Seoritae was mixed with 70% ethanol in a ratio of 1 to 10 and extracted at room temperature for 24 h. The resulting extract was filtered through filter paper (AF-31H, HM, Seoul, Korea), and then concentrated in a rotary vacuum concentrator (EYELA N-2100, Tokyo Rikakikai Co., Tokyo, Japan).

Seoritae powder was dissolved in DMSO for the cell-based experiments. DHT, H₂O₂, and biotin were purchased from Sigma-Aldrich (St Louis, MO, USA). Human follicle dermal papilla cells (HFDPC) were purchased from PromoCell (C-12071, Heidelberg, Germany).

Cell proliferation was assessed using the WST-1 assay (EZCytox, DoGenBio, Seoul, Korea) according to the manufacturer's instructions. Briefly, WST-1 analysis was performed as described above. Unlike generally cell viability assays, cells were incubated for 72 h after treatment with test samples (50 ㎍/㎖ - 400 ㎍/㎖) and positive control biotin (150 ㎍/㎖) cell for proliferation assays.

To assess the ability of seoritae extract to protect HFDPC against DHT- or H₂O₂-induced apoptosis, we dispensed HFDPC to 96-well plates (0.6 × 10⁴ cells in 100 ㎕/well), treated each well with 200 μM DHT or 400 μM H₂O₂ with various concentrations of seoritae extract (3.125 ㎍/㎖ - 50 ㎍/㎖), and incubated the plates for 24 h.

The supernatant was removed, WST-1 reagent was added to a final concentration of 10%, and the plate was further incubated at 37℃ with 5% CO₂. After 1 h, the absorbance was measured at 450 ㎚ using a microplate reader.

Cells in culture dishes were directly lysed with RIPA buffer (Thermo Fisher Scientific, MA, USA) supplemented with a proteinase and phosphatase inhibitor cocktail mixture (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates (30 ㎍) were resolved by 4% - 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Invitrogen, Waltham, MA, USA).

The blots were washed with Tris-buffered solution containing Tween-20 [TBST; 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween-20], blocked with 5% skimmed milk in TBST for 1 h at room temperature, and incubated for 12 h at 4℃ with primary antibodies against Bax, β-actin, and β-catenin (1 : 1000 dilutions, Cell Signaling Technology Inc., Danvers, MA, USA).

The membranes were washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG antibodies (1 : 10,000 dilutions, Cell Signaling Technology Inc., Danvers, MA, USA) for 1 h at room temperature.

The bands were visualized using an enhanced chemiluminescence detection system (GE Healthcare, ORD, USA) according to the manufacturer’s protocols.

A DPPH discoloration assay was carried out to examine the scavenging effect of the seoritae extract or ascorbic acid (positive control). Briefly, the seoritae extract or ascorbic acid and 1 mM DPPH solution (Sigma Aldrich, St Louis, MO, USA) were mixed at a ratio of 4 : 1 and incubated at room temperature for 30 min.

The absorbance of each sample at 517 ㎚ was measured by spectrophotometry. The DPPH-scavenging effect was expressed as percent inhibition, as follows: (%) = [(A0-A1)/A0] × 100, where A0 is the absorbance of DPPH and A1 is the absorbance of the test material.

All analytical values were obtained by repeating 3 times and expressed as mean and standard deviation (Means ± SD). Comparisons between samples were statistically processed based on Student's t-test via the Microsoft Office (Raymond, WA, USA) Excel T-TEST function and determined to be significant if the p-value was less than 0.05 (p < 0.05).

To rule out the possibility that seoritae could have cytotoxic effects on HFDPC that would impact our experiments, we treated HFDPC with various concentrations of seoritae extract (50 ㎍/㎖ - 800 ㎍/㎖) and assessed cell viability using the WST reagent. We observed cell survival of 90% or more at concentrations of 400 ㎍/㎖ or less, indicating that these concentrations were not toxic to HFDPC. We performed our subsequent experiments in HFDPC using sub-toxic doses of seoritae extract.

Next, to assess the proliferative effect of seoritae extract on HFDPC, we applied sub-toxic concentrations of the extract to HFDPC, cultured the cells for 3 days, and examined cell viability. Indeed, seoritae extract effectively enhanced the cell proliferation of HFDPC (Fig. 1C). by 20 to 45% depending on the concentration of seoritae extract.

These results indicate that seoritae extract can enhance the proliferation of a human dermal papilla cell line, confirming its potential as a material for alleviating hair loss.

To assess the ability of seoritae extract to suppress the DHT-or H₂O₂-induced apoptosis of HFDPC, we applied sub-toxic doses of the extract and assessed cell viability. Our results confirmed that the applied concentrations of H₂O₂ and DHT induced apoptosis in HFDPC under our experimental conditions (Fig. 2A).

Fig. 2.  The effect of seoritae extract on the H₂O₂- and DHT-induced apoptosis of HFDPC.

(A) Optimized stimulators concentration by using cytotoxicity assay on HFDPC. (B) Anti-apoptotic effect of seoritae in HFDPC by DHT. (C) Anti-apoptotic effect of seoritae in HFDPC by H₂O₂. Cell viability was estimated using a colorimetric WST-1 assay kit. The results are given as the percentage compared to control group (Means ± SD). Positive controls: Vit. C and biotin. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the stimulator (DHT, H₂O₂). ^##p < 0.005, ^###p < 0.001 compared to the CTL.

The concentrations of DHT and H₂O₂ that maintain cell viability at 70% were all about 200 μM, and this concentration was applied throughout this study. Then, the data showed that the seoritae extract suppressed apoptosis in our system, depending on the stimulant and applied concentration of extract (Fig. 2B and 2C). In DHT-treated HFDPC, searitae extract slightly recovered the cytotoxicity at relatively low concentrations. On the other side, searitae extract dramatically recovered the cytotoxicity at relatively high concentrations. Thus, we found that the anti-cytotoxic concentration of seoritae extract differed apparently, depending on the stimulant.

These results confirm that seoritae extract can suppress the H₂O₂- or DHT-induced apoptosis of HFDPC, and thus may be a hair loss-easing substance.

Whereas a variety of growth factors and signaling pathways are involved in hair growth, the activation of wnt/β-catenin signaling plays a central role in hair follicle regeneration (Choi, 2020).

We determined if seoritae extract could induce the regulation of wnt/β-catenin signaling molecules in HFDPC under apoptotic conditions. The HFDPC were treated with the indicated concentrations of DHT and seoritae extract for 4 h, and the Western blot analysis of cell lysates was performed to evaluate the levels of selected proteins involved in β-catenin signaling (Bax, β-catenin).

As shown in Fig. 3, 200 ㎍/㎖ and 400 ㎍/㎖ seoritae extract increased β-catenin and suppresed Bax expression in DHT-treated HFDPC, although DHT (200 μM) did not affect on β-catenin and Bax expression in our experimetal system. These results suggest that seoritae extract modulates the levels of β-catenin and Bax proteins in human follicular dermal papilla cells even if the cells are under apoptotic conditions.

Fig. 3.  The effects of seoritae extract on the protein expression levels of β-catenin and Bax in DHT-treated HFDPC cells.

The cells were co-treated with DHT (200 μM) and seoritae (50, 100, 200, 400 ㎍/㎖) for 4 h, and whole-cell lysates were subjected to immunoblotting with antibodies against the indicated proteins.

Oxidative stress cause the apoptosis of hair follicle cells, resulting in the hair loss (Naito et al., 2009; Seo et al., 2012). Thus, we determined the anti-oxidative efficacy of seoritae extract.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which is commonly used to assess the antioxidant activity of plant extracts, was used to examine the radical-scavenging effect of seoritae extract. As shown in Fig. 4, the seoritae extract showed a significant DPPH scavenging effect.

Fig. 4.  DPPH radical-scavenging activity of seoritae extract.

The given values represent the means ± SD of three independent measurements. Positive control: Ascorbic acid. **p < 0.01, ***p < 0.001 compared to the control.

In recent years, interest in appearance has spread to various fields. Hair loss is increasing not only in middle-aged and elderly people, but also in younger people. Agents capable of exerting cell-protective effects against aspects of skin inflammation and aging can be important contributors to ameliorating such conditions, and this may be extended to preventing hair loss. An ultimate solution for hair loss would be to address its most common cause by suppressing the synthesis of 5α-DHT.

Much research has been already done in this regard, but many of the well-known chemical agents for treating hair loss, such as minoxidil, cause side effects such as erythema and itching.

Currently, researchers are actively working to identify effective natural product-derived substances that can address hair loss with few side effects (Park et al., 2015; Jain et al., 2016; Dhariwala and Ravikumar, 2019; Jung, 2019), and to assess their underlying action mechanisms.

In this study, we show that seoritae extract has antioxidant, cell proliferation-enhancing, and anti-apoptotic effects in a human follicle dermal papilla cells. In addition, we further use protein analysis to show that alterations of β-catenin signaling may be a mechanism underlying this anti-apoptotic effect of seoritae extract, because there are multiple lines of evidence suggesting that various wnts promote hair cycling and regeneration via the activation of β-catenin signaling (Choi, 2020).

Based on these results, we propose that seoritae extract may be a candidate agent for effectively alleviating hair loss. Additional studies are needed to further explore the potential hair loss-relieving effect and underlying action mechanisms of seoritae extract. In addition, we consider that it is necessary to conduct further research to analyze the components of seoritae extract and to identify a single compound that exhibits efficacy.