I. Introduction
Alopecia is a common dermatological condition affecting both men and women, and androgenetic alopecia (AGA) is the most prevalent clinical form. AGA can adversely affect psychological well-being, self-image, and social quality of life. The two best-known pharmacological options, minoxidil and finasteride, require continued use and may be associated with limitations such as relapse after discontinuation, local irritation, hypotension-related concerns, sexual adverse effects, or restricted use in specific populations (Trüeb 2002;Iamsumang et al. 2020;Sankhwar & Khan 2025;Zhou et al. 2025). Accordingly, there is increasing demand for safe, naturally derived materials that can be developed as functional foods or cosmeceuticals for hair loss prevention and improvement.
The dermal papilla (DP), located at the base of the hair follicle, functions as a specialized mesenchymal signaling center that controls hair follicle morphogenesis, cyclic regeneration, and epithelial-mesenchymal interactions. Human hair follicle dermal papilla cells (HFDPCs) release multiple paracrine factors that regulate the proliferation, differentiation, and survival of surrounding epithelial cells during anagen. These factors include vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF7 and FGF10), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) (Madaan et al. 2018;Morgan 2014;Zhang et al. 2024).
Among the intracellular pathways involved in DP activation and hair cycle regulation, the Wnt/β-catenin and PI3K/Akt pathways are particularly important. Wnt/β-catenin signaling is essential for hair follicle formation and anagen induction. Stabilized β-catenin translocates into the nucleus and interacts with TCF/LEF transcription factors, leading to the expression of genes involved in cell proliferation and hair growth, including Cyclin D1 and VEGF (Enshell-Seijffers et al. 2010;Kim et al. 2024). The PI3K/Akt pathway also supports cell survival, proliferation, and de novo hair follicle regeneration (Manning & Cantley 2007;Chen et al. 2020). Although many plant-derived hair growth-promoting agents have been interpreted primarily through Wnt/β-catenin activation (Shin et al. 2020;Kim et al. 2021;Ryu et al. 2021;Kim et al. 2024), PI3K/Akt-centered mechanisms in HFDPCs remain less fully characterized.
Polygonum cuspidatum Siebold & Zucc. (Polygonaceae) is a perennial plant widely used in East Asian traditional medicine. Extracts of P. cuspidatum have been reported to modulate oxidative stress, inflammation, lipid metabolism, and cell signaling pathways, including PI3K/Akt-related signaling, in several experimental models (Tao et al. 2021;Ke et al. 2023). These biological activities may be associated with its diverse phytochemical constituents, as the rhizome contains stilbenes such as resveratrol and piceid, anthraquinones such as emodin, and various flavonoids (Peng et al. 2013;Ke et al. 2023). Among these constituents, resveratrol is a non-glycosylated stilbene with antioxidant and anti-inflammatory activities, while piceid is a glycosylated resveratrol derivative and a major stilbene form in P. cuspidatum with higher aqueous solubility that can be hydrolyzed to resveratrol. Emodin, a hydroxylated anthraquinone, has low aqueous solubility and oral bioavailability but has been reported to exert anti-inflammatory and anticancer effects (Zhang et al. 2022;Guerrero-Rubio et al. 2023). However, the effect of P. cuspidatum extract on HFDPC proliferation and the expression of hair growth-related factors has not been systematically evaluated.
Therefore, this study aimed to identify a hair growth-promoting candidate material through screening of six plant-derived extracts and to elucidate the cellular and molecular effects of the selected extract. Based on the screening results, P. cuspidatum aqueous extract (PCAE) was selected for further investigation. Dose-response analysis, Western blotting, and RT-qPCR were performed to assess the proliferative activity of PCAE and its effects on Akt phosphorylation, β-catenin/Cyclin D1 protein expression, DKK1 expression, and hair growth-related paracrine factor expression in HFDPCs.
II. Materials and Methods
1. Plant materials and preparation of extracts
Dried rhizome of Polygonum cuspidatum Siebold & Zucc. was purchased from an online commercial supplier, and the product was labeled as originating from Jecheon, Chungcheongbuk-do, Republic of Korea. Three P. cuspidatum extracts were prepared using distilled water, 50% ethanol, or 70% ethanol as extraction solvents. The dried powder was mixed with each solvent at a 10-fold volume and extracted at 80°C for 2 h with stirring. The extracts were filtered, concentrated, and freeze-dried. The dried extracts were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions at 100 mg/mL and diluted in culture medium immediately before use. The final DMSO concentration in cell culture did not exceed 0.1% (v/v). The aqueous extract is hereafter referred to as PCAE (P. cuspidatum aqueous extract). Extracts of Inula japonica flower, Castanea crenata flower, Glycyrrhiza uralensis, and Justicia procumbens were included as comparative or positive-control samples in the primary screening assay. The sample codes and extract names used in the screening are listed in Table 1.
2. Cell culture
HFDPCs were purchased from CEFObio (Seoul, Korea). Cells were cultured in CEFOgro™ Human Dermal Papilla Growth Medium supplemented with 10% fetal bovine serum and 0.5% penicillin-streptomycin at 37°C in a humidified incubator containing 5% CO2. The culture medium was replaced every 2–3 days, and cells were passaged at 80–90% confluence. Cells within passage 6 were used for all experiments.
3. Cell proliferation assay
Cell proliferation was measured using the MTT assay. HFDPCs were seeded in 96-well plates at 1 × 104 cells/well and allowed to stabilize for 24 h. For the primary screening, the plant-derived extracts listed in Table 1 were treated at 100 µg/mL for 24 h. Justicia procumbens aqueous extract, previously shown to promote hair growth-related responses, was used as a positive control (Kim et al. 2024). For dose-response analysis, PCAE, 50% ethanol extract, and 70% ethanol extract of P. cuspidatum were treated at 0, 12.5, 25, 50, 100, 200, 300, and 400 µg/mL for 24 h. After MTT reaction, absorbance was measured at 570 nm using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA). Cell proliferation was expressed as a percentage of the untreated control group.
4. Western blot analysis
To evaluate molecular signaling responses, HFDPCs were treated with PCAE at 0, 50, 100, and 200 µg/mL for 24 h. Cells were lysed using RIPA lysis buffer (Thermo Fisher Scientific Pierce, Rockford, IL, USA), and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (10–20 µg) were separated by 8–12% SDS-PAGE and transferred to PVDF membranes. Following protein transfer, nonspecific antibody binding was minimized by incubating the PVDF membranes in 5% skim milk prepared in TBST for 1 h. Subsequently, the membranes were exposed to the appropriate primary antibodies overnight at 4°C. The primary antibodies targeted Vinculin (Sigma-Aldrich; V9131), β-catenin (Cell Signaling Technology; #8480S), Akt (Cell Signaling Technology; #9272S), phospho-Akt (Ser473; Cell Signaling Technology; #4366S), and Cyclin D1 (Santa Cruz Biotechnology; sc-753). The membranes were then incubated with goat anti-rabbit IgG-HRP (Novus Biologicals; NB7160) or goat anti-mouse IgG-HRP (Novus Biologicals; NB7539). Protein bands were visualized using an ECL substrate and a Syngene G:BOX Chemi XX6 system. Band intensity was quantified using ImageJ software (version 1.54, NIH) and normalized to Vinculin or total Akt as appropriate.
5. Quantitative real-time PCR (RT-qPCR)
HFDPCs were treated with PCAE at 0, 50, 100, and 200 µg/mL for 24 h. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized using ReverTra Ace™ qPCR RT Master Mix (Toyobo, Osaka, Japan; FSQ-201). RT-qPCR was performed using SYBR® Green Realtime PCR Master Mix (Toyobo; QPK-201) and a ViiA 7 Real-Time PCR System (Applied Biosystems). The mRNA expression levels of IGF-1, VEGF, EGF, FGF7, FGF10, and DKK1 were normalized to GAPDH and calculated using the method. Primer sequences are listed in Table 2.
6. Statistical analysis
Statistical analysis was conducted using GraphPad Prism software (version 11.0; GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test was used to compare treatment groups. Statistical significance was defined as p < 0.05. In the figures, *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the untreated control group. Data are presented as mean ± standard error (SE).
III. Results and Discussion
1. Screening of plant-derived extracts for HFDPC proliferation-promoting activity
Six plant-derived extracts were screened at a single concentration of 100 µg/mL to identify candidate materials that promote HFDPC proliferation (Figure 1). The extract codes were as follows: A, Inula japonica flower 70% EtOH extract; B, Castanea crenata flower 50% EtOH extract; C, P. cuspidatum aqueous extract (PCAE); D, P. cuspidatum 50% EtOH extract; E, P. cuspidatum 70% EtOH extract; F, Glycyrrhiza uralensis aqueous extract; and PC, Justicia procumbens aqueous extract as the positive control.
The positive control markedly increased HFDPC proliferation, confirming the validity of the screening system. Among the test samples, PCAE (sample C) showed the strongest proliferative activity and significantly increased cell proliferation to approximately 125% of the untreated control. The 70% EtOH extract of P. cuspidatum (sample E) also significantly enhanced HFDPC proliferation, whereas the 50% EtOH extract (sample D) showed only a modest, non-significant increase under the screening condition. Inula japonica, Castanea crenata, and Glycyrrhiza uralensis extracts did not show statistically significant proliferative activity in this assay.
Because PCAE displayed robust activity and was prepared through a food-compatible aqueous extraction process, it was selected for subsequent dose-response and mechanistic studies. Water extraction is generally advantageous for functional food and cosmeceutical development because of lower solvent-related safety concerns, easier scale-up, and compatibility with traditional herbal preparation methods.
2. Dose-dependent effects of P. cuspidatum extracts on HFDPC proliferation
The effects of three P. cuspidatum extracts on HFDPC proliferation were examined over a concentration range of 0–400 µg/mL (Figure 2). PCAE significantly increased HFDPC proliferation at 50, 100, 200, and 300 µg/mL, with the strongest response observed around 100–200 µg/mL. Importantly, PCAE did not show apparent cytotoxicity even at 400 µg/mL, indicating a comparatively wide usable concentration range.
In contrast, the 50% and 70% EtOH extracts showed proliferative effects mainly at lower concentrations. The 50% EtOH extract significantly increased cell proliferation at 25–100 µg/mL, whereas marked decreases in cell viability were observed at 200 µg/mL or higher. Similarly, the 70% EtOH extract significantly increased proliferation at 12.5–100 µg/mL but reduced cell viability at higher concentrations. This pattern suggests that hydroethanolic extraction may enrich constituents that promote proliferation at low concentrations but become cytotoxic at higher levels, potentially including anthraquinone-related compounds, although chemical profiling is required to confirm this possibility (Peng et al. 2013).
Based on the overall results between efficacy and cytotoxicity, PCAE was selected as the safest and most suitable extract for mechanistic studies. Concentrations of 50, 100, and 200 µg/mL were used in subsequent Western blot and RT-qPCR analyses.
3. PCAE activates Akt signaling and reduces β-catenin protein abundance
To investigate the signaling mechanism underlying PCAE-induced HFDPC proliferation, Western blot analysis was performed after PCAE treatment at 0, 50, 100, and 200 µg/mL for 24 h (Figure 3A and 3B). PCAE increased the p-Akt/Akt ratio in a concentration-dependent manner, indicating activation of Akt signaling. In contrast, β-catenin protein levels were significantly reduced at 100 and 200 µg/mL. Cyclin D1 showed a modest increase at 50 µg/mL but did not reach statistical significance.
Because β-catenin was reduced by PCAE, DKK1 mRNA expression was further examined as a Wnt/β-catenin-related regulatory factor (Figure 3C). PCAE strongly increased DKK1 mRNA expression at all tested concentrations. This result is consistent with the observed reduction in β-catenin protein, as DKK1 is a secreted antagonist of canonical Wnt signaling that binds LRP5/6 and inhibits Frizzled/LRP receptor complex formation, thereby promoting β-catenin destabilization. In AGA pathology, DKK1 is considered a DHT-inducible factor in balding dermal papilla cells and can induce apoptosis in follicular keratinocytes (Kwack et al. 2008).
This protein and mRNA expression pattern differs from the mechanism commonly described for many natural hair growth-promoting extracts, in which Wnt/β-catenin activation and β-catenin stabilization are central events (Shin et al. 2020;Kim et al. 2021;Ryu et al. 2021;Kim et al. 2024). The present results suggest that PCAE may promote HFDPC proliferation primarily through PI3K/Akt-related signaling rather than through direct activation of the canonical Wnt/β-catenin pathway. Akt activation is known to support cell survival and proliferation by regulating downstream targets involved in cell-cycle progression, metabolism, and stress resistance (Manning & Cantley 2007). In hair biology, PI3K/Akt signaling has been reported to be essential for de novo hair follicle regeneration, and pharmacological inhibition of PI3K or Akt can suppress neogenic hair formation in experimental models (Chen et al. 2020). However, because this study did not include pathway inhibitor experiments or other loss-of-function analyses, the observed increase in Akt phosphorylation should be interpreted as a correlative finding rather than direct mechanistic evidence of PI3K/Akt-driven HFDPC proliferation in response to PCAE.
The concomitant decrease in β-catenin and induction of DKK1 raise an important mechanistic question. Rather than indicating a lack of biological activity, these findings suggest that PCAE-induced Akt activation may drive proliferation independently of canonical Wnt/β-catenin stabilization, as Akt can inactivate GSK-3β and stabilize Cyclin D1 without requiring β-catenin nuclear translocation. Moreover, the reduction in total β-catenin protein does not preclude residual transcriptional activity, and DKK1 up-regulation may reflect a compensatory feedback response rather than a primary suppressive event. However, causal interpretation requires additional experiments, such as PI3K/Akt inhibition, Akt knockdown, β-catenin localization analysis, DKK1-neutralizing experiments, and downstream transcriptional reporter assays. Thus, the present study identifies a distinct signaling pattern but does not yet fully define the upstream molecular target of PCAE.
4. PCAE selectively remodels the expression profile of hair growth-related paracrine factors
Because Akt signaling was activated by PCAE, RT-qPCR was performed to determine whether PCAE altered the paracrine factor profile of HFDPCs (Figure 4). PCAE markedly decreased IGF-1 mRNA expression at all tested concentrations. Although IGF-1 is generally considered supportive of hair follicle survival and growth (Trüeb 2002;Madaan et al. 2018), the increase in HFDPC proliferation observed despite IGF-1 suppression suggests that PCAE-induced proliferation may not require IGF-1 mRNA up-regulation. Because P. cuspidatum contains resveratrol-related stilbenes, this result may be partly consistent with reports that resveratrol can interfere with IGF-1R-associated signaling, although this possibility requires direct validation in HFDPCs (Vanamala et al. 2010).
VEGF mRNA was significantly increased at selected concentrations, particularly at 50 and 200 µg/mL, whereas the 100 µg/mL group showed an increasing tendency without statistical significance. VEGF is an important angiogenic factor that supports perifollicular vascularization during anagen and has been implicated in the hair growth-related action of minoxidil (Dai et al. 2023;Yum et al. 2018). PCAE also strongly induced FGF7 mRNA expression at all tested concentrations. FGF7 signaling through FGFR2b is a key paracrine axis by which DP cells communicate with hair matrix keratinocytes and support hair follicle growth and differentiation (Danilenko et al. 1995;Kawano et al. 2005). Therefore, the robust induction of FGF7 suggests that PCAE may enhance the epithelial-supportive function of HFDPCs.
EGF and FGF10 showed upward trends, especially at 50 µg/mL, but statistical significance was not evident under the tested conditions. Therefore, these responses were interpreted as trends rather than definitive inductions. Future validation using additional biological replicates and protein-level assays such as ELISA would clarify whether EGF and FGF10 are reproducibly induced by PCAE.
Taken together, PCAE activated Akt signaling, reduced β-catenin protein abundance, strongly induced DKK1, and enhanced selected paracrine growth factor transcripts, particularly VEGF and FGF7. This profile differentiates PCAE from natural extracts whose hair growth-promoting activity is explained mainly by Wnt/β-catenin activation. The current results support the potential of PCAE as a functional material for hair loss prevention and improvement. Nevertheless, several limitations should be addressed before translation. First, the extract must be chemically standardized using marker compounds such as resveratrol, piceid, and emodin to ensure reproducibility and practical applicability. In this regard, the present study did not include chemical profiling or quantitative analysis of these constituents. Therefore, future studies should establish a standardized chemical profile and quantify representative marker compounds to strengthen the translational relevance of PCAE. Second, although PCAE increased Akt phosphorylation, the functional involvement of the PI3K/Akt pathway should be directly verified using pathway inhibitors or gene-silencing approaches. Third, the changes in hair growth-related factors, including FGF7, VEGF, IGF-1, EGF, and FGF10, were mainly demonstrated at the mRNA level. Therefore, protein-level validation using methods such as ELISA or Western blotting remains necessary. Finally, the biological effects of PCAE should be further confirmed in hair follicle organ culture systems or in vivo models.
IV. Summary and Conclusion
This study screened six plant-derived extracts and identified P. cuspidatum aqueous extract (PCAE) as a promising candidate that promotes HFDPC proliferation. Compared with the 50% and 70% EtOH extracts of P. cuspidatum, PCAE showed a wider effective concentration range and no apparent cytotoxicity under the tested conditions. PCAE significantly increased HFDPC proliferation at 50–300 µg/mL and maintained cell viability at 400 µg/mL.
Mechanistic analyses showed that PCAE increased Akt phosphorylation while decreasing β-catenin protein levels. DKK1 mRNA was strongly up-regulated by PCAE, providing a plausible association with the decrease in β-catenin. RT-qPCR analysis further revealed strong up-regulation of FGF7, significant induction of VEGF at selected concentrations, and suppression of IGF-1. EGF and FGF10 showed upward tendencies but were interpreted as non-significant trends under the tested conditions. These results suggest that PCAE promotes HFDPC proliferation in association with Akt phosphorylation and PI3K/Akt-related signaling and selective regulation of hair growth-related paracrine factor expression rather than through conventional Wnt/β-catenin activation. The distinctive molecular profile of PCAE, characterized by Akt activation, DKK1 induction, reduced β-catenin abundance, and increased expression of selected paracrine growth factors, provides a rationale for developing P. cuspidatum aqueous extract as a functional food ingredient or natural hair care material for hair loss prevention and improvement. Further research is required for the chemical standardization of the extract and verification of its efficacy in in vitro and in vivo hair growth models.
