18-β-Glycyrrhetinic Acid Promotes Hair Growth by Stimulating the Proliferation of Dermal Papilla Cells and Outer Root Sheath Cells, and Extends the Anagen Phase by Inhibiting 5α-Reductase

Abstract

18-β-Glycyrrhetinic acid, a major component of licorice, stimulated the proliferation of both dermal papilla cells and outer root sheath cells isolated from human hair follicles. Thus, suggesting that this compound promotes hair growth. Furthermore, this compound inhibited the activity of testosterone 5α-reductase, an enzyme responsible for converting androgen to dihydroandrogen, with an IC₅₀ of 137.1 µM. 18-β-Glycyrrhetinic acid also suppressed the expression of transforming growth factor-β1 (TGF-β1), which shifts the hair cycle from the anagen phase to the telogen phase. It suggested that this compound may prolong the anagen phase. Based on these findings, this compound could be a potentially effective treatment for androgenetic alopecia.

INTRODUCTION

Licorice is one of the commonly used herbs in traditional medicine and has a long history of use.^1,2) This plant has been used for a long time, especially in countries like China and India.^3,4) 18-β-Glycyrrhetinic acid (GA) is the main active ingredient in licorice (Glycyrrhiza glabra L.) root extract⁵⁾ (Supplementary Fig. S1). While the extract contains 2 to 25% GA, it is primarily in the form of its glycoside, glycyrrhizic acid.⁵⁾ GA represents the active form of glycyrrhizin and has various biological activities. Pharmacological effects of GA include anti-inflammatory, antiviral, immunomodulatory, antiallergic, and anticancer effects.⁶⁾

When inflammation occurs on the scalp, it has been reported that the quality of hair produced from that area diminishes.⁷⁾ Therefore, calming scalp inflammation is crucial for growing healthy hair. The Japanese Ministry of Health, Labour, and Welfare acknowledges GA’s anti-inflammatory effect on the skin (scalp), its role in maintaining a healthy skin (scalp) condition.⁸⁾ For this purpose, many hair care products contain GA in Japan.

In other words, the reason GA is effective for hair growth is not because GA directly acts on the hair follicle cells to promote human hair growth. Rather, it is believed that its anti-inflammatory effects improve the scalp conditions, which in turn indirectly promote hair growth. There are hardly any reports of GA directly acting on human hair follicle cells; the only reports concern in vivo studies related to the back hair of mice. In mice treated with GA, there was a significant increase in the number of hair follicles.⁹⁾ Does GA not possess activity that directly influences human hair follicle cells to promote hair growth?

Androgenetic alopecia (AGA) is the primary cause of male hair loss.¹⁰⁾ Inhibiting the AGA is the primary cause of male hair loss.¹⁰⁾ Inhibiting the enzyme, testosterone 5α-reductase (5αR), and preventing the conversion of androgen to dihydrotestosterone (DHT) is effective against AGA.¹⁰⁾ Therefore, substances with 5αR inhibitory activity are being sought as anti-AGA drugs. Among them, dutasteride and finasteride are the representative drugs.^10,11)

While there are hardly any reports on the effects of GA on AGA, there has been a reported in vivo study where licorice extract was applied to rats. In male rats induced with alopecia by intramuscular injection of testosterone, when treated topically with dutasteride and finasteride and the petroleum ether extract of G. glabra roots, neither developed alopecia due to the testosterone.¹²⁾ This report suggests that licorice extract may contain components effective against AGA. As an example of GA acting on steroid conversion enzymes, it has been reported that GA inhibits the activity of 17β-hydroxysteroid dehydrogenase, which catalyzes the conversion of androstenedione to testosterone.¹³⁾ However, the effect of GA on 5αR which catalyzes the conversion of testosterone to DHT remains unclear.

In dermal papilla cells, DHT, the active form of androgen, binds to the androgen receptor, leading to the upregulation of one of its target genes, transforming growth factor-β1 (TGF-β1).¹⁴⁾ TGF-β1 induces apoptosis in hair follicle epithelial cells, promoting the transition from the anagen phase to the telogen phase.¹⁵⁾ As a result, the hair cycle concludes prematurely, leading to AGA.¹⁶⁾ Therefore, inhibiting the expression of TGF-β1 could be a promising therapeutic approach for AGA. There have been no reports on the effects of GA on the expression of TGF-β1.

In this study, we investigated the effects of GA, the main component of licorice, on the proliferation of human hair follicle cells and its impact on 5αR, a key to the onset of AGA. Furthermore, we investigated the impact of GA on the expression of TGF-β1, a promoting factor for the transition to the telogen phase of the hair cycle, which is a direct cause of AGA.

MATERIALS AND METHODS

Chemicals

GA was provided by Maruzen Pharmaceuticals (Hiroshima, Japan).

Cell Culture

Cell lines of human dermal papilla cell (hDPC) and human outer root sheath cell (hORSC) immortalized with large T antigen were used.^17,18) 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 hDPCs. As the nutrient medium for hDPCs, basal DMEM with 10% fetal bovine serum (FBS) was used. Keratinocyte serum-free medium, Keratinocyte-SFM; K-SFM (Thermo Fisher Scientific), with antibiotics was used as the basal medium for hORSCs. As the nutrient medium for hORSCs, basal K-SFM was supplemented with epidermal growth factor (5 ng/mL) and bovine pituitary extract (50 µg/mL). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. 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).

Cell Proliferation Assays

Cells (hDPCs or hORSCs; 7000 cells each) were seeded onto 96-well culture plates (Iwaki Glass, Chiba, Japan). The cells were then pre-incubated in the respective nutrient media for 2 d. After pre-incubation, the medium was replaced with basal media containing various concentrations of GA. The cells were then cultured for 3 d, and proliferation was evaluated by AlamarBlue assay (Bio-Rad Laboratories, Hercules, CA, U.S.A.).

Real-Time PCR for TGF-β1 Expression in DPCs

Sub-confluent DPCs were cultured on 6-well plates with or without GA for 4 h. 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.

Measurement of 5αR Inhibitory Activity

The following 4 solutions were mixed and incubated at 37 °C for 30 min: 0.02 mL of 14.56 mM testosterone (Sigma-Aldrich, St. Louis, MO, U.S.A.); 0.825 mL of 5 mM Tris–HCl (pH 7.13) containing 1 mg/mL of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (Wako Pure Chemical Corporation, Osaka, Japan); 0.08 mL of the test sample with or without GA; and 0.075 mL of the S-9 mixture. The S-9 mixture (Oriental Yeast, Tokyo, Japan) which containing enriched with 5αR and 3α-hydroxysteroid dehydrogenase (3αHSD) from rat liver homogenate, was used as the source of the enzymes.¹⁹⁾ The 5αR enzyme catalyzes a conversion from testosterone to DHT, and the 3αHSD enzyme catalyzes a conversion from DHT to 3α-androstanediol.²⁰⁾ In this evaluation system, two reaction products, DHT and 3α-androstandiol, can be formed from testosterone. However, since DHT is produced only by 5αR, by measuring the amounts of both DHT and 3α-androstandiol, the conversion rate by 5αR can be calculated. After incubation, 1 mL of dichloromethane was added to extract testosterone and other compounds. Then, the mixture was centrifuged at 1600 × g for 10 min. The lower phase was taken for analysis with a gas chromatography (GC) system. The conditions for GC analysis are shown in Supplementary Table S2. A mixture containing equal amounts of 3α-androstanediol, DHT, and testosterone was used as the standard reference. Methylene chloride solutions of the standard substances, namely 3α-androstanediol, DHT, and testosterone, were subjected to GC under identical conditions. This allowed for the calculation of the amount of substance per peak area for these three compounds. The formula for calculating the percentage inhibition of 5αR activity is given in the supplementary materials.

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 GA on the Proliferation of hDPCs and hORSCs

The effect of GA on the proliferation of hDPCs and hORSCs are shown in Fig. 1. For proliferation assay, the cells were cultured for 3 d. In Fig. 1, “PC” represents the positive control, in which cells were cultured in nutrient medium without test compound. GA significantly enhanced the proliferation of hDPCs at concentrations of 25 µM or greater (Fig. 1a). GA also significantly enhanced the proliferation of hORSCs at concentrations of 10 µM or greater (Fig. 1b). These results indicate that GA can promote the proliferation of both hDPCs and hORSCs.

Fig. 1. The Effect of GA on the Proliferation of (a) hDPCs and (b) hORSCs

The activity of GA is expressed as the cell proliferation index relative to the negative control. “PC” indicates cells cultured in the nutrient medium for hDPCs or hORSCs. For proliferation assay, the cells were cultured for 3 d. Data are given as the mean ± S.D. of eight replicates. * p < 0.05, *** p < 0.001.

The Effect of GA on the Activity of 5αR

The effect of GA on the activity of 5αR is shown in Fig. 2. In the evaluation system used, the inhibition rate of 5αR activity by GA increased linearly, and in a concentration-dependent manner between 26.6 and 212.5 µM. The inhibition rate was 4.2% when GA was at a concentration of 26.6 µM and 78.3% at 212.5 µM. Based on these results, the IC₅₀ for 5αR inhibition by GA was determined to be 137.1 µM. These results indicate that GA inhibits the activity of 5αR.

Fig. 2. Inhibition Rate of 5αR Activity at Each GA Concentration

The relationship between GA concentration and the inhibition rate of 5αR activity can be expressed by a linear equation with R² = 0.995, represented as y = 0.397x − 4.363. From this equation, the IC₅₀ is suggested to be 137.1 µM.

The Effect of GA on the Expression of TGF-β1 by hDPCs

The effects of GA on TGF-β1 expression in hDPCs are shown in Fig. 3. GA significantly down regulated TGF-β1 expression by hDPCs at concentrations of 25 and 50 µM (Fig. 3). This result suggests that GA has the potential to improve AGA by directly inhibiting the expression of TGF-β1 or indirectly through the androgen signaling pathway.

Fig. 3. The Effects of GA on the Expression of TGF-β1 in hDPCs

Data are given as the mean ± S.D. of four replicates. * p < 0.05, ** p < 0.01.

DISCUSSION

GA promoted the proliferation of both hDPCs and hORSCs (Fig. 1). hDPCs in thick hair shafts are larger than those in thin hair shafts.^21–23) Additionally, hDPCs isolated from AGA hair follicles are smaller than those from non-AGA hair follicles.²³⁾ DPCs from AGA patients proliferate more slowly than DPCs isolated from non-AGA follicles.²⁴⁾ Thus, stimulating the proliferation of hDPCs is expected to result in the formation of thicker hair. The current study demonstrates that GA increases the proliferation of hDPCs, suggesting that GA might stimulate the growth of thicker hair.

Compounds that promote the proliferation of hORSCs can extend the anagen phase.²⁵⁾ Consequently, the proportion of hair in the anagen phase increases, and their efficacy in improving AGA in vivo has been reported.²⁵⁾ Given that GA increases the proliferation of hDPCs and hORSCs, it is suggested that GA might also elongate the anagen phase of the human hair cycle. This also suggests that GA may cause the formation of thicker and longer hairs in human.

5αR is an enzyme that plays a significant role in AGA. DHT which is an active form of testosterone is produced from testosterone by 5αR, leading to the increased expression of TGF-β1.¹⁰⁾ Therefore, inhibiting the activity of 5αR is crucial for the improvement of AGA.¹⁰⁾ Dutasteride and finasteride, the medication for AGA treatments, are also an inhibitors of 5αR.^10,11) This study showed that GA inhibited the activity for 5αR in a dose dependent manner (Fig. 2). In other words, GA can be expected to extend the anagen phase by inhibiting the activity of 5αR, thereby suppressing the expression of TGF-β1.

TGF-β1 is a target gene of DHT and androgen receptor complex, and induces apoptosis in hair epithelial cells such as ORSCs.¹⁴⁾ Thus, TGF-β1 stimulates the transition human hair cycle from the anagen phase to the telogen phase.¹⁵⁾ This study demonstrated that GA inhibits the expression of TGF-β1 in human dermal papilla cells (Fig. 3). One mechanism is that GA inhibits DHT production by inhibiting 5αR activity, thereby reducing TGF-β1 expression. Another possibility is that GA affects a step in the regulatory mechanism of TGF-β1 expression, leading to reduced TGF-β1 expression (Fig. 3). A further possibility is that both effects may occur simultaneously. Regardless, GA might extend the anagen phase by inhibiting the expression of TGF-β1, a factor that induces the regression phase. This indicates that GA might elongate the anagen phase, influencing long and thick hair formation. This suggests that GA might also be an excellent tool for treating human AGA.

Acknowledgments

The authors are grateful to Mr. Shogo Ueno for technical help.

Author Contributions

Tokuro Iwabuchi contributed to study conception and design as well as writing of manuscript. Kenta Hagiwara and Akinori Kiso equally contributed to perform the experiments and analyzed data. Shogo Ono, Hiroaki Kitamura, Haruyo Yamanishi and Yuki Tsunekawa also contributed the experiments.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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