Abstract Melasma is a pigmentation disease with refractory and high recurrence risk. Therefore, finding effective treatment has become the focus of research. This study aimed to reveal the mechanism of Licorice rose beverage (LRB) in treating melasma from the perspective of network pharmacology and in vitro and in vivo experimental techniques. Network pharmacological studies have shown that Isolicoflavonol, quercetin, and kaempferol are the main active components of anti-melasma and tyrosinase is the main target. Molecular docking studies have shown that these compounds have a good affinity for these targets. In vitro tyrosinase inhibition experiments showed that LRB could significantly inhibit tyrosinase activity. In vivo studies showed that LRB could significantly improve skin damage and skin pigmentation, reduce the activities of serum and skin tyrosinase in model mice, increase the activity of SOD in serum, and reduce the content of MDA in mice, showing a good effect of anti-melasma. In conclusion, these findings reveal the molecular mechanism of LRB in treating melasma and provide the scientific basis for this product's development and clinical application. Keywords: Licorice rose beverage, Network pharmacology, Melasma, Tyrosinase Graphical abstract Image 1 [51]Open in a new tab Abbreviations LRB Licorice rose beverage SOD superoxide dismutase MDA malondialdehyde l-DOPA levodopa solution TCM traditional Chinese medicine 1. Introduction Melasma is a common pigmented skin disease, which is common in women [[52]1]. It has a long course and is easy to relapse [[53]2]. In recent years, the incidence of melasma has been increasing year by year, and the prevention and treatment of melasma have attracted wide attention [[54]3]. However, the pathogenesis of melasma has not been fully elucidated. Studies show that there are many pathogenic factors of melasma, among which ultraviolet radiation, endocrine dysregulation, oxygen free radicals, and improper use of drugs and cosmetics are the main causes of melasma [[55]4]. In addition, tyrosinase also plays a significant role in the formation of melasma. When the activity of tyrosinase is too high or overactive, an excessive amount of tyrosine is converted into melanin, leading to the formation of melasma [[56]5]. At present, there are many methods for the clinical treatment of melasma, but the overall efficacy is not ideal [[57]6]. According to traditional Chinese medicine, the liver, spleen, and kidney are closely related to melasma, and it is believed that melasma arises from liver stagnation, spleen dampness, and kidney deficiency [[58]7]. Therefore, treating melasma based on traditional Chinese medicine theory holds promise as a potential approach. Traditional Chinese medicine (TCM) is a medical system with a long history and unique theories and techniques [[59]8,[60]9]. In recent years, the use of Chinese herbal medicine treatment of diseases associated with melasma has attracted wide attention. For example, Zhang et al. found that the cream containing camellia, mulberry, Dauphin oil, and purslane could improve melasma by various mechanisms such as anti-inflammatory, anti-oxidant, improving microcirculation, inhibiting melanin production and improving the skin permeability barrier [[61]7]. The Licorice rose beverage (LRB) includes Licorice, Rose flowers, Tangerine peel, Wolfberry, Poria cocos, Mulberry leaf, and Cinnamon. Among them, Licorice is a very famous ancient herb and one of the most commonly used in TCM, such as antibacterial, antiviral, anti-inflammatory, antidiabetic, immunomodulatory, liver protection, etc. [[62]10]. In addition to glycyrrhizic acid, flavonoids, saponins, triterpenes, isoflavones, and chalcones, the major active ingredients in licorice also contain large amounts of iron (Fe), manganese (Mn), and cobalt (Co). These chemical elements are the reasons for the comprehensive utilization of glycyrrhiza in health [[63]11,[64]12]. The beauty and fragrance of rose flowers have been known since ancient times, known as the “gift of angels” [[65]13]. Rose flowers are rich in beneficial components, such as flavonoids (flavonols and anthocyanins) and aromatic components (essential oils), which can be used as disinfectants, anti-inflammatory agents, and antioxidants, and are widely used in the food industry, perfume, and cosmetics [[66]14]. Tangerine peel is the mature peel of Rutaceae and its cultivated varieties. It is rich in phenolic compounds and carotenoids. It is an excellent source of dietary fiber and minerals. It is widely used in the food, pharmaceutical, and cosmetic industries [[67]15]; Wolfberry has been used in TCM for thousands of years as a healthy food and a treatment for diseases. According to TCM theory and practice, wolfberry can function on both the liver meridian and kidney meridian, wolfberry's main health effect is to nourish the liver and kidney. According to China's State Food and Drug Administration, medlar is one of 87 kinds of TCM ingredients that can be used as both normal food and functional food [[68]16]; Poria cocos (Polyporaceae) is a saprophytic fungus that grows in different species of pine. TCM believes that Poria cocos has a diuretic, sedative, and nourishing effect. Modern medical research shows that Poria Cocos has anti-inflammatory, anti-tumor, and other pharmacological activities, widely used in medicine and the health food field [[69]17]. Mulberry leaves are very valuable food plants for nutrition and nutrient composition. In some Asian countries, it is widely used as ethnic medicine and functional food, such as tea, drinks, noodles, etc., due to its biological and nutritional value [[70]18]. Cinnamon is an eternal tree used in tropical medicine and is one of the most important spices used daily. Cinnamon contains manganese, iron, dietary fiber, cinnamaldehyde, cinnamic acid, and polyphenols. It has antioxidant, anti-inflammatory, anti-diabetic, anti-bacterial, and anti-cancer effects [[71]19]. LRB can regulate qi, tonify the spleen and kidney, promote blood circulation, and regulate menstruation through the combined action of seven medicines, thereby promoting beauty, nourishing the skin, and removing melasma. In general, although the pharmacological effects of these seven drugs are significant, the molecular mechanism of the combination of these drugs to treat melasma remains unclear. Network pharmacology is an emerging discipline to explores the mechanism of action of TCM with multi-components, multi-targets, and multi-pathways [[72]20]. It is helpful to study the active components, targets, and molecular mechanisms of TCM in the intervention of diseases and provides direction for the development of new drugs. Molecular docking technology is a further analysis method based on the prediction of network pharmacology, which can explain the molecular mechanism of drug molecules acting on disease-related targets [[73]21]. In order to reveal the mechanism of action of LRB in treating melasma, this study carried out a strategy based on TCM network pharmacology combined with calculation prediction and experimental verification. Firstly, network pharmacology and molecular docking technology were used to explore the molecular mechanism of LRB in the treatment of melasma, and in vitro experiments and mouse melasma model were used to verify it, to provide the scientific basis for the future development and clinical application of this product. 2. Materials and methods 2.1. Materials Licorice rose beverage (LRB) was purchased from Guangzhou Tianzhiyuan Beauty & Health Products Co., Ltd. The names and formulations of 7 herbs in LRB were provided ([74]Table 1). Levodopa was purchased from Shanghai Aladdin Biotech Co., Ltd. Progesterone injection (20 mg/mL) was purchased from Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd. Tyrosinase Reagent was purchased from Beijing Soleb Technology Co., Ltd. l-ascorbic acid, polyformaldehyde fixative, superoxide dismutase (SOD) and malondialdehyde (MDA) were purchased from Shanghai McLean Biochemical Technology Co., Ltd. Table 1. Names and formulations ratio of seven herbs in LRB. Herbal name Plant scientific name Branch Genus Application area Weight ratio Licorice Glycyrrhiza uralensis Fisch. Leguminosae Glycyrrhiza Dried root 5 Rose flowers Rosa rugosa Thunb. Rosaceae Rosa L. Dried flower 3 Tangerine peel Citrus Reticulata Rutaceae Citrus spp. Dried bark 5 Wolfberry Lycii Fructus Solanaceae Genus Lycium Dried ripe fruit 5 Poria cocos Poria Cocos(Schw.) Wolf. Polyporaceae Poria genus Dried Mycorrhiza 5 Mulberry leaf Mori Follum moraceae Morus genus Dried leaf 5 Cinnamon Cinnamomum cassia (L.) J.Presl Lauraceae Cinnamomum Dried bark 2 [75]Open in a new tab 2.2. Network pharmacology data analysis The chemical constituents of licorice, rose, tangerine peel, wolfberry, poria cocos, mulberry leaves, and cinnamon [[76]22,[77]23] in LRB were collected using the TCMSP database ([78]http://tcmspw.com/tcmsp.php) and published literature. According to the pharmacokinetic characteristics given by the TCMSP data platform, oral bioavailability (OB ≥ 30 %) and drug-like (DL ≥ 0.18) were selected as the screening parameters for the chemical components of traditional Chinese medicine ([79]Table S1) [[80]24,[81]25]. The two-dimensional structure of the compound is obtained from the PubChem database ([82]www./pubchem.ncbi.nlm.nih.gov). Through GeneCards ([83]https://www.genecards) and DisGeNET ([84]https://www.disgenet.org/) in the database for “melasma” related targets. These common targets were calculated using a Venn diagram ([85]Fig. S1). The interactions between LRB anti-melasma related targets were analyzed by the STRING database ([86]https://string-db.org/). GO (Gene Ontology) analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis using the DAVID database ([87]https://david.ncifcrf.gov/). Finally, the Cytoscape software (version 3.9.1) was used to construct the active compound-target network. 2.3. Molecular docking Tyrosinase, the core target of network pharmacology, was used as a receptor protein, and the top 3 compounds of H–C-T-P was used as ligand molecule. The three-dimensional structure of protein tyrosinase (2Y9W) was retrieved from the RCSB Protein Data Bank ([88]https://www.rcsb.org) and ligand molecules were obtained from the PubChem database ([89]https://pubchem.ncbi.nlm.nih.gov/) [[90]26,[91]27]. Among them, tyrosinase is a protein-encoding gene. The gene can regulate the catalytic activity of tyrosine hydroxylase and dopa oxidase, regulate the formation of melanin, and play a key role in the pathogenesis of melasma [[92]28,[93]29]. The Full Minimization module of Discovery Studio is used to minimize the energy of small molecules and set the CHARMM force field to be assigned to the structure. Molecular docking using the LibDock module on the Discovery Studio 2019 software. Make minor modifications according to the method reported. Among them, Libdockscore≥90 indicates that the active component has a strong affinity with the target protein [[94]30]. 2.4. Tyrosinase inhibition assay The inhibitory activity of LRB on tyrosinase in vitro was studied [[95]31]. Briefly, the inhibition of tyrosinase was detected using the levodopa solution (l-DOPA) as the substrate [[96]32]. Glabridin was determined as a positive control. Add 40 μL LRB of different concentrations (0.125, 0.25, 0.5, 0.75, and 1.0 g/mL) and substrate solution containing 40 μL l-DOPA (0.453 g/L) and 80 μL potassium phosphate buffer (pH 6.8) to 96 hole plate, water bath at 37 °C for 10 min at a constant temperature, then add 200 U/mL tyrosinase 40 μL in each hole, and shake and mix for 15 min under room temperature and light protection. Then, the absorbance was measured and recorded at 475 nm by Multiskan Go, and the inhibition rate was calculated 3 times in parallel. 2.5. Animal research 2.5.1. Modeling A melasma mouse model has been established. Female mice from Kunming weighed about 20 g each and all experiments were conducted at the Animal Laboratory Center of the Southern Medical University (Guangzhou, China, quality certificate number: SCXK [Yue] 20160041). The ambient temperature (25 ± 5 °C) and humidity (55 ± 5 %) of mice remained stable during the experiment. During the study, mice were given standard diets and drinking water. After adapting to 2 days, 60 mice were randomly divided into six groups: blank, model, positive control, LRB high (1 g/mL), medium (0.5 g/mL), low concentration (0.25 g/mL), each group 10 mice. The melasma model mice were established by injecting progesterone (20 mg/kg) into the hind leg muscle and irradiating it with ultraviolet for 60 min. The control group was given Vitamin C (Vit C, 0.1 g/kg), and the other three groups were given an oral solution of the corresponding concentration for 30 consecutive days (1 dose per day). 2.5.2. Characterization of skin status and pathological sections After 24 h of the last dose, the skin condition on the backs of the mice was carefully observed and photographed. The mice were then anesthetized with pentobarbital sodium and weighed using an electronic balance. Blood samples were collected from the eyeballs and centrifuged at 3000 rpm for 30 min to obtain serum, which was subsequently stored at −80 °C. The back skin of each mouse was divided into two parts. Additionally, two portions of the dorsal skin of each mouse were collected, fixed with polyformaldehyde fixative for hematoxylin-eosin (HE) staining, and stored as frozen skin tissue homogenate material. Throughout the experiment, none of the mice showed any signs of toxicity and all of them survived. 2.5.3. Effects on tyrosinase content in serum and skin tissue of melasma mice Tyrosinase levels in serum and skin tissues were measured. The back skin of each mouse was homogenized in 0.9 % NaCl cold solution (10 %), centrifuged at 3000 rpm for 15 min, and stored at −80 °C. The serum samples were obtained as mentioned in step “2.4.2". The determination of tyrosinase was carried out according to the operation requirements and steps of the tyrosinase kit. Finally, the tyrosinase content was calculated by measuring and recording the absorbance value at 450 nm wavelength with a Multiskan Go enzyme micrograph. 2.5.4. Effects on serum antioxidant enzymes in mice In order to analyze the effects on serum antioxidant enzymes in mice, serum samples were collected from the same procedure as described in section “2.4.2". The activity of Superoxide Dismutase (SOD) and the content of Malondialdehyde (MDA) in the serum were determined using appropriate kits. The SOD activity was measured at a wavelength of 450 nm, while the content of MDA was assessed at a wavelength of 532 nm. Both measurements were performed using a Multiskan Go spectrophotometer. 2.6. Statistical analysis Data were expressed as mean ± standard deviation (SD). Pictures were drawn using GraphPad Prism 6.0. Statistical analysis was performed by one-way analysis of variance (ANOVA), and P < 0.05 was statistically significant. 3. Results and discussion 3.1. Network pharmacology analysis 3.1.1. GO and KEGG pathway enrichment analysis [97]Fig. 1 shows the GO functional enrichment analysis of LRB anti-melasma related targets, including biological process (BP), cell composition (CC), and molecular function (MF). The results of the GO analysis showed that the biological processes involved mainly include of processes negative regulation of gene expression, negative regulation of transcription from RNA polymerase II promoter and positive regulation of transcription from RNA polymerase II promoter, positive regulation of gene expression, etc. The cellular components involved mainly include cytoplasm, cytosol, nucleus and nucleoplasm, etc. The molecular function involved mainly includes protein binding, enzyme binding, zinc ion binding, DNA binding and RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding, etc. Fig. 1. [98]Fig. 1 [99]Open in a new tab The bar chart of the results of GO functional enrichment for the top ten biological processes (BP), cell components (CC), and molecular functions (MF). The KEGG pathway of 13 candidate targets was enriched by DAVID 6.8 database, and the first ten signal pathways of LRB anti-melasma were obtained according to the P value, as shown in [100]Fig. 2. The bubble area size represents the number of target genes, the bubble color represents enrichment significance (i.e., the P value), and the Y axis represents the pathway name. The main pathways related to melasma include Metabolic ways, Chemical Carcinogenesis-Receptor activation, Relaxin signaling pathway, etc. It can be found that the anti-melasma effect of LRB is realized by multi-pathway and multi-target. Fig. 2. [101]Fig. 2 [102]Open in a new tab Bubble diagram of the results of KEGG pathway enrichment of the top ten for LRB treatment of melasma. 3.1.2. "H–C-T-P" network construction and analysis To better explore the molecular mechanism of LRB in treating melasma, the “H–C-T-P" topological network with 227 nodes and 1320 edges was created using Cytoscape 3.9.1 software ([103]Fig. 3). Nodes are made up of different colors and shapes. Edges are used to represent the correlation between different nodes. The active components in LRB interact with different targets and pathways, which is the same as the concept of TCM multi-target and multi-way cooperative treatment of diseases. Fig. 3. [104]Fig. 3 [105]Open in a new tab LRB “H–C-T–P" network diagram. The degree values of the active ingredients isolicoflavonol, quercetin, kaempferol, norartocarpetin, jaranol, tetramethoxy luteolin, quercetin der, semilicoisoflavone B, glepidotin A and hedysarimcoumestan B were high, suggesting that these active ingredients play an important role in the anti-melasma process ([106]Table 2). The potential targets of LRB anti-melasma are TYR (Tyrosinase), ESR2 (Estrogen Receptor 2), ESR1 (Estrogen Receptor 1), AR (Androgen Receptor), and EGFR (Epidermal Growth Factor Receptor), suggesting that these targets may be key targets for the treatment of melasma. Table 2. The top ten potentially effective compounds in prescriptions were screened by network pharmacology. Pubchem ID Compounds Degree 5318585 Isolicoflavonol 14 5280343 quercetin 14 5280863 kaempferol 13 5481970 Norartocarpetin 13 5318869 Jaranol 13 631170 Tetramethoxy luteolin 13 5316900 Quercetin der. 12 5481948 Semilicoisoflavone B 12 5281619 Glepidotin A 12 11558452 Hedysarimcoumestan B 11 [107]Open in a new tab 3.1.3. PPI network construction and analysis The intersection targets were uploaded to the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database, and protein-protein interaction (PPI) networks were constructed, as shown in [108]Fig. 4. In the PPI networks, the nodes represent proteins, while the edges indicate the interactions between proteins. The strength of the association is represented by the number of lines on the edges, with more lines indicating a stronger interaction. In these PPI networks, TYR (tyrosinase), ESR1, and VEGFA have higher degree values, suggesting their prominent roles in the network. Notably, tyrosinase emerges as a central player in both the “H–C-T-P" and PPI networks. This observation leads us to speculate that LRB may influence downstream protein expression by modulating the activity or expression of tyrosinase. Fig. 4. [109]Fig. 4 [110]Open in a new tab PPI network. 3.2. Molecular docking verification In order to verify the interaction between the components and targets screened by network pharmacology, and to explore the molecular mechanism of LRB in the treatment of melasma, molecular docking technology was used to verify the top three active components and target proteins. [111]Table 3 shows the docking scores and energies of Isolicoflavonol, quercetin, kaempferol, glabridin, and Vitamin C with tyrosinase (2Y9W) target proteins. The results showed that these compounds had a good binding activity with target proteins. The results indicate that these compounds exhibit good binding activity with the target protein. Isolicoflavonol, quercetin, and kaempferol showed dock scores greater than 90 with the target protein, indicating that these three compounds exhibit a certain level of affinity in their interaction with the target protein, and may play a crucial role in LRB anti-melasma activity. [112]Fig. 5 shows the visualized molecular docking results of these active compounds with target proteins. It is found that the binding of these compounds to the target protein occurs mainly through hydrogen bonds, van der Waals forces, and π bonds. Among them, the hydrogen atom on the hydroxyl group of the Isolicoflavonol compound acts as a hydrogen bond donor, and the amino acid residues ASP312 on the tyrosinase target protein form hydrogen bond interactions with distances of 2.47 Å ([113]Fig. 5a). The hydrophobic region of the benzene ring on the quercetin compound formed π bond interactions with amino acid residues PHE368 and TRP358 on tyrosinase target protein with the spacing of 4.44 Å, 5.85 Å, and 5.16 Å ([114]Fig. 5b). Kaempferol compounds and amino acid residues ASP357, ASN310, GLN307, and LYS376 on tyrosinase target proteins form van der Waals forces ([115]Fig. 5c). In addition, the amino acid residues Glu356 and Lys379 on the tyrosinase target protein show significant interactions with these three compounds. Mahdavi et al. have shown that Glu356 is an important residue in tyrosinase activity, and certain tyrosinase inhibitors, such as α-arbutin, interact with this binding site to inhibit tyrosinase activity [[116]33]. Additionally, Lys379 is considered to play a decisive role in the binding of substrates/ligands to the tyrosinase enzyme [[117][33], [118][34], [119][35]]. This further suggests that these three compounds may have good inhibitory effects on tyrosinase. The molecular docking results show that these compounds have a good affinity with target proteins, further verifying the rationality of network pharmacological prediction. In addition, glabridin and vitamin C also showed good docking activity with tyrosinase target proteins, which could be used as positive drugs for experimental verification ([120]Figs. S2a–b). Table 3. Molecular docking results of main active ingredients in LBR and positive drugs with tyrosinase target protein. Compound Protein Libdock score Binding Energy（kcal/mol） Ligand Energy （kcal/mol） Protein Energy （kcal/mol） Complex Energy （kcal/mol） Entropic Energy （kcal/mol） Isolicoflavonol [121]Tyrosinase (2Y9W) 105.516 181.8753 114.4414 −42160.4665 −41864.1498 19.9345 quercetin [122]Tyrosinase (2Y9W) 99.4469 4038.3635 1797.7582 −42160.4665 −36324.3448 19.4212 kaempferol [123]Tyrosinase (2Y9W) 101.213 189.8574 8690.0941 −42160.4665 −33280.5152 19.2834 glabridin (positive drug) [124]Tyrosinase (2Y9W) 116.697 1185.5550 45.0593 −4216.4665 −40929.8521 19.6204 Vitamin C (positive drug) [125]Tyrosinase (2Y9W) 91.0794 776.4857 51.0197 −42160.4665 −41326.9614 17.7626 [126]Open in a new tab Fig. 5. [127]Fig. 5 [128]Open in a new tab Molecular docking of tyrosinase with Isolicoflavonol (a), quercetin (b), and kaempferol (c). 3.3. Inhibition of LRB on tyrosinase in vitro Tyrosinase is a key enzyme in the skin melanin synthesis process, so it can directly affect the rate of melanin synthesis [[129]36]. By inhibiting tyrosinase, melanin production in cells is specifically inhibited because tyrosinase is produced only by melanocytes [[130]37]. In this experiment, l-DOPA was used as a substrate, LRB as an effector, and glabridin was used as a positive control to determine its inhibition on tyrosinase [[131]38]. As shown in [132]Fig. 6, different concentrations of LRB can significantly inhibit tyrosinase activity, and with the increase of the concentration of LRB, the inhibition rate of tyrosinase increased gradually, showing a significant dose dependence. The inhibition rate of tyrosinase at a high dose (1.0 g/mL) of LRB was similar to that of glabridin. This phenomenon suggests that the content of licorice in LRB may have a significant effect on tyrosine kinase inactivation. Therefore, this study shows that LRB can significantly inhibit tyrosinase activity, and its inhibition effect is positively correlated with concentration. Fig. 6. [133]Fig. 6 [134]Open in a new tab In vitro tyrosinase inhibition rate statistical analysis graph of licorice rose drink (**p＜0.01 indicates a very significant difference compared with the control group). 3.4. In vivo validation 3.4.1. Characterization of the skin condition of experimental animals The effect of LRB on the skin of melasma model mice was evaluated by measuring the skin state of the mice. As shown in [135]Fig. 7, after 30 days of intramuscular injection of progesterone and UV irradiation. Compared with the control group, the model group had obvious surface damage, redness, dryness, scab, and coloration ([136]Fig. 7a and b). However, compared with the model group, the skin surface of the positive group was smooth without skin pigmentation ([137]Fig. 7b and c). Similarly, after administration of low, medium, and high doses of LRBs ([138]Fig. 7d–f), compared with the model group, the skin surface of mice in the administration group improved significantly, and the effect increased with the increase of doses. The results showed that LRB could significantly improve the skin color, redness, dry peeling, sunburn, and pigmentation of melasma model mice. It showed a good effect in preventing melasma. Fig. 7. [139]Fig. 7 [140]Open in a new tab Effect of LRB on local appearance of skin in melasma model mice. Control group (a), model group (b), Vit C positive control group (c), LRB low-dose group (0.25 g/mL) (d), LRB medium-dose group (0.50 g/mL) (e), LRB high-dose group (1.0 g/mL) (f). 3.4.2. Histopathological analysis of skin The histopathological characteristics of the skin of mice with the melasma models were evaluated by observing the histopathological sections of mouse skin. As shown in [141]Fig. 8, the epidermis and dermis of the blank group were intact without thickening, edema, inflammation, necrosis, and so on. However, compared with the control group, the epidermal epithelial cells in the model group proliferated significantly, with more layers of cells, local necrosis, inflammatory cells, edema, and obvious inflammatory phenomenon, hair and sebaceous glands proliferated ([142]Fig. 8a and b). Compared with the model group, the epithelial cells in the epidermis of the positive group were arranged neatly without obvious hyperplasia or thickening, and most of them showed no obvious inflammation, necrosis, edema, etc. Similarly, compared with the model group, the epidermis and dermis of the mice in the treatment group were basically intact, without obvious necrosis, edema, and inflammation, and the effect was more obvious with the increase in the dosage ([143]Fig. 8a and b). The results showed that the LRB could repair the skin injury and skin pigmentation of melasma model mice, and improve the skin thickening, inflammation, necrosis, and congestion of the model mice. In addition, the effective rate of skin pathology experiments was 100 % by statistical analysis. Fig. 8. [144]Fig. 8 [145]Open in a new tab HE staining to observe the effect of LRB on skin pathology of melasma model mice at 100× (a) and 200× (b). 3.4.3. Effect of LRB on tyrosinase content in vivo Tyrosinase plays an important role in the formation of melanin [[146]39]. By reducing the activity of tyrosinase, the synthesis and deposition of melanin can be blocked, thereby alleviating or improving the appearance of melasma [[147]36]. For example, clinical trial results by Hantash et al. demonstrate that a proprietary oligopeptide called Lumixyl, through competitive inhibition of tyrosinase activity, has the potential to be effective in treating melasma [[148]28]. The inhibitory activity of LRB on tyrosinase in vivo was studied. As shown in [149]Fig. 9, the tyrosinase activity in the serum and skin of the model group was significantly higher than that of the control group. Compared with the model group, tyrosinase activity in serum and skin decreased significantly in the positive group ([150]Fig. 9a and b). Similarly, different doses of LRB were given after intervention. In comparison to the model group, the tyrosinase activity of serum and skin decreased, and the effect was more significant with the increase in dosage. Among them, LRBs inhibited the activity of serum tyrosinase strongly ([151]Fig. 9a and b). The results showed that LRB could significantly inhibit the tyrosinase activity in the serum and skin of melasma model mice, especially the activity of serum tyrosinase. It is suggested that LRB can prevent and cure melasma by inhibiting melanin formation and precipitation. Fig. 9. [152]Fig. 9 [153]Open in a new tab Effect of LRB on serum (a) and skin (b) tyrosinase content in melasma model mice (Note: # #p < 0.01 indicates extremely significant difference compared with the control group, * *p < 0.01 indicates extremely significant difference compared with the model group). The studies of in vitro and in vivo tyrosinase both indicate that LRB can significantly inhibit tyrosinase activity during the process of melanin synthesis, and the inhibitory effect is dose-dependent. In vitro study, the inhibitory effect of LRB on tyrosinase gradually increases with the increase of LRB concentration and even approaches the positive control glabridin. In vivo study shows that LRB significantly reduces the activity of tyrosinase in both serum and skin of the mouse model of melasma, particularly in serum. The results showed that LRB significantly inhibited tyrosinase in vitro and in vivo, and showed good biological activity. This finding provides a scientific basis for further research on the application of LRB in the prevention and treatment of melasma and offers guidance for optimizing its formulation and dosage. 3.4.4. Analysis of antioxidant enzymes in serum of LRB When the balance of oxidizing reactions is disrupted, an excess of oxygen free radicals will be generated in the body, reducing the activity of antioxidant enzymes such as SOD and leading to the formation of lipid peroxidation products (LPO) through lipid peroxidation. LPO is unstable and rapidly decomposes, producing aldehydes. The end product MDA also increases, rapidly attacking phospholipids and proteins, causing oxidative damage to melanocytes [[154]40]. This further promotes the oxidation reaction of tyrosinase, leading to an increase in melanin production and its accumulation in the basal layer of the skin, which is one of the mechanisms underlying pigmentary skin diseases such as melasma [[155][41], [156][42], [157][43]]. Therefore, enhancing the activity of the antioxidant enzyme SOD, and reducing the level of MDA are of great significance for the prevention and treatment of melasma. SOD plays a vital role in the balance of oxidation and antioxidation [[158]44]. This enzyme can scavenge oxygen free radicals to protect cells from damage [[159]45]. The effect of LRB on SOD activity in the serum of melasma model mice is shown in [160]Fig. 10a. Compared with the control group, the SOD activity in the serum of the model group was significantly lower, indicating that the model was basically successful in terms of SOD activity. Compared with the model group, the SOD activity of the LRB and Vit C positive control group was significantly enhanced, and the SOD activity of the high dose group was the most significant. Fig. 10. [161]Fig. 10 [162]Open in a new tab Effect of LRB on serum SOD activity (a) and MDA content (b) in melasma model mice (Note: # #p < 0.01 indicates extremely significant difference compared with the blank control group, * *p < 0.01 indicates extremely significant difference compared with the model group). The content of MDA reflects the severity of the free radical attack [[163]46]. The effect of LRB on serum MDA content in melasma model mice is shown in [164]Fig. 10b. In comparison to the control group, the serum MDA content of the model group was significantly increased, indicating that the model was basically successful. Compared with the model group, the content of serum MDA in every dose group was significantly lower, and the content of serum MDA in the high dose group was lower than that in the positive control group Vit C. In conclusion, LRB could significantly increase the activity of SOD and decrease the content of MDA in the serum of melasma model mice and the effect is more significant with the increase in the dose. This provides a scientific basis for using LRB as a preventive and therapeutic strategy for melasma. 4. Conclusions In this study, the molecular mechanism of LRB in the treatment of melasma was studied by network pharmacology, molecular docking and in vitro and in vivo experiments. Network pharmacological studies have shown that isolicoflavonol, quercetin, and kaempferol are the main active ingredients in LRB for the treatment of melasma. Tyrosinase is the core target of LRB drink anti-melasma. The molecular docking results show that the three representative compounds interact with tyrosinase target proteins through hydrogen bonds and π bonds. In vitro tyrosinase assay showed that the activity of tyrosinase was inhibited by LRB, and the inhibition rate of high-dose LRB to tyrosinase was close to that of the positive control glabridin. In vivo animal experiments showed that LRB could significantly reduce the tyrosinase activity in the serum and skin of melasma model mice, increase the SOD activity and decrease the MDA content. The experimental results of skin state and pathological section showed that LRB had a good repair effect on skin damage and skin pigmentation in melasma model mice, and could significantly improve the symptoms of epidermal thickening, inflammation, necrosis, and congestion in model mice, showing a good therapeutic effect. In conclusion, LRB can inhibit the tyrosinase activity of melanocytes and melanoma cells in local skin tissue by increasing the activity of the SOD enzyme and decreasing the content of MDA. LRB can promote the oxidation and reduction of skin cells, reduce the production of free radicals, inhibit the formation of melanin, and then effectively treat melasma. Ethical approval and consent to participate All animal experiments were performed in accordance with the “Guiding Principles in the Care and Use of Animals” (China), and approved by the Ethics Committee of Southern Medical University (L2019036, date of approval: April 13, 2019). Consent for publication The manuscript is published with the consent of all authors. Funding This work was financially supported by the National Natural Science Foundation of China [grant number 82074023, 81874346]. Data availability Data supporting the results of this study may be obtained from the corresponding author upon reasonable request. Institutional review board statement All animal experiments were performed in accordance with the “Guiding Principles in the Care and Use of Animals” (China), and approved by the Ethics Committee of Southern Medical University (L2019036, date of approval: 13 April 2019). CRediT authorship contribution statement Dan Zhai: Writing – original draft. Yi Hu: Writing – review & editing. Li Liu: Visualization. Zhuxian Wang: Methodology. Peiyi Liang: Methodology, Investigation. CuiPing Jiang: Supervision, Methodology. Hui Li: Data curation. Quanfu Zeng: Formal analysis, Data curation. Hongkai Chen: Resources, Data curation. Yufan Wu: Validation, Software, Data curation. Yinglin Guo: Formal analysis, Data curation. Yankui Yi: Supervision, Data curation. Chunyan Shen: Supervision, Conceptualization. Qiang Liu: Supervision, Conceptualization. Hongxia Zhu: Supervision, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement