrossrefGOOGLE SCHOLAR
  23. 23.Zhou, Y., Gu, J., Yu, H., Chen, F., Long, C., Maihemuti, M. Screening and identification of ESR1 as a target of icaritin in hepatocellular carcinoma: evidence from bibliometrics and bioinformatic analysis. Curr Mol Pharmacol 2024;17:e18761429260902. https://doi.org/10.2174/0118761429260902230925044009CrossrefGOOGLE SCHOLAR
  24. 24.Xiao, X., Liu, Y., Huang, Y., Zeng, W., Luo, Z. Identification of the NF-κB Inhibition Peptides in Asthma from Pheretima aspergillum Decoction and Formula Granules using Molecular Docking and Dynamics Simulations. Curr Pharm Anal 2024;20(3):202-11. https://doi.org/10.2174/0115734129298587240322073956CrossrefGOOGLE SCHOLAR
  25. 25.Chaudhary, A., Patel, M., Singh, S. Current debates on etiopathogenesis and treatment strategies for vitiligo. Curr Drug Targets 2022;23(13):1219-38. https://doi.org/10.2174/1389450123666220406125645CrossrefGOOGLE SCHOLAR
  26. 26.Flydal, M. I., Martinez, A. Phenylalanine hydroxylase: function, structure, and regulation. IUBMB Life 2013;65(4):341-9. https://doi.org/10.1002/iub.1150CrossrefGOOGLE SCHOLAR
  27. 27.Huang, H., Chen, J., Ren, J., Zhang, C., Ji, F. Palladium(II)-catalyzed efficient synthesis of wedelolactone and evaluation as potential tyrosinase inhibitor. Molecules 2019;24(22):4130. https://doi.org/10.3390/molecules24224130CrossrefGOOGLE SCHOLAR
  28. 28.Lin, V. C., Ding, H. Y., Tsai, P. C., Wu, J. Y., Lu, Y. H., Chang, T. S. In vitro and in vivo melanogenesis inhibition by biochanin A from Trifolium pratense. Biosci Biotechnol Biochem 2011;75(5):914-8. https://doi.org/10.1271/bbb.100878CrossrefGOOGLE SCHOLAR
  29. 29.Joh, E. H., Gu, W., Kim, D. H. Echinocystic acid ameliorates lung inflammation in mice and alveolar macrophages by inhibiting the binding of LPS to TLR4 in NF-κB and MAPK pathways. Biochem Pharmacol 2012;84(3):331-40. https://doi.org/10.1016/j.bcp.2012.04.020CrossrefGOOGLE SCHOLAR
  30. 30.Huo, S. X., Wang, Q., Liu, X. M., Ge, C. H., Gao, L., Peng, X. M. The effect of Butin on the vitiligo mouse model induced by hydroquinone. Phytother Res 2017;31(5):740-6. https://doi.org/10.1002/ptr.5794CrossrefGOOGLE SCHOLAR
  31. 31.Tsai, Y. T., Chen, P. Y., To, K. Y. Induction of polyploidy and metabolic profiling in the medicinal Herb Wedelia chinensis. Plants (Basel, Switzerland) 2021;10(6):1232. https://doi.org/10.3390/plants10061232CrossrefGOOGLE SCHOLAR
  32. 32.Yang, Y., Cheng, R., Liu, J., Fang, J., Wang, X., Cui, Y. Linarin protects against cadmium-induced osteoporosis via reducing oxidative stress and inflammation and altering RANK/RANKL/OPG pathway. Biol Trace Elem Res 2022;200(8):3688-700. https://doi.org/10.1007/s12011-021-02967-wCrossrefGOOGLE SCHOLAR
  33. 33.Guo, H., Zeng, H., Fu, C., Huang, J., Lu, J., Hu, Y. Identification of sitogluside as a potential skin-pigmentation-reducing agent through network pharmacology. Oxid Med Cell Longev 2021;2021:4883398. https://doi.org/10.1155/2021/4883398CrossrefGOOGLE SCHOLAR
  34. 34.Leis Ayres, E., Dos Santos Silva, J., Eberlin, S., Facchini, G., Vasconcellos, C., Da Costa, A. In vitro effect of pine bark extract on melanin synthesis, tyrosinase activity, production of endothelin-1, and PPAR in cultured melanocytes exposed to ultraviolet, infrared, and Visible light radiation. J Cosmet Dermatol 2022;21(3):1234-42. https://doi.org/10.1111/jocd.14202CrossrefGOOGLE SCHOLAR
  35. 35.Hu, S., Liu, Y., Yang, S., Ji, K., Liu, X., Zhang, J. The effects of IGF1 on the melanogenesis in alpaca melanocytes in vitro. In vitro Cell Dev Biol Anim 2016;52(8):806-11. https://doi.org/10.1007/s11626-016-0052-yCrossrefGOOGLE SCHOLAR

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Sun, M., & Guan, S. (2026). Analysis on the Active Components and Mechanisms of Action of Ecliptae Herba Intervention on Melanin Synthesis Applying UHPLC-Q-Orbitrap HRMS and Network Pharmacology in Combination with Molecular Docking Screening. Indian Journal of Pharmaceutical Education and Research, 61(1), 399-410. https://doi.org/10.5530/ijper.20271427

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