Ginsenosides in Panax ginseng Extract Promote Anagen Transition by Suppressing BMP4 Expression and Promote Human Hair Growth by Stimulating Follicle-Cell Proliferation

Abstract

Studies showing that Panax ginseng promotes hair growth have largely been conducted using mice; there are few reports on how P. ginseng affects human hair growth. In particular, little is known about its effect on the telogen to anagen transition. To determine the effect of P. ginseng on human hair growth and the transition from the telogen to the anagen phase. The effects of P. ginseng extract (PGE) and the three major ginsenoside components, Rb1, Rg1, and Re, on the proliferation of human dermal papilla cells (DPCs) and human outer root sheath cells (ORSCs) were investigated. The effects of these compounds on the cell expression of bone morphogenetic protein 4 (BMP4), fibroblast growth factor 18 (FGF18) and Noggin were assessed by real-time PCR. The effect of PGE on hair-shaft elongation was determined in a human hair follicle organ-culture system. PGE and the three ginsenosides stimulated the proliferation of DPCs and ORSCs and suppressed BMP4 expression in DPCs but did not affect FGF18 expression in ORSCs and Noggin expression in DPCs. PGE stimulated hair-shaft growth. PGE and the ginsenosides Rb1, Rg1, and Re stimulate the transition from the telogen phase to anagen phase of the hair cycle by suppressing BMP4 expression in DPCs. These compounds might be useful for promoting the growth of human hair.

INTRODUCTION

Panax ginseng is a herbal medicine that has been used since ancient times in east Asia. It contains a variety of bioactive components, the most abundant being a group of saponins called ginsenosides, which have diverse bioactivities.^1,2)

P. ginseng extract (PGE) and its major component, ginsenosides, stimulate hair growth in mice, and several studies suggest these compounds also stimulate the growth of human hair.^3,4) For example, PGE stimulates the proliferation of human dermal papilla cells (DPCs).⁵⁾ PGE and several ginsenosides (Rb1, Rg1, Re; Supplementary Fig. S1) stimulate hair-shaft growth in an ex vivo organ-culture system of human hair follicles and red ginseng extract stimulates the proliferation of hair matrix keratinocytes in the same culture system.^2,6)

Several studies indicate that compounds from Panax ginseng have hair growth-promoting effects in mice. Red ginseng oil promotes hair regeneration and increases the density and bulb diameter of hair follicles in mice.⁷⁾ Ginsenoside Ro stimulates hair growth in several mouse models by preventing apoptosis of hair follicle cells and inhibiting 5α-reductase activity.⁸⁾ Ginsenoside Re stimulates the growth of the hair shaft in an organ culture of mouse vibrissae follicles.⁹⁾ However, there are few studies on the effect of ginsenosides on human hair, particularly studies investigating the effect of ginsenosides on promoting the transition from the resting phase (telogen) to the growing phase (anagen) of the hair cycle.

Ginsenosides can be classified as protopanaxadiol (PPD) type and protopanaxatriol (PPT) type. Common PPD-type ginsenosides include ginsenosides Rb1, Rb2, Rc, Rd, Rg3, F2, Rh2, compound K (cK), and PPD. PPT-type ginsenosides include Re, Rf, Rg1, Rg2, F1, Rh1, and PPT.¹⁰⁾ The ginsenosides Rb1, Rg1, and Re are the major ginsenoside components in PGE.^6,8) There are few reports on whether these three ginsenosides regulate the human hair cycle.

In this study, the effects of PGE, Rb1, Rg1, and Re on the proliferation of human DPCs and human outer root sheath cells (ORSCs) were investigated. The effects of these compounds on the expression of bone morphogenetic protein 4 (BMP4) in DPCs, fibroblast growth factor 18 (FGF18) in ORSCs and Noggin in DPCs were also investigated.

MATERIALS AND METHODS

Chemicals

The P. ginseng extract prepared by extraction from red ginseng using 20% ethanol was provided by Maruzen Pharmaceuticals (Hiroshima, Japan). Ginsenoside Rb1 was purchased from Wako Pure Chemical Corporation (Osaka, Japan). Ginsenoside Rg1 and Re were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan).

Cell Culture

Cell lines of DPCs and ORSCs immortalized with SV40 large T antigen were used in this study.¹¹⁾ Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing antibiotics (100 µg/mL of penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of amphotericin B) was used as the basal medium for DPCs, and basal DMEM containing 10% fetal bovine serum was used as the nutrient medium. Keratinocyte serum-free medium (K-SFM) (Thermo Fisher Scientific) containing antibiotics was used as the basal medium for ORSCs, and K-SFM supplemented with epidermal growth factor (5 ng/mL) and bovine pituitary extract (50 µg/mL) was used as the nutrient medium. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. PGE was added to the medium at a dry weight concentration.

Cell Proliferation Assays

Ten thousand cells were seeded onto 96 well-plastic cell-culture plates (Iwaki Glass, Chiba, Japan) and preincubated in the nutrient medium for 2 d. After preincubation, the medium was replaced with basal media containing various concentrations of PGE or ginsenosides. The cells were then cultured for 3 d, and proliferation was evaluated using an AlamarBlue assay system (Bio-Rad Laboratories, Hercules, CA, U.S.A.).

Tissue and Human Hair Follicle Organ Culture

Scalp skin samples were provided by volunteers with informed consent. The anagen hair follicles were isolated from the skin according to previously published methods.¹²⁾ This study was approved by the Tokyo University of Technology Ethics Committee to ensure subject protection and adhered to the principles of the Declaration of Helsinki (the Approval Number was E22BS-026).

Isolated human hair follicles were cultured according to the methods described by Philpott et al. and Iino et al.^12,13) For the preculture, hair follicles were incubated in William’s E medium (Thermo Fisher Scientific) containing of 10 ng/mL sodium selenite, 10 mg/mL transferrin, 10 ng/mL hydrocortisone, and 10 mg/mL insulin, as the supplements. After 24 h, only the follicles with elongated hair shafts were selected. The selected follicles were then continued to be cultured in basal William’s E medium with or without PGE and was then replaced the medium every day for 8 d. The length of the hair shaft was measured on day 9 using an inverted microscope (Olympus, Tokyo, Japan) equipped with an eyepiece micrometer under the ×40 magnification.

Real-Time PCR

Subconfluent cells were cultured with or without PGE or ginsenosides for 4 h. The mRNA was then extracted with ISOGEN II (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. The mRNA was reverse-transcribed with SuperScript III (Thermo Fisher Scientific). Real-time quantitative PCR was performed on a LightCycler rapid thermal cycler system (Roche Diagnostics, Tokyo, Japan) using Thunderbird Next SYBR qPCR mix (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a reference gene. All PCR primers used in this study are listed in Supplementary Table S1.

Statistical Analysis

In vitro experiments were analyzed by comparing groups using unpaired t-tests. In all cases, p < 0.05 was considered statistically significant, and bar shown in graphs indicate the mean ± standard deviation (S.D.).

RESULTS

The Effect of PGE on the Proliferation of DPCs and ORSCs

PGE promoted the proliferation of DPCs and ORSCs in a concentration-dependent manner, as shown in Fig. 1. PGE significantly enhanced the proliferation of DPCs and ORSCs at concentrations of 50 µg/mL or greater (Figs. 1a, b). These results indicated that PGE promotes thick hair fiber formation and growth.

Fig. 1. The Effects of PGE on the Proliferation of DPCs and ORSCs

(a) The effect of PGE on DPCs, (b) the effect of PGE on ORSCs. The activity of PGE is expressed as the cell proliferation index (growth rate %) relative to the negative control. Data are given as the mean ± S.D. of eight replicates. Statistical analyses of these data were carried out using Student’s t-test. *** p < 0.001.

The Effect of Rb1, Rg1, and Re on the Proliferation of DPCs and ORSCs

Ginsenosides Rb1, Rg1, and Re are the major component ginsenosides in P. ginseng.^6,8) These three ginsenosides promoted the proliferation of DPCs and ORSCs in a concentration-dependent manner (Fig. 2). All three ginsenosides significantly enhanced the proliferation of DPCs at concentrations of 25 µM or greater (Figs. 2a–c). Ginsenosides Rb1 and Re significantly enhanced the proliferation of ORSCs at concentrations of 10 µM or greater (Figs. 2d, f). Ginsenoside Rg1 also significantly enhanced the proliferation of ORSCs at concentrations of 25 µM (Fig. 2e). These results indicated that the three ginsenosides promote the proliferation of cell types that are important in thick hair fiber formation and growth.

Fig. 2. The Effect of Rb1, Rg1, and Re on the Proliferation of DPCs and ORSCs

(a) Ginsenoside Rb1 on DPCs, (b) ginsenoside Rg1 on DPCs, (c) ginsenoside Re on DPCs, (d) ginsenoside Rb1 on ORSCs, (e) ginsenoside Rg1 on ORSCs, (f) ginsenoside Re on ORSCs. The activities of the ginsenosides are expressed as the cell proliferation index (growth rate %) relative to the negative control. Data are given as the mean ± S.D. of eight replicates. Statistical analyses of these data were carried out using Student’s t-test. * p < 0.05, ** p < 0.01.

PGE Stimulates Hair-Shaft Elongation in Human Hair Follicle Organ Culture

The effect of PGE on shaft elongation of human hair follicles was evaluated using an organ-culture system. PGE (100 µg/mL) significantly increased the elongation of the hair shaft after 8 d of incubation (Fig. 3). This result suggests that in an ex vivo system, PGE can promote the anagen phase of the hair cycle.

Fig. 3. The Effects of PGE on Hair-Shaft Elongation in Follicle Organ Cultures from Human Scalp

Anagen hair follicles were isolated from human scalp and incubated with PGE at concentrations of 0, 50, and 100 µg/mL (n = 12 for each concentration). Increases in the length of the hair shafts were measured over 8 d with an inverted microscope fitted with an eyepiece graticule. The results represent the mean ± S.D. of the increase in hair-shaft length, compared with the 0 µg/mL PGE data. Statistical analyses of these data were carried out using Student’s t-test. ** p < 0.01.

The Effect of PGE and the Three Ginsenosides on the Expression of BMP4 and Noggin in DPCs

BMP4 is expressed in DPCs and is a potent telogen maintenance factor that regulates the hair cycle.^14–16) Noggin is also expressed in DPCs. Noggin affects BMP4 signaling, as it binds to BMP4 and inhibits its binding to the receptors.^14–16) The effects of PGE and the three ginsenosides (Rb1, Rg1, and Re) on BMP4 and Noggin expression in DPCs are shown in Fig. 4 and Supplementary Fig. S3, respectively.

Fig. 4. The Effects of P. ginseng Extract (PGE), Ginsenosides Rb1, Rg1, and Re on the Expression of BMP4 in DPCs

(a) PGE, (b) ginsenoside Rb1, (c) ginsenoside Rg1, (d) ginsenoside Re. Data are given as the mean ± S.D. of four replicates. Statistical analyses were carried out using Student’s t-test. * p < 0.05, ** p < 0.01.

PGE significantly suppressed BMP4 expression in DPCs at concentrations of 50 µg/mL and 100 µg/mL (Fig. 4a). Ginsenosides Rb1 and Rg1 significantly suppressed BMP4 expression in DPCs at concentrations of 25 µM or more (Figs. 4b, c). Ginsenoside Re also significantly suppressed BMP4 expression in DPCs at a concentration of 50 µM (Fig. 4d). However, PGE, Rb1, Rg1, and Re did not affect the expression of Noggin at any of the tested concentrations in DPCs, as shown in Supplementary Fig. S3. These results suggest that PGE and the three ginsenosides can shift the human hair cycle from the telogen phase to the anagen phase by suppressing BMP4 expression.

The Effect of PGE and the Three Ginsenosides on the Expression of FGF18 in ORSCs

FGF18 is expressed in ORSCs and is another potent telogen maintenance factor that regulates the hair cycle.¹⁷⁾ PGE, Rb1, Rg1, and Re did not affect the expression of FGF18 at any of the tested concentrations in ORSCs, as shown in Supplementary Fig. S2. These results indicate that the tested compounds altered BMP4 expression but not FGF18 and Noggin expression in human hair follicle.

DISCUSSION

Many studies have described the effects of P. ginseng and ginsenosides on hair growth; however, most studies have used mice rather than human-derived samples.¹⁾ To help overcome this limitation, the current study examined the effects of PGE and three ginsenosides (Rb1, Rg1, and Re) on the regulation of the human hair cycle.

It is well documented that human DPCs with thick hair shafts are larger than those with thin hair shafts.^18–20) DPCs from patients with androgenetic alopecia (AGA) also proliferate more slowly than DPCs isolated from non-AGA hair follicles.²¹⁾ The current study showed that PGE and three ginsenosides (Rb1, Rg1, and Re) increase the proliferation of human DPCs (Figs. 1a, 2a–c). Thus, PGE and the three ginsenosides might stimulate the growth of thick hair.

ORSCs incorporate BrdU when organ culture of anagen hair follicles of human are maintained in medium containing growth factors and other supplements.²²⁾ Thus, the increased proliferation of hair keratinocytes is considered an index of the activation and elongation of the anagen phase.²²⁾ Sophora flavescens Aiton root extract promoted hair growth in a human clinical trial; furthermore, S. flavescens extract stimulated human ORSC proliferation and elongation of the hair shaft in a human hair follicle organ-culture system.²³⁾ In the current study, PGE and the three ginsenosides stimulated the proliferation of human ORSCs (Figs. 1b, 2d–f). In addition, PGE stimulated hair-shaft growth in a human hair follicle organ-culture system (Fig. 3). These results indicate that PGE and the three ginsenosides might enhance the growth of human hair.

In male AGA, hair thickness is more important than hair density in the appearance and progression of baldness.²⁴⁾ Moreover, hair density in males with AGA is not significantly different from that in males without AGA.²⁴⁾ Thus, increasing hair thickness is key to addressing male AGA. In this study, PGE and the three ginsenosides promoted the proliferation of DPCs and stimulated hair-shaft elongation (Figs. 1a, 2a–c). Furthermore, PGE and the three ginsenosides stimulated the proliferation of hair keratinocytes (Figs. 1b, 2d–f). Thus, PGE and the three ginsenosides may be suitable for treating AGA, in which hairs become extremely thin.

The main cause of female pattern hair loss is an increase in the level of resting hairs and decreased hair thickness.^25–27) Reducing the level of hairs in the telogen phase and increasing hairs in the anagen phase are important considerations for treating female pattern hair loss.

BMP4 (expressed in DPCs) and FGF18 (expressed in hair keratinocytes) are factors to maintain the telogen phase of the hair cycle.^14–17) In the current study, PGE and the three ginsenosides suppressed the expression of BMP4 in DPCs (Fig. 4) but did not affect the expressions of FGF18 in ORSCs and Noggin in DPCs (Supplementary Figs. S2, S3). It is known that BMP4 signaling contributes to the maintenance of telogen phase.^14–16) Essentially, when BMP4 signaling weakens, the hair cycle transitions to anagen phase.²⁸⁾ Noggin is a factor that affects BMP4 signaling, as it binds to BMP4 and inhibits its binding to the receptors.^14–16) PGE and three ginsenosides suppressed BMP expression in dermal papilla cells (Fig. 4). However, they had no effect on Noggin expression in dermal papilla cells (Supplementary Fig. S3). Therefore, it is likely that PGE and the three ginsenosides promote the transition from telogen to anagen by suppressing BMP expression in dermal papilla cells. These results suggest that PGE and the three ginsenosides shift the hair cycle from the telogen phase to the anagen phase by suppressing BMP4 expression in DPCs. Therefore, PGE and the three ginsenosides might be effective treatments for female pattern hair loss.

In all our experiments, the three ginsenosides exhibited the same effects in DPCs and ORSCs. This finding suggests that the structural differences between PPD-type and PPT-type compounds did not impact their cellular effects. We found that PGE extracted with 20% ethanol promoted hair-shaft elongation to the same extent as PGE extracted with 100% methanol. This finding facilitates the use of the active extract (obtained with a low alcohol concentration) in a wide range of cosmetic products.

Acknowledgments

The authors are grateful to Mr. Shogo Ono and Mr. Daiki Endo for their technical help.

Author Contributions

Tokuro Iwabuchi contributed to study conception and design as well as writing of manuscript. TI also performed the experiments and analyzed data. Kazuki Ogura, Kenta Hagiwara, Shogo Ueno, Hiroaki Kitamura, Haruyo Yamanishi and Yuki Tsunekawa performed the experiments. Akinori Kiso contributed to study conception and design.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Choi BY. Hair-growth potential of ginseng and its major metabolites: a review on its molecular mechanisms. Int. J. Mol. Sci., 19, 2703 (2018).
• 2) Park GH, Park KY, Cho HI, Lee SM, Han JS, Won CH, Chang SE, Lee MW, Choi JH, Moon KC, Shin HS, Kang YJ, Lee DH. Red ginseng extract promotes the hair growth in cultured human hair follicles. J. Med. Food, 18, 354–362 (2015).
• 3) Shin HS, Park SY, Hwang ES, Lee DG, Mavlonov GT, Yi TH. Ginsenoside F2 reduces hair loss by controlling apoptosis through the sterol regulatory element-binding protein cleavage activating protein and transforming growth factor-β pathways in a dihydrotestosterone-induced mouse model. Biol. Pharm. Bull., 37, 755–763 (2014).
• 4) Suzuki A, Matsuura D, Kanatani H, Yano S, Tsunakawa M, Matsuyama S, Shigemori H. Inhibitory effects of polyacetylene compounds from Panax ginseng on neurotrophin receptor-mediated hair growth. Biol. Pharm. Bull., 40, 1784–1788 (2017).
• 5) Park SJ, Shin WS, Ho JY. Fructus Panax ginseng extract promotes hair regeneration in C57BL/6 mice. J. Ethnopharmacol., 138, 340–344 (2011).
• 6) Kim SN, Kim SH, Hong YD, Park HO, Shin SH, Kim AR, Park BC, Shin SS, Park JS, Park MY, Park YH, Lee HK, Lee JH, Park WS. The ginsenosides of Panax ginseng promote hair growth via similar mechanism of minoxidil. J. Dermatol. Sci., 77, 132–134 (2015).
• 7) Truong VL, Keum YS, Jeong WS. Red ginseng oil promotes hair growth and protects skin against UVC radiation. J. Ginseng. Res., 45, 498–509 (2021).
• 8) Murata K, Takeshita F, Samukawa K, Tani T, Matsuda H. Effects of ginseng rhizome and ginsenoside Ro on testosterone 5α-reductase and hair re-growth in testosterone-treated mice. Phytother. Res., 26, 48–53 (2012).
• 9) Li Z, Ryu SW, Lee JS, Choi KS, Kim SC, Choi CH. Protopanaxatirol type ginsenoside Re promotes cyclic growth of hair follicles via inhibiting transforming growth factor β signaling cascades. Biochem. Biophys. Res. Commun., 470, 924–929 (2016).
• 10) Kim DH. Herbal medicines are activated by intestinal microflora. Nat. Prod. Sci., 8, 35–43 (2002).
• 11) Iwabuchi T, Ogura K, Tamba K, Tsunekawa Y, Sugano M, Hagiwara K, Kiso K. Cepharanthine induces the proliferation of human dermal papilla cells and stimulates vascular endothelial growth factor expression through increased intracellular calcium mobilization and hypoxia-inducible factor activation. Clin. Exp. Dermatol., 46, 694–703 (2021).
• 12) Iino M, Ehama R, Nakazawa Y, Iwabuchi T, Ogo M, Tajima M, Arase S. Adenosine stimulates fibroblast growth factor-7 gene expression via adenosine A2b receptor signaling in dermal papilla cells. J. Invest. Dermatol., 127, 1318–1325 (2007).
• 13) Philpott MP, Green MR, Kealey T. Human hair growth in vitro. J. Cell Sci., 97, 463–471 (1990).
• 14) Botchkarev VA, Botchkareva NV, Roth W, Nakamura M, Chen LH, Herzog W, Lindner G, McMahon JA, Peters C, Lauster R, McMahon AP, Paus R. Noggin is a mesenchymally derived stimulator of hair-follicle induction. Nat. Cell Biol., 1, 158–164 (1999).
• 15) Botchkarev VA, Botchkareva NV, Nakamura M, Huber O, Funa K, Lauster R, Paus R, Gilchrest BA. Noggin is required for induction of the hair follicle growth phase in postnatal skin. FASEB J., 15, 2205–2214 (2001).
• 16) Botchkarev VA, Botchkareva NV, Sharov AA, Funa K, Huber O, Gilchrest BA. Modulation of BMP signaling by noggin is required for induction of the secondary (nontylotrich) hair follicles. J. Invest. Dermatol., 118, 3–10 (2002).
• 17) Kimura-Ueki M, Oda Y, Oki J, Komi-Kuramochi A, Honda E, Asada M, Suzuki M, Imamura T. Hair cycle resting phase is regulated by cyclic epithelial FGF18 signaling. J. Invest. Dermatol., 132, 1338–1345 (2012).
• 18) Chase HB. Growth of the hair. Physiol. Rev., 34, 113–126 (1954).
• 19) Jahoda CAB. Induction of follicle formation and hair growth by vibrissa dermal papillae implanted into rat ear wounds: vibrissa-type fibres are specified. Development, 115, 1103–1109 (1992).
• 20) Elliott K, Stephenson TJ, Messenger AG. Differences in hair follicle dermal papilla volume are due to extracellular matrix volume and cell number: implications for the control of hair follicle size and androgen responses. J. Invest. Dermatol., 113, 873–877 (1999).
• 21) Randall VA, Hibberts NA, Hamada K. A comparison of the culture and growth of dermal papilla cells from hair follicles from non-balding and balding (androgenetic alopecia) scalp. Br. J. Dermatol., 134, 437–444 (1996).
• 22) Soma T, Ogo M, Suzuki J, Takahashi T, Hibino T. Analysis of apoptotic cell death in human hair follicles in vivo and in vitro. J. Invest. Dermatol., 111, 948–954 (1998).
• 23) Takahashi T, Ishino A, Arai T, Hamada C, Nakazawa Y, Iwabuchi T, Tajima M. Improvement of androgenetic alopecia with topical Sophora flavescens Aiton extract, and identification of the two active compounds in the extract that stimulate proliferation of human hair keratinocytes. Clin. Exp. Dermatol., 41, 302–307 (2016).
• 24) Ishino A, Takahashi T, Suzuki J, Nakazawa Y, Iwabuchi T, Tajima M. Contribution of hair density and hair diameter to the appearance and progression of androgenetic alopecia in Japanese men. Br. J. Dermatol., 171, 1052–1059 (2014).
• 25) Tajima M, Hamada C, Arai T, Miyazawa M, Shibata R, Ishino A. Characteristic features of Japanese women’s hair with aging and with progressing hair loss. J. Dermatol. Sci., 45, 93–103 (2007).
• 26) Messenger AG, Sinclair R. Follicular miniaturization in female pattern hair loss: clinicopathological correlations. Br. J. Dermatol., 155, 926–930 (2006).
• 27) Williams R, Westgate GE, Pawlus AD, Sikkink SK, Thornton MJ. Age-related changes in female scalp dermal sheath and dermal fibroblasts: how the hair follicle environment impacts hair aging. J. Invest. Dermatol., 141 (4S), 1041–1051 (2021).
• 28) Plikus MV, Mayer J, de la Cruz D, Baker RE, Maini PK, Maxson R, Chuong CM. Cyclic dermal BMP signaling regulates stem cell activation during hair regeneration. Nature, 451, 340–344 (2008).