Kaempferol, AAPH, superoxide dismutase (SOD), malondialdehyde (MDA), and cytotoxicity assay (CCK-8) kits were obtained from Solarbio (Beijing, China). 3,5-dimethylphenol was obtained from Life Technologies (Carlsbad, CA, United States). Phosphate-buffered saline (PBS) was procured from Biological Industries (Ridgefield, CT, United States). Penicillin–streptomycin solution was obtained from ScienCell (San Diego, CA, United States). Dulbecco’s modified Eagle’s medium (DMEM) and trypticase were purchased from HyClone (Logan, UT, United States). Fetal bovine serum (FBS) was procured from Gibco (Waltham, MA, United States). A BCA Protein Quantitation Kit was obtained from Thermo Fisher Scientific (Waltham, MA, United States).

Vernonia anthelmintica (L.) Willd. was procured from the Eric Uygur medical room in Urumqi (Xinjiang Province, China). Subsequently, the chief pharmacist of the Pharmaceutical Research Institute of the Xinjiang Uygur Autonomous Region identified this sample as dried mature seeds of V. anthelmintica (L.) Willd. The extraction process involved placing the seed powder in a sealed contained and adding 50 mL of 60% ethanol solution. This mixture was then subjected to ultrasonic extraction at 40°C for 50 min, followed by filtration. Subsequently, 40 mL of 60% ethanol solution was added to the residue before further ultrasonic extraction at 40°C for 20 min, followed by filtration. The filtrates from both steps were then combined, before a final filtration with 10 mL of hot ethanol solution (60% volume fraction, 56°C preheat). The resulting filtrate was then cooled down to 25°C and a 60% ethanol solution was adjusted to 100 mL. Finally, the sample was shaken and left to stand for future use.

First, we analyzed the V. anthelmintica (L.) Willd. extract using SRM/MRM. Initially, 500 µL of the sample was placed in a 2-mL centrifuge tube, for centrifugation at 4000 rpm for 5 min. The supernatant was then removed, with 5 µL aliquots being placed into a 96 well plate for mass analysis. The sample was then placed in an ACQUITY UPLC I-Class system (Waters, American), for SRM/MRM under the following conditions: An autosampler temperature of 4°C; Mobile phase A and B of 0.1% formic acid in water and acetonitrile, respectively. The gradient elution procedure used was as follows: B increased from 5% to 20% between 0 and 3 min; B was then maintained at 20% between 3 and 4.3 min; at 4.3–9 min B increased from 20% to 45%; then, B increased from 45% to 98% between 9 and 11 min; at 11–13 min B was maintained at 98%; B then decreased from 98% to 5% between 13 and 13.1 min; and, finally, B was maintained at 5% at 13.1–15 min. The flow rate of this system was set at 400 μL/min, with an injection volume of 5 µL.

For mass spectrometry (MS), a Sciex 5500 QTRAP, mass spectrometer (AB SCIEX, American) was used, which was capable of operating in both positive and negative ion switching modes. The positive ion source parameters were as follows: source temperature, 550°C; Gas 1, 55 psi; Gas 2, 50 psi; curtain gas (CRU), 30 psi; ion spray voltage floating (ISVF), 5–500 V. The negative ion source parameters were as follows: source temperature, 550°C; Gas 1, 55 psi; Gas 2, 50 psi; CRU, 30 psi; ISVF, 4500 V.

In this study, human keratinocytes (HaCaT; Kunming Cell Bank, Chinese Academy of Sciences, China) were cultured in DMEM supplemented with 10% FBS and a 1% penicillin–streptomycin solution. These cells cultured grown at 37°C in a humidified atmosphere with 5% CO₂ and 10% a copper sulfate solution. Subsequent experiments were performed 24 h after the cells were seeded. Specifically, HaCaT cells were seeded at a density of 8×10⁴ cells/well into 96-well plates for 12 h and treated with various concentrations (10, 15, 20, 25, 30, 40, 50, or 60 mM) of AAPH. Alternatively, a subset of HaCaT cells was pre-treated with various concentrations (10, 20, 30, 40, 60, 80, or 100 µM) of kaempferol within the medium for 12 h to determine the optimal treatment concentration. Subsequently, the pre-treated cells were cultured for an additional 12 h with 25 mM AAPH to induce oxidative damage. Three replicates were conducted per group for all the experiments; untreated cells and cells treated solely with AAPH were used as control groups.

To evaluate the cellular toxicity of AAPH and the protective effects of kaempferol, we employed the CCK-8 assay on HaCaT cells. After treatment with the aforementioned concentrations of AAPH and kaempferol, 10 µL of the CCK-8 kit solution was added to each well. Subsequently, the cells were incubated for 2 h at 37°C. The absorbance of each sample was measured using a microplate reader (Thermo, Multiskanm FC, American).

Cold PBS was used to collect kaempferol-treated and untreated HaCaT cell samples following centrifugation at 157 g for 3 min, according to the instructions provided in the respective enzyme kits. These cells were then homogenized with cold PBS, and corresponding commercial kits were used to extract the supernatant; this supernatant was then analyzed for MDA and SOD activity levels.

To assess the protective effect of kaempferol against oxidative stress–induced apoptosis in HaCaT cells, we employed a Hoechst assay. Cells pre-treated with 10, 20, and 30 µM kaempferol were used as the experimental groups for this assay. After cell treatment, 5 μg/mL Hoechst staining solution was added to the samples, before incubation for 10–15 min at 25°C in the dark. Subsequently, 400 µL of 1× binding buffer was added to the samples, ensuring thorough mixing before being placed on ice. Finally, cellular images were taken within 1 h under an Olympus fluorescence microscope. This experiment was repeated thrice.

In this study, SOD activity in HaCaT cells was evaluated using the tetrazolium nitro blue method. First, HaCaT cells in the logarithmic growth stage were prepared by digestion and PBS washing. The cell suspensions were then centrifuged at 157 g for 5 min. HaCaT cells were seeded in 6-well plates at a density of 1×10⁶ cells/well, and cultured for 12 h at 37°C in a humidified atmosphere with 5% CO₂. When the cells had adhered to the wells and reached a density of approximately 80%, they were treated with various concentrations (10, 20, or 30 µM) of kaempferol for 12 h. Subsequently, the pre-treated cells were cultured for an additional 12 h with 25 mM AAPH. The cell suspensions were then mixed thoroughly using a vortex mixer (ice bath; power, 20%/200 W; ultrasound, 3-s, 10-s intervals, 30 repeats) before centrifugation at 8000 g at 4°C for 10 min. Subsequently, the corresponding reagents were added to the cell suspension according to the manufacturer’s instructions. Finally, the mixture was thoroughly mixed before culturing at 37°C for 30 min. Cell suspension samples were then placed in a 1-mL glass cuvette, and maximal absorbance was measured at 560 nm using a spectrophotometer (Thermo, Multiskanm FC, American). One unit of SOD activity was defined as the amount of enzyme required for 50% activity inhibition.

In this study, MDA concentration was used to assess lipid peroxidation in the HaCaT cells. First, the cell suspensions were mixed thoroughly using a vortex mixer (ice bath; power, 20%/200 W; ultrasound 3-s, 10-s intervals, 30 repeats) before centrifugation at 8000 g at 4°C for 10 min. The corresponding reagents were then added to the cell suspensions according to the manufacturer’s instructions. Subsequently, the mixture was thoroughly mixed and incubated at 100°C for 60 min, cooled in an ice bath, and centrifuged at 10,000 ×g for 10 min. Finally, 200 µL of each supernatant was transferred into a microglass cuvette or 96-well plate; maximal absorbance was then determined at 450 nm, 532 nm, and 600 nm using a spectrophotometer. MDA content was calculated according to the identified protein concentration.

All statistical analyses were conducted using SPSS (version 22.0; IBM Corp., Armonk, NY, United States). Statistical significance was set at p < 0.05.

Ethanolic extracts obtained from V. anthelmintica (L.) Willd. Seeds were subjected to quantitative flavonoid testing, which revealed a total flavonoid content of 0.97 g per 100 g. Then, we conducted SRM MS analysis following high-performance liquid chromatography and SRM/MRM; this analysis identified 36 flavonoid compounds. These compounds were subsequently categorized according to their structure: flavonoid glycosides (9/36), isoflavonoids (9/36), flavans (7/36), flavones (5/36), and others (6/36). Notably, all of the resultant curves exhibited excellent linear regressions with coefficients of determination (R ²) ranging from 0.9963 to 0.9999 (Figure 1). The top five compounds with the highest contents were saccarol (12650.98 ng/100 g), isquercitrin (7293.86 ng/100 g), meltein (4503.36 ng/100 g), violet (4320.83 ng/100 g), and naringenin (3390.59 ng/100 g).

Overall, 36 flavonoid compounds were obtained from the ethanolic extract of V. anthelmintica (L.) Willd. Seeds. To identify the active ingredient of this herb, we utilized databases to screen bioactive compounds and ADME properties in V. anthelmintica (L.) Willd. When the screening criteria (OB ≥ 30%; DL ≥ 0.18) were applied, we identified 14 compounds with druggability in V. anthelmintica (L.) Willd. (Table 1).

In this study, we obtained a total of 1286 vitiligo-related genes from various databases, including GeneCards, OMIM, and TTD. Subsequently, we identified a total of 211 target genes associated with the 14 compounds selected for further analysis, eliminating any overlapping targets obtained from the GeneCards, PubChem, and Swiss target prediction databases. After identifying the compound-target vitiligo-related genes, we visualized the corresponding compound-target interaction network, constructing a network that consisted of 227 nodes and 963 edges (Figure 2A). The five compounds with the highest degree of interaction in this network were kaempferol, isorhamnetin, kaempferide, quercetin, and liquiritigenin (degree = 101). Further, by taking the intersection between compound–target genes and vitiligo-related genes from the Wayne database, we identified the compounds target and vitiligo-related gene set (Figure 2B); this consisted of 43 common targets, which were used for further analysis.

As shown in Figure 2C, the PPI network contained candidate targets of pharmaceutical ingredients for treating vitiligo and its interacting proteins. This network comprehensively summarizes the internal network of the pharmaceutical ingredients of V. anthelmintica (L.) Willd. involved in treating vitiligo. Finally, the NetworkAnalyzer and CytoNCA functions in Cytoscape were used to identify the core targets by analyzing various key properties, including the closeness, degree, and betweenness centrality (BC). In this analysis, a greater degree value indicates a more significant role in the network. r. The top five nodes with the highest degree values were determined to be AKT1 (d = 60), VEGF-A (d = 50), ESR1 (d = 44), IL2 (d = 30), and MPO (d = 24). The degree values of the top 10 nodes are shown in Figure 2D. Therefore, these core targets are postulated to play a major role in the treatment of vitiligo using V. anthelmintica (L.) Willd.

GO enrichment analysis revealed that the biological processes associated with the targets of the active components in V. anthelmintica (L.) Willd. (Figure 2E), and vitiligo primarily involve responses to inorganic substances, regulation of mitochondrial membrane potential, and mitochondrial organization. The cell components identified were predominantly enriched in the phosphatidylinositol 3-kinase complex, the extrinsic component of the membrane, and the protein–DNA complex. The associated molecular functions primarily involved protein homodimerization, steroid binding, and transcription coactivator binding.

Biological pathway enrichment analysis of the target genes was also performed using the KEGG database. The enriched pathways identified by KEGG analysis included the PI3K−Akt signaling pathway, chemical carcinogenesis receptor activation, the estrogen signaling pathway, endocrine resistance, and C-type lectin receptor signaling pathways. Among them, the PI3K-AKT signaling pathway was determined to be particularly important. As shown in Figure 2F, the PI3K-AKT signaling pathway was linked to several key functions, including apoptosis, cell proliferation, cell cycle regulation, protein synthesis, and metabolism. Furthermore, it exhibits synergistic crosstalk with various signaling pathways, including the VEGF, MAPK, JAK/STAT, NFκB signaling pathways.

Kaempferol (d = 101) was identified as one of the compounds with the highest degree of association with the constructed networks, according to the network pharmacology analysis, with OB and DL values of 41.88% and 0.24, respectively. Furthermore, in this study, we used detected a kaempferol content of 62.96 ng per 100 g of V. anthelmintica (L.) Willd.seed ethanolic extract by MRM approach. However, the specific mechanism of action remains unclear. Next, we conducted molecular docking simulations to explore the binding interactions between kaempferol and five target proteins (AKT1, VEGFA, ESR1, PTGS2, and IL2) that exhibited the highest degree of association with the established networks (Figure 3).

The molecular docking analysis used in this study simulated the binding of kaempferol to the potential key targets, AKT1, MMP2, ESR1, IL2, PTGS2, and MPO. Diagrams of the ligand and potential target docking results are shown in Figure 3. Overall, kaempferol was able to bind to the ARG-86, GLU-85, GLY-16, THR-87, and ARG-67 residues of AKT1 with a binding energy of −6.42 kcal·mol-1. The binding energy of Kaempferol to MMP2 residues, including CYS-29, SER-25, ARG-39, and THR-31, was −5.14 kcal·mol-1.

To investigate the role of kaempferol in the treatment of vitiligo, we constructed a vitiligo oxidative stress model. We first treated HaCaT cells with different concentrations of AAPH for 24 h in vitro. In terms of cell morphology, cells in the oxidative stress model group compare with control group displayed slightly elliptical, oval, or round morphology, with blurred edges and reduced adherence (Figure 4B). Nonetheless, after intervention with kaempferol at different concentrations (10, 20, and 30 mM), there was partial restoration of cell morphology (Figures 4C–E). The cell viability results revealed a concentration-dependent reduction in cell viability as the concentration of AAPH increased (Figure 5A). Moreover, cell viability in the AAPH-alone group (>25 mM) was significantly lower than that of the control group (no AAPH treatment), indicating that AAPH induces cytotoxic damage in HaCaT cells (p < 0.001). Additionally, the half-maximal inhibitory concentration (IC₅₀) value of AAPH was determined to be approximately 25 mM. Therefore, 25 mM AAPH was used in future experiments to establish a HaCaT cell oxidative stress model. Subsequently, we confirmed that the concentration of kaempferol treatment can effect HaCaT cell viability (Figure 5B).

The CCK-8 results revealed the application of kaempferol at concentrations ranging from 10 to 30 µM led to a significant increase in cell viability within AAPH-treated cells, without any apparent cytotoxic effects; in particular, treatment with 20 µM kaempferol exhibit the highest increase in cell viability and provide the highest cytoprotective effect (Figure 5C).

To investigate the effects of kaempferol pretreatment on AAPH-induced apoptosis in HaCaT cells, we conducted propidium iodide (PI) staining (Figure 5D). The corresponding results indicated that the apoptosis rates for the control group, AAPH-alone group, AAPH with 10 μM, 20 µM kaempferol and 30 µM Kaempferol were 14.87% ± 0.35%, 28.03% ± 0.24%, 32.53% ± 0.42%, 14.47% ± 0.42% and 20.37% ± 0.32%, respectively. Thus, when compared to the AAPH-alone group, pre-treatment with 20 µM kaempferol yielded the most significant reduction in apoptosis.

Next, we evaluated the effects of different concentrations of AAPH on MDA content, SOD enzyme activity, and ROS activity in HaCaT cells. In comparison to the control group, ROS activity (Figure 5E) and SOD level (Figure 5F) were significantly reduced, while MDA content (Figure 5G) was significant increased in HaCaT Cells that were exposed to 25 mM AAPH for 24 h. Nonetheless, pretreatment with 10, 20, or 30 µM kaempferol led to a significant reduction in the elevated MDA content and ROS activity, while increasing the decreased SOD level induced by AAPH. These findings suggest that kaempferol may ameliorate the oxidative and antioxidant imbalances induced by AAPH through its effects on MDA, SOD, and ROS in HaCaT cells.

Finally, we evaluated the effects of kaempferol on the expression of HO-1, GCLC, GCLM, Nrf2, AKT, NQO1, Keap1 and PI3K. Compared with the control group, 25 mM AAPH can increase the cellular expression levels of HO-1, GCLC, GCLM, Nrf2, NQO1 and Keap1 mRNA, and decrease AKT and PI3K mRNA (Figure 6). Low doses of kaempferol increased Nrf 2, HO-1 and GCLC mRNA expression levels in HaCaT cells, and high doses of KP only increased the intracellular Nrf2 mRNA expression levels. Nrf two protein expression in HaCaT compared with the control group. Different doses of KP can increase the intracellular Nrf two protein expression in a concentration-dependent manner as compared to the model group (Figure 6I).