Mechanistic study on the treatment of androgenetic alopecia with Platycladus orientalis leaf extract via regulation of the VEGFA/Bcl2 pathway: An in silico and in vitro study

Abstract

To identify the active compounds of Platycladus orientalis leaf extract (POE) and quantify its efficacy against androgenetic alopecia (AGA) via vascular endothelial growth factor A (VEGFA)/B-cell lymphoma 2 (Bcl-2) signaling. Active compounds and their potential targets in POE were first identified. A compound–target interaction network was constructed using network pharmacology approaches, and molecular docking was employed to evaluate the binding affinities of key compounds to their predicted targets. Furthermore, in vitro experiments were conducted to assess the effects of key bioactive constituents on the expression levels of proteins involved in the VEGFA/Bcl2 pathway. POE promotes dermal papilla cell proliferation and angiogenesis by activating VEGFA/Bcl-2 signaling. Network pharmacology screened 28 compounds against 506 androgenetic alopecia targets; the top 6 (amentoflavone, isoquercitrin, afzelin, phloretin, pinusolide, and quinic acid) were docked to VEGFA (threshold Vina score < −7.0). POE at 3.13 µg mL⁻¹ increased dermal papilla cell number by 15.6 ± 1.2% versus control (P < .01) and up-regulated VEGFA (0.76 ± 0.02 vs 0.64 ± 0.03), Bcl-2 (0.86 ± 0.01 vs 0.79 ± 0.01) and phosphorylated B-cell lymphoma 2 (0.81 ± 0.01 vs 0.71 ± 0.01); all P < .01. Efficacy matched 10 µg mL⁻¹ minoxidil (P > .05).

1. Introduction

Hair loss is a prevalent global health concern, with androgenetic alopecia (AGA) representing the most common form. AGA is a progressive condition characterized by the miniaturization of hair follicles, typically beginning during or after puberty. Epidemiological studies indicate that AGA affects approximately 70% of men over the age of 70 and up to 50% of women.^[1–3] In recent years, a noticeable trend toward earlier onset has emerged, with increasing incidence among individuals aged 20 to 30, significantly impacting patients’ quality of life and psychological well-being.^[4,5] The pathogenesis of AGA is multifactorial, involving complex interactions among genetic predisposition, hormonal regulation, and secondary influences from various systemic conditions.^[6–8] A hallmark pathological feature of AGA is follicular miniaturization, primarily driven by the action of dihydrotestosterone. Dihydrotestosterone binds to androgen receptors located in the dermal papilla of hair follicles, triggering a cascade of molecular events.^[9] Through modulation of multiple signaling pathways, androgens shorten the anagen (growth) phase, prolong the telogen (resting) phase, accelerate the senescence of hair follicle stem cells, and promote apoptosis via regulation of B-cell lymphoma 2 (Bcl-2) family proteins, ultimately leading to hair thinning and loss.^[10–12] Angiogenesis, in addition to inflammation, oxidative stress, and cellular aging, plays a critical role in the pathogenesis of AGA by influencing its blood supply and nutritional support.^[13,14] A clinical trial confirmed that oral minoxidil (MXD) elevates serum VEGF levels, correlating with improved hair growth in AGA patients.^[15] This modifiable role of VEGF is further supported by in vitro studies, where compounds like Kyoh and Ageratum conyzoides extract significantly upregulate its expression.^[16–18] Collectively, targeting VEGF presents a promising therapeutic strategy to ameliorate the follicular microenvironment and promote angiogenesis in AGA.

Current management of AGA includes pharmacological, surgical, and physical therapies. Topical MXD remains first-line due to its favorable safety profile, though efficacy is limited – only 30 to 40% of patients achieve moderate improvement after 4 to 6 months. Common limitations include variable response, initial shedding, and local adverse effects like hypertrichosis and irritation. These drawbacks have driven the adoption of combination therapies (e.g., MXD with finasteride or microneedling) and motivate the exploration of natural extracts as potential adjuncts or alternatives. In contrast, oral 5α-reductase inhibitors, though effective, are limited by sexual side effects.^[19–22] Traditional Chinese medicine (TCM) has long utilized Platycladus orientalis leaf for hair loss. According to the Compendium of Materia Medica, its powder mixed with sesame oil and applied topically can promote hair growth. Traditionally, it has been used both orally (e.g., as a decoction at 6–12 g) and topically (in washes or ointments) to address underlying patterns like “blood heat” and inflammation, which are linked to hair loss in TCM theory. Modern research further supports the potential of P orientalis and its compound formulations in managing alopecia.^[23–25] P orientalis leaf is considered a promising medicinal herb due to its abundance of bioactive constituents, including flavonoids, essential oils, and polysaccharides. These compounds, either alone or in combination with other herbs, have been shown to interfere with the pathological processes underlying AGA.^[26,27] However, current research on the pharmacological mechanisms and standardized preparations of P orientalis leaf remains limited. There is an urgent need to elucidate the active constituents and molecular regulatory mechanisms of P orientalis leaf through integrated approaches combining in vitro/in vivo experimentation with data mining and systems pharmacology. It is important to note that the interactions identified through network pharmacology are predictive in nature and served as the basis for our subsequent in vitro experimental design to confirm these proposed mechanisms. Such efforts will provide a scientific foundation for the development of novel plant-derived anti-alopecia therapeutics. The research workflow is illustrated in Figure 1.

Figure 1.

Research workflow diagram. AGA = androgenetic alopecia, KEGG = Kyoto Encyclopedia of Genes and Genomes.

2. Materials and methods

2.1. Network pharmacology study of P orientalis leaf in treating AGA

2.1.1. Prediction of drug targets for P orientalis leaf

The active compounds of P orientalis leaf were screened and imported into the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) to obtain the simplified molecular-input line-entry system structures of each active ingredient. These simplified molecular-input line-entry system structures were then used to predict targets on the Swiss Target Prediction platform (http://www.swisstargetprediction.ch/). We limit the Homo sapiens and use a Probability Score > 0 as the screening threshold.

2.1.2. Retrieval of disease targets for AGA

Using “Androgenic alopecia” as the keyword, 6 disease target databases were searched: DrugBank (https://go.drugbank.com/), GeneCards (https://www.genecards.org/), MalaCards (https://www.malacards.org/), OMIM (https://omim.org/), NCBI Gene (https://www.ncbi.nlm.nih.gov/gene), and DisGeNET (https://www.disgenet.org/home/). We use EXCEL to merge various database targets and search, annotate, and delete duplicate targets.

2.1.3. Mapping drug-component-shared targets and network construction

Through the aforementioned process, we obtained the active compounds and targets associated with P orientalis leaf, as well as the disease-related targets for AGA. The shared targets between the 2 sets indicate the potential targets of P orientalis leaf for AGA treatment. The “Drug-Component-Shared Targets” network was constructed and visualized using Cytoscape version 3.7.1.

2.1.4. Construction and analysis of protein–protein interaction (PPI) network

The shared targets were imported into the STRING database (https://string-db.org/) to obtain the PPI mapping using the medium confidence threshold (confidence value > 0.4). The network was visualized using Cytoscape software. Additionally, the Cytoscape plugin CytoHubba was employed for topological analysis of the PPI network to identify key targets as the critical targets of P orientalis leaf in treating AGA.

2.1.5. Biological enrichment analysis

Biological enrichment analysis was conducted on the shared targets of the drug and disease, as well as on key submodule targets. This included Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The Gene Ontology enrichment analysis was categorized into 3 aspects: molecular function, biological process, and cellular component. This analysis was performed using the R package clusterProfiler, with a significance threshold of P < .05. Enrichment analysis was conducted by screening for significant terms using adjusted P-values, and Benjamin–Hochberg was used for multiple testing correction. The top-ranked enriched terms were visualized using R software.

2.1.6. Molecular docking

Molecular docking was employed in this study to further analyze the interactions between the active components of P orientalis leaf and core target proteins, predicting binding modes and affinities. The crystal structures of the core target proteins were downloaded from the RCSB Protein Data Bank (https://www.rcsb.org/). The active ingredients of P orientalis leaf were exported in SDF format from the PubChem database. Molecular docking was performed using the CB-DOCK2 platform (https://cadd.labshare.cn/cb-dock2/php/index.php). CB-Dock2 is a blind docking tool for protein–ligand interactions based on AutoDock Vina, in which the Vina score corresponds to the docking energy. According to this study, Vina score < −7.0 suggests favorable binding between the receptor and ligand.^[28]

2.2. Experimental study on the effect of P orientalis leaf extract (POE) on the proliferation of dermal papilla cells (DPCs)

2.2.1. Experimental materials

The P orientalis extract used in this study is characterized as a brown powder with batch number 2024042201. Its physicochemical properties indicate that it is soluble in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL (20°C). The extract should be stored in a cool, dry place, protected from light. The positive control used was MXD, a product from Shanxi Zhendong Anxin Biopharmaceutical Co., Ltd., presented as a colorless to pale yellow clear liquid (specification: 4.5 g/90 mL or 50 mg/mL). This control should also be stored in a light-protected, sealed container at room temperature. The cell line used in this experiment was primary human DPCs, which were passaged in our laboratory. Additional reagents and materials are listed in Table S1, S2 and S3, Supplemental Digital Content 1–3.

2.2.2. Experimental method for assessing the effect of P orientalis leaf on DPC proliferation

2.2.2.1. Toxicity testing of the extract on DPCs

DPCs were obtained from Hefei Wanwu Biology, Product number: Delf-10698. This study utilized commercially available cell lines and did not involve human subjects or human tissue samples. Therefore, ethical review and approval were waived by the Institutional Ethics Committee. All in vitro experiments were conducted in accordance with relevant guidelines and regulations. Frozen primary DPCs were rapidly thawed at 37°C and resuspended in complete fibroblast culture medium (88% basal fibroblast culture medium + 1% fibroblast culture supplement + 10% fetal bovine serum [FBS] + 1% antibiotics). The cells were centrifuged at 1000 rpm for 5 minutes to obtain a pellet, which was then resuspended in the corresponding complete culture medium and incubated at 37°C in a 5% CO2 incubator. The growth of the cells was monitored, and upon reaching approximately 90% confluence, the cells were passaged. The existing culture medium was discarded, and the cells were washed once with PBS. Subsequently, 0.25% trypsin was added to digest the cells, which were monitored until rounded. The addition of complete culture medium halted the digestion process, and the cells were triturated to form a single-cell suspension, ensuring uniformity. The cells were plated at a specific ratio and cultured in an appropriate volume of medium at 37°C in a 5% CO2 incubator. Once confluent, the cell concentration was adjusted to 5 × 10⁴ cells/mL and seeded in a 96-well plate at 90 µL per well, and incubated for 24 hours at 37°C in a 5% CO2 incubator. The mother solution of the test extract was diluted 100-fold in complete culture medium to prepare the first test concentration, followed by sequential 2-fold dilutions to achieve final concentrations of 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, and 0.20 µg/mL. Each well received 10 µL of the corresponding sample or medium, and after 48 hours, CCK-8 assays were performed. Ten microliters of CCK-8 solution were added to each well, and the plate was incubated for 1 hour at 37°C in a 5% CO2 incubator, protected from light. The optical density values were measured at 450 nm using a microplate reader to analyze cell proliferation capacity.

Cell survival rate (%) = [(As–Ab)/(Ac–Ab)] ×100%; inhibition rate (%) = [(Ac–As)/(Ac–Ab)]×100%; As: absorbance of experimental hole (including cell, medium, CCK-8 solution, and subject solution); Ac: control air absorbance (medium containing cells, CCK-8, and without subject); and Ab: Blank hole absorbance (including culture medium, CCK-8 solution, without cells, and subject).

2.2.2.2. Experimental study on the proliferative effects of POE on DPCs

The maximum nontoxic concentration was used as the highest test concentration, which was then diluted in a 2-fold gradient. The mother solution of the test extract was prepared by accurately weighing 100 mg of P orientalis extract powder and dissolving it in 1 mL of DMSO, resulting in a concentration of 100 mg/mL, equivalent to 4630 mg/mL (1 g of extract powder = 46.3 g of liquid extract). This mother solution was then diluted 100-fold with culture medium, followed by a 4-fold dilution to prepare the first test concentration of 250 µg/mL. The positive control solution was prepared by adding 100 µL of MXD to culture medium to a final volume of 6.25 mL, creating a mother solution of 800 µg/mL, which was subsequently diluted in a 2-fold gradient with culture medium.

Frozen primary DPCs were rapidly thawed at 37°C, then resuspended in complete fibroblast culture medium (88% basal fibroblast culture medium + 1% fibroblast culture supplement + 10% FBS + 1% antibiotics). The cells were centrifuged at 1000 rpm for 5 minutes to obtain a pellet, which was resuspended in the corresponding complete culture medium and incubated at 37°C in a 5% CO2 incubator. The growth of the cells was monitored, and upon reaching approximately 90% confluence, the cells were passaged. The existing culture medium was discarded, and the cells were washed once with PBS. Subsequently, 0.25% trypsin was added to digest the cells, which were monitored until they became rounded. The addition of complete culture medium halted the digestion process, and the cells were triturated to form a single-cell suspension, ensuring uniformity. The cells were plated at a specific ratio and cultured in an appropriate volume of medium at 37°C in a 5% CO2 incubator. Once confluent, the cell concentration was adjusted to 5 × 10⁴ cells/mL and seeded in a 96-well plate at 90 µL per well, and incubated for 24 hours at 37°C in a 5% CO2 incubator. The mother solution of the test extract was diluted 100-fold with complete culture medium to prepare the first test concentration, followed by sequential 2-fold dilutions to achieve final concentrations of 250, 125, 62.5, 31.25, 15.63, 7.81, 3.9, and 1.95 µg/mL. The positive control mother solution was used as the first test concentration, and subsequently diluted in a 2-fold gradient to achieve final concentrations of 80, 40, 20, 10, 5, 2.5, 1.25, and 0.625 µg/mL. Each well received 10 µL of the corresponding sample or medium, and after 48 hours, CCK-8 assays were performed. Ten microliters of CCK-8 solution were added to each well, and the plate was incubated for 1 hour at 37°C in a 5% CO2 incubator, protected from light. The optical density values were measured at 450 nm using a microplate reader to analyze cell proliferation capacity.

Cell survival rate (%) = [(As–Ab)/(Ac–Ab)] ×100%; proliferation rate (%) = [(As–Ac)/(Ac–Ab)]×100%; As: absorbance of experimental hole (including cell, medium, CCK-8 solution, and subject solution); Ac: control air absorbance (medium containing cells, CCK-8, and without subject); and Ab: Blank hole absorbance (including culture medium, CCK-8 solution, without cells, and subject). The proliferation rate (%) was calculated relative to the untreated control group, which was set as 100% baseline.

2.2.2.3. Experimental study on the mechanism of proliferative effects of POE on DPCs

Accurately weigh 100 mg of P orientalis extract powder and dissolve it in 1 mL of DMSO to prepare a mother solution at a concentration of 100 mg/mL, equivalent to 4630 mg/mL (1 g of extract powder = 46.3 g of liquid extract). This mother solution was then diluted to the dosing concentrations of 3.12, 1.56, and 0.78 µg/mL using culture medium.

Frozen primary DPCs were rapidly thawed at 37°C and resuspended in complete fibroblast culture medium (88% basal fibroblast culture medium + 1% fibroblast culture supplement + 10% FBS + 1% antibiotics). The cells were centrifuged at 1000 rpm for 5 minutes to obtain a pellet, which was then resuspended in the corresponding complete culture medium and incubated at 37°C in a 5% CO2 incubator. Cell growth was monitored, and upon reaching approximately 90% confluence, the cells were passaged. The existing culture medium was discarded, and the cells were washed once with PBS. Subsequently, 0.25% trypsin was added to digest the cells, which were monitored until they became rounded. The addition of complete culture medium halted the digestion process, and the cells were triturated to form a single-cell suspension, ensuring uniformity. The cells were plated at a specific ratio and cultured in an appropriate volume of medium at 37°C in a 5% CO2 incubator. Once confluent, the cell concentration was adjusted to 1 × 10⁴ cells/mL and seeded in a 6-well plate at 2 mL per well, incubated for 24 hours at 37°C in a 5% CO2 incubator. The cells were divided into 4 groups: control, high-dose group (3.12 µg/mL), medium-dose group (1.56 µg/mL), and low-dose group (0.78 µg/mL). After discarding the culture medium from each well, 2 mL of fresh medium or corresponding sample medium was added, and the cells were cultured for 48 hours before protein extraction.

The cell culture supernatant was discarded, and the cells were washed with PBS. Lysis buffer (containing protease and phosphatase inhibitors) was added, and the cells were scraped off and collected into a 1.5 mL EP tube placed on ice for 30 minutes. Cells were then disrupted using an ultrasonic cell disruptor for 2 minutes at low temperature. After sonication, the lysate was centrifuged in a precooled centrifuge at 4°C, and the supernatant was collected. The pellet containing cell debris was discarded, and the protein-containing supernatant was transferred to a new 1.5 mL EP tube. A 5 µL aliquot of the protein solution was used to measure protein concentration using the BCA method. Standard solutions were prepared by serial dilution (2000, 1500, 1000, 750, 500, 250, 125, 25, and 0 μg/mL). The BCA working solution was prepared by mixing solution A and solution B in a 50:1 ratio. Both the protein standards and samples were added to a clear 96-well plate, with 200 µL of freshly prepared BCA working solution added to each well. The plate was incubated at 37°C for 30 minutes, and the absorbance at 562 nm was measured using a microplate reader to calculate the protein concentration. Protein samples mixed with 5 × SDS protein loading buffer were boiled in a metal bath at 95°C for 10 minutes and stored at −20°C for later use.

After the samples were thawed at room temperature, they were vortexed to mix. A total of 15 wells were prepared for gel electrophoresis, with 6 µL of sample loaded per well. The gel was run at 80 V for 30 minutes, followed by 120 V for 30 minutes to separate the target proteins. The separated proteins were transferred to a PVDF membrane at 300 mA for 60 minutes and then blocked for 60 minutes. The primary antibody was incubated overnight at 4°C, followed by 6 washes with TBST for 5 minutes each. The secondary antibody was incubated at room temperature for 1.5 hours, followed by additional washes. ECL chemiluminescence solution was prepared, and the membrane was immersed in this solution for 1 minute at room temperature before scanning.

2.2.3. Data statistics and analysis

Data analysis involved measuring the grayscale values of protein bands using ImageJ software, which served as an indicator of target protein expression levels. Relative quantification was performed using glyceraldehyde-3-phosphate dehydrogenase as an internal control, with the ratio of target protein to glyceraldehyde-3-phosphate dehydrogenase used as the relative expression level for statistical analysis. Relative expression levels were presented as mean ± SD, and one-way ANOVA was conducted on the results using GraphPad Prism 9.5 software.

3. Results

3.1. Network pharmacology study of P orientalis leaf in treating androgenic alopecia

3.1.1. Screening of active components and target prediction for P orientalis leaf

A total of 456 data points were obtained. By removing duplicates based on similar retention times and identical molecular weights from both positive and negative ion modes, 381 unique compounds were identified. Active components of P orientalis leaf were prioritized based on comparisons with the PubChem database, with a preference for those selected from TCM libraries. Compounds consistently reported in the literature regarding P orientalis leaf were identified, favoring components with relatively higher concentrations. The selection of 28 active components from P orientalis leaf was guided by multiple criteria, including relative abundance, citation frequency in the literature, inclusion in established TCM databases, and pharmacological relevance (Table 1). The total ion flow diagram is presented in Figure 2. Using the Swiss Target Prediction platform, target predictions for each component were made, resulting in 612 unique drug targets after removing duplicates. Figure 3A illustrates the network of active components and their corresponding targets of P orientalis leaf.

Table 1.

Information table of active components in Platycladus orientalis leaf.

Name	Formula	PubChemID	CAS	Content
Quinic acid	C7H12O6	6508	77-95-2	4.473
Pinusolide	C21H30O4	161721	31685-80-0	2.534
Quercetin 7-rhamnoside	C21H20O11	5748601	22007-72-3	2.206
Isoneobavachalcone	C17H14O5	5318608	/	1.174
Astragalin	C21H20O11	5282102	480-10-4	1.157
Isopimaric acid	C20H30O2	442048	5835-26-7	1.102
Citric acid	C6H8O7	311	77-92-9	0.795
Myricetin	C15H10O8	5281672	529-44-2	0.595
Isoquercitrin	C21H20O12	5280804	482-35-9	0.566
Kaempferol	C15H10O6	5280863	520-18-3	0.469
Afzelin	C21H20O10	5316673	482-39-3	0.379
Amentoflavone	C30H18O10	5281600	1617-53-4	0.257
Reynoutrin	C20H18O11	5320861	549-32-6	0.247
Neocryptomerin	C31H20O10	5320065	20931-36-6	0.198
(-)-Caryophyllene oxide	C15H24O	1742210	1139-30-6	0.142
Emodin-3-methyl ether/Physcion	C16H12O5	10639	521-61-9	0.137
Apigenin-7-O-β-d-glucoside	C21H20O10	5280704	578-74-5	0.120
Quercitrin	C21H20O11	5280459	522-12-3	0.091
Apigenin	C15H10O5	5280443	520-36-5	0.085
Shikimic acid	C7H10O5	8742	138-59-0	0.083
Quercetin	C15H10O7	5280343	117-39-5	0.071
Rutin	C27H30O16	5280805	153-18-4	0.059
Kaempferol-7-O-glucoside	C21H20O11	5480982	16290-07-6	0.014
Phloretin	C15H14O5	4788	60-82-2	0.009
2,4,6-Trihydroxy-2-(4-hydroxybenzyl)-1-benzofuran-3(2H)-one	C15H12O6	160803	5989-16-2	0.008
Cedrol	C15H26O	65575	77-53-2	0.005
Homoorientin	C21H20O11	114776	4261-42-1	0.004
EPICEDROL	C15H26O	522667	19903-73-2	0.003

Figure 2.

Total ion flow diagram of Platycladus orientalis leaf in positive and negative ion modes.

Figure 3.

(A) Network of active components and targets of Platycladus orientalis Leaf; (B) Venn diagram of common targets between Platycladus orientalis leaf and AGA; and (C) “Drug-Component-Common Targets” network. AGA = androgenetic alopecia, POE = Platycladus orientalis leaf extract.

3.1.2. Construction of disease target library for AGA

A total of 620 AGA-related targets were retrieved from 6 databases, including 402 from the GeneCards database, 112 from DisGenet, 48 from OMIM, 33 from NCBI Gene, 14 from MalaCards, and 11 from DrugBank. After removing duplicates among the targets identified from these 6 databases, 506 unique AGA disease targets were obtained.

3.1.3. Mapping of drug-component-common targets and network construction

The 612 targets identified from P orientalis leaf were mapped against the 506 AGA targets, resulting in 48 common targets (Fig. 3B). Additionally, a “Drug-Component-Common Targets” network was constructed, which includes 28 active components and 48 common targets (Fig. 3C).

3.1.4. Construction and analysis of PPI network

The 48 common targets were imported into the STRING platform to obtain the PPI network mapping. This network was further visualized using Cytoscape software (Fig. 4A), containing 47 nodes and 212 edges. The nodes were differentiated based on their Degree values; larger nodes and those with a redder hue indicate higher Degree values. Using the CytoHubba plugin, the top 10 key targets from the PPI network were identified based on their MCC values, ranked from highest to lowest as follows: vascular endothelial growth factor A (VEGFA), TP53, TNF, PPARG, IL1B, IGF1R, IGFBP3, VDR, AHR, and HPGDS, with VEGFA having the highest MCC value (Fig. 4B).

Figure 4.

(A) PPI network of common targets; (B) key targets in the PPI network; and (C) bar chart of KEGG pathway enrichment. PPI = protein–protein interaction, KEGG = Kyoto Encyclopedia of Genes and Genomes.

3.1.5. KEGG pathway enrichment analysis of common targets

The KEGG pathway analysis revealed that the top 5 significantly enriched pathways were: steroid hormone biosynthesis, MAPK signaling pathway, ovarian steroidogenesis, endocrine resistance, and Hedgehog signaling pathway. Among the top 20 pathways, VEGFA was involved in 9 (Fig. 4C). The adjusted p-values and enrichment scores of KEGG enrichment were showed in Table S4, Supplemental Digital Content 2.

3.1.6. Molecular docking

A total of 6 active components from TCM were selected for docking: pinusolide, phloretin, isoquercitrin, amentoflavone, afzelin, and quinic acid. The docking was performed against VEGFA, which had the highest MCC value. Figure 5A shows a heatmap of the best docking energies for each component, indicating that except for quinic acid, the other 5 components exhibited favorable docking interactions with VEGFA (vina score < −7.0), with amentoflavone showing the best docking performance. Figure 5B–G illustrate the optimal docking poses for each component with VEGFA. The specify docking grid, vina score were showed in Table S5, Supplemental Digital Content 3.

Figure 5.

(A) Docking energy heatmap; (B) Pinusolide-VEGFA; (C) Phloretin-VEGFA; (D) Isoquercitrin-VEGFA; (E) Amentoflavone-VEGFA; (F) Afzelin-VEGFA; and (G) Quinic acid-VEGFA. VEGFA = vascular endothelial growth factor A.

3.2. Study on the proliferative effects of POE on DPCs

3.2.1. Toxicity results of POE on DPCs

The effects of POE on cell viability and inhibition rates in DPCs are presented in Figure 6A and B. The maximum nontoxic concentration of the extract was determined to be 25 µg/mL, which was used as the starting concentration for subsequent proliferation experiments.

Figure 6.

(A) Effect of Platycladus orientalis leaf extract on cell viability; (B) effect of P orientalis leaf extract on cell inhibition rate; (C) effect of P orientalis leaf extract on cell viability; (D) effect of P orientalis leaf extract on cell proliferation Rat; (E) Effects of different concentrations of P orientalis leaf extract and Minoxidil on cell viability; and (F) effects of different concentrations of P orientalis leaf extract and Minoxidil on cell proliferation.

3.2.2. Proliferative effects of POE on DPCs

The impact of POE on cell viability and proliferation rates is summarized in Figure 6C and D. The results indicate that 5 tested concentrations of the extract (12.5–0.78 µg/mL) significantly increased cell viability compared to the control group, showing a significant difference (**P < .01, *P < .05). The highest cell proliferation rate was observed at 3.13 µg/mL of P orientalis extract, reaching 15.63%. Figure 6E shows that both 20 and 10 µg/mL of MXD significantly enhanced DPC viability. Figure 6F indicates that the proliferation rates of DPCs treated with various concentrations of P orientalis extract were lower than that of the 20 µg/mL MXD group (**P < .01, *P < .05), but there was no significant difference compared to the 10 µg/mL MXD group (P > .05).

3.2.3. Mechanistic insights of POE on DPCs

This study determined the high, medium, and low doses of POE (3.13, 0.78, and 1.56 µg/mL) through toxicity and proliferation experiments. Following treatment with different doses, Western Blot analysis was conducted to explore the effects of the extract on key target protein VEGFA and apoptosis-related proteins Bcl2 and phosphorylated B-cell lymphoma 2 (p-Bcl-2). The results indicated that in the high-dose group (3.13 µg/mL), the expression levels of VEGFA, Bcl2, and p-Bcl-2 proteins were significantly elevated compared to the control group (P < .01, P < .05), while medium and low doses did not exhibit significant effects on the target proteins (Table 2 and Fig. 7A–D).

Table 2.

Comparison of target protein expression among different groups.

Group	VEGF/GAPDH	Bcl-2/GAPDH	P-Bcl-2/GAPDH
Normal group	0.64 ± 0.03	0.79 ± 0.01	0.71 ± 0.01
Low dose group	0.66 ± 0.04	0.81 ± 0.02	0.75 ± 0.01
Medium dose group	0.68 ± 0.02	0.80 ± 0.02	0.73 ± 0.05
High dose group	0.76 ± 0.02**	0.86 ± 0.01**	0.81 ± 0.01*

Bcl-2 = B-cell lymphoma 2, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, p-Bcl-2 = phosphorylated B-cell lymphoma 2, VEGFA = vascular endothelial growth factor A.

^*P < .05 (compared to the normal group).

^**P < .01 (compared to the normal group).

Figure 7.

(A) Effect of Platycladus orientalis leaf extract on VEGFA protein expression levels in human dermal papilla cells; (B) effect of P orientalis leaf extract on Bcl-2 protein expression levels in human dermal papilla cells; (C) effect of P orientalis leaf extract on p-Bcl-2 protein expression levels in human dermal papilla cells; and (D) Western Blot (WB) analysis of target proteins (compared to the normal group, **P < .01, *P < .05). Bcl2 = B-cell lymphoma 2, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, p-Bcl-2 = phosphorylated B-cell lymphoma 2, VEGFA = vascular endothelial growth factor A.

4. Discussion

This study employed a combination of network pharmacology and molecular docking techniques to preliminarily explore the key active components, protein targets, and molecular pathways of POE in the treatment of AGA. Additionally, through cell experiments, we confirmed the effects of the extract on DPCs, providing a comprehensive analysis of its therapeutic effects on AGA.

The results from the network pharmacology analysis indicated that VEGFA had the highest co-occurrence among the core common targets of P orientalis leaf and AGA. Notably, VEGFA was involved in 8 out of the top 20 KEGG enrichment pathways, playing a critical role in the pathogenesis of AGA.^[29] VEGFA is a key factor regulating microvascular permeability and angiogenesis. Previous studies have confirmed that the expression levels and protein content of VEGFA are significantly reduced in the hair follicle stem cells of AGA patients.^[30,31]

Our findings demonstrate that P orientalis extract exhibits significant potential in treating AGA, with key active components including amentoflavone, isoquercitrin, afzelin, pinusolide, phloretin, and quinic acid. These components primarily act through mechanisms such as antioxidant stress response, anti-inflammation, antiaging, and metabolic regulation. Prior research has indicated that P orientalis can protect hair affected by AGA from ultraviolet-induced damage by scavenging hydroxyl radicals and reducing melanin free radicals generated by UV radiation.^[32] This study suggests that these chemical components may regulate VEGFA, exerting antiapoptotic effects, which expands the understanding of the mechanisms by which P orientalis treats AGA. Improving microcirculation in hair follicles and modulating the microenvironment of DPCs emerges as an important direction for future research.

Molecular docking results indicated that amentoflavone, isoquercitrin, and afzelin exhibited significantly better docking results compared to other compounds. These 3 compounds are flavonoids, which are important secondary metabolites in P orientalis. Amentoflavone, with its unique biflavonoid structure, may enhance molecular binding capacity to targets. Previous studies have shown that amentoflavone acts as a prostaglandin D2 inhibitor and possesses favorable pharmacokinetic properties with minimal skin adverse reactions, indicating substantial potential for further in vitro and in vivo studies, thus paving the way for its development as a safe and effective treatment for hair loss.^[33]

Both afzelin and isoquercitrin are flavonoid glycosides, and their glycosidic bonds provide binding sites for target interaction, though their intermolecular forces are relatively weaker compared to biflavonoids. This suggests that attention should be focused on flavonoids with multiple hydroxyl groups in P orientalis. Research has shown that isoquercitrin can promote hair growth by targeting MAPK and IGF-1R pathways, inducing autophagy and angiogenesis.^[34] Afzelin demonstrates strong antioxidant capacity through the Nrf2 pathway.^[35] Additionally, compounds such as pinusolide, phloretin, and quinic acid also show potential to improve pathological aspects of AGA, providing more active components for treatment options.^[36–39]

The extract of P orientalis demonstrated significant proliferative effects in DPC experiments, with a cell proliferation rate increase of 15.63% at a concentration of 3.13 µg/mL (P < .01), showing a clear dose-dependent relationship. Notably, the inhibitory effects of the extract exhibited a dose–response relationship at higher concentrations, suggesting its potential practical application in local administration or as part of compound formulations. The absence of significant changes in VEGFA and Bcl-2 protein levels at low and medium doses may indicate a threshold effect, where a minimum effective concentration is required to activate the VEGFA/Bcl-2 signaling pathway. Further dose–response studies are needed to confirm this hypothesis. Western blot analysis further confirmed that in the high-dose group (3.13 µg/mL), the expression of VEGFA, Bcl2, and p-Bcl-2 proteins was significantly upregulated (P < .01), consistent with the predictions from network pharmacology. VEGFA provides nutritional support to DPCs by promoting angiogenesis around hair follicles, while the upregulation and phosphorylation of Bcl2 family proteins (increased p-Bcl-2/Bcl2 ratio) indicate that the extract may promote hair growth by inhibiting apoptosis and extending the survival period of DPCs.^[40,41] Importantly, the proliferative effect of the extract at 3.13 µg/mL was comparable to that of MXD (10 µg/mL; P > .05), suggesting that it may serve as an alternative or complementary treatment to MXD at medium to high doses, highlighting its potential clinical application value.

In summary, this study suggests that POE, by regulating VEGFA expression and promoting the levels of Bcl2 and p-Bcl-2 proteins, inhibits apoptosis and thereby exerts therapeutic effects on androgenic alopecia (AGA). The specific mechanism is illustrated in Figure 8, where VEGFA supports DPCs through enhanced angiogenesis, while the upregulation and phosphorylation of Bcl2 family proteins promote hair growth by extending the survival period of DPCs. Further research indicates that flavonoids such as amentoflavone, isoquercitrin, and afzelin in P orientalis are key active components in treating AGA, working through synergistic mechanisms to regulate the aforementioned molecular pathways and demonstrating significant anti-hair loss potential. These findings provide theoretical and experimental support for the development of P orientalis as a natural anti-hair loss medication.

Figure 8.

Mechanism diagram of Platycladus orientalis regulating the VEGFA/Bcl2 pathway. Bcl2 = B-cell lymphoma 2, VEGFA = vascular endothelial growth factor A.

While our in vitro findings demonstrate that P orientalis up-regulates VEGFA and Bcl-2 in DPCs, these results do not guarantee similar efficacy in vivo. Translation to clinical benefit requires validation in appropriate animal models and human trials. Only the top-ranked VEGFA/Bcl-2 axis predicted by network pharmacology was experimentally tested. The remaining targets and pathways remain putative and await experimental confirmation. Synergism was inferred from network pharmacology (multi-compound and multi-target topology) but was not experimentally tested. Future work should apply fixed-ratio combination assays (e.g., Chou–Talalay method) to quantify synergistic, additive, or antagonistic interactions among the major flavonoids identified. No in vivo animal or human data. Exposure limited to 48 hour; chronic effects unknown. Batch-to-batch variability in POE flavonoid content (±15% observed in-house) may influence reproducibility; standardization markers (≥2% amentoflavone and ≥0.5% isoquercitrin) are proposed for future studies. P orientalis is a dark-brown hygroscopic powder prone to oxidation; micro-encapsulation (liposomes or cyclodextrins) could improve stability and skin deposition. Amentoflavone is classified as a generally recognized as safe flavonoid, yet any novel topical formulation must comply with ICH Q3A guidelines and undergo sensitization testing (OECD TG 406/442). As a botanical mixture, P orientalis may qualify as a botanical drug product under FDA 21 CFR 310.3(h), requiring standardized manufacturing, QC release specs, and a well-defined IND-enabling package.

5. Conclusion

This study reveals that the extract of P orientalis, particularly its flavonoid compounds amentoflavone, isoquercitrin, and afzelin, significantly promotes the proliferation of DPCs by regulating the VEGFA/Bcl2 signaling pathway and improving microcirculation in hair follicles, laying the foundation for new plant-based anti-hair loss medications for the treatment of androgenic alopecia. These preliminary in vitro data suggest that P orientalis flavonoids may promote DPC proliferation via VEGFA/Bcl-2 signaling; however, clinical efficacy remains to be established and should not be inferred from the current study. Reproducible results will require batch-to-batch standardization of P orientalis with quantitative specifications for amentoflavone, isoquercitrin, and afzelin as primary analytical markers. The identified flavonoids represent starting scaffolds for lead optimization, including structure-activity refinement and pharmacokinetic enhancement, before any therapeutic candidate can be advanced.

Author contributions

Conceptualization: Yan Jia, Changyong Luo.

Data curation: Siqi Zhao, Yujing Shi.

Methodology: Yueheng Liu.

Writing – original draft: Yijie Du.