1. Introduction
Androgen alopecia (AGA) is a common form of hair loss in men and women of different ages [
1,
2]. AGA is associated with high levels of dihydrotestosterone (DHT) released from testosterone by the enzymatic activity of 5α-reductase (5α-R). DHT binds to the androgenic receptor (AR) to form a hormone-receptor complex. The expressions of 5α-R and AR are enhanced in patients with AGA [
3]. In addition to its action on follicular androgen receptors, DHT stimulates growth factor TGF-β in the dermal papillary cells of cellular apoptosis, which cause hair follicles to die [
4].
DHT is mainly degraded in the liver by hydroxysteroid dehydrogenases (HSDs), such as 3α-hydroxysteroid dehydrogenase (3α-HSD), 3β-HSD, and 17β-HSD [
5,
6,
7]. Aldo-keto reductases (ARKs), as one of two protein superfamilies within the 3α-HSD, can bind to DHT [
8] and degrade DHT by acting as a 3-ketosteroid reductase in the presence of dihydronicotinamide adenine dinucleotide phosphate (NADPH) [
9]. Currently, 5α-reductase inhibitors that suppress the conversion of testosterone into DHT are widely used to treat AGA [
5,
6]. However, the effect of the DHT-degrading enzymes on hair growth is less clear and has not been clinically tested.
Many active ingredients of synthetic origin are available for the treatment of baldness [
7,
10]. Only two drugs, finasteride and minoxidil, have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of AGA. Finasteride is primarily used as an oral drug for the treatment of androgen-related hair disorders due to its inhibitory activity on 5α-R [
11,
12], as shown in
Figure 1.
Little is known about the mechanisms of other synthetic substances that are used to decrease hair loss. In addition to its frequent side effects, the length of treatment is often uncertain. Some synthetic substances should be taken for at least 6 months to decrease hair loss and 12 months to restore hair growth [
7,
10]. For these reasons, there is a need for alternative treatments, including cosmetic treatments, supplements, and the use of herbal extracts [
8,
13,
14]. Recently, attention has been given toward the development of new alternative treatments that promote the relief of hair loss symptoms, which has focused on the discovery of new and safer remedies, which were often provided by natural functional products.
For new alternative pharmacological treatments, plant preparations have many advantages over synthetic substances, considering their higher compliance, fewer side effects, wider activity spectrum, and lower cost of treatment. Due to the diverse composition of plant extracts, plants often possess more than one biochemical action that is effective for treating hair loss [
15]. Many researchers have searched continually for new medicinal plants with active ingredients that could present a viable natural alternative to already established hair loss treatments using synthetic substances.
Sulforaphane, which is an isothiocyanate isolated from broccoli, is a potential modulator of DHT degradation and possesses anti-oxidative effects through the activation of the nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) pathway [
9,
16]. It has been reported that sulforaphane induces DHT-degradation enzymes, such as aldo-keto reductase 1c1 (AKR1C1) [
17] and aldo-keto reductase 1c2 (AKR1C2) [
18] in human cultured cells. It was previously reported that sulforaphane increased the mRNA and protein levels of two forms of 3α-hydroxysteroid dehydrogenase (3α-HSD), AKR1C1 and dehydrogenase/reductase (SDR family) member 9 (DHRS 9) in the liver of the mice and in cultured murine hepatocyte Hepa1c1c7 cells. [
19,
20].
This study aims to provide insight into the possibility of using sulforaphane as a potent and functional hair cosmetic ingredient as an alternative pharmacological treatment. The potential effect of sulforaphane is its ability to promote the relief of hair loss symptoms by the expression of two isozymes of 3α-HSD, AKR1C2 and DHRS9, which are the murine homologous of human 3α-HSD isozymes that are encoded by Akr1c2 [
21] and Dhrs9 [
22]. To the best of our knowledge, sulforaphane has not been reported as a functional cosmetic material that induces DHT-degrading enzymes to promote the relief of hair loss symptoms in humans.
To further understand the underlying mechanism, our laboratory recently tested whether sulforaphane could enhance the mRNA and protein levels of Akr1c2 and Dhrs9 in murine Hepa1c1c7 cells. In these experiments, we employed RAW 264.7 macrophage cells and Hepa1c7c7c cells for the cytotoxicity and genetic identification of Ak1c21 and Dhrs9 at the RNA and protein levels, respectively. As negative controls, four common ingredients were also employed, including dexpanthenol, biotin, l-menthol, and zinc pyrithione, which are known as functional cosmetic materials that help relieve hair loss symptoms, according to the Ministry of Food and Drug Safety Food and Drug Administration (MFDS) in Korea. Subsequently, based on a clinical application test for hair loss, we evaluated whether potential functional cosmetics containing sulforaphane, l-menthol, and dexpanthenol as active ingredients promoted the relief of hair loss symptoms.
2. Materials and Methods
2.1. Chemicals and Reagents
Sulforaphane, biotin, dexpanthenol, L-menthol, zinc pyrithione, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Murine RAW264.7 macrophage cells and murine Hepa1c1c7 cells were purchased from the Korea Cell Line Bank (Seoul, Korea). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the cell proliferation assay kit (Quanti-MaxTM) were purchased from Biomax (Seoul, Korea).
2.2. Cell Culture
RAW264.7 macrophage cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, USA) supplemented with 10% fetal bovine serum (FBS, GIBCO, Grand Island, USA) and 1% penicillin/streptomycin (P/S) (100 U/mL, 100 mg/mL, respectively). In the logarithmic growth phase, RAW264.7 macrophage cells were maintained in DMEM at 37 °C. The Hepa1c7c7 cells were also cultivated in DMEM supplemented with 10% FBS and 1% P/S (100 U/mL and 100 mg/mL, respectively). The cells were maintained in a minimum essential medium eagle-alpha modifier (α-MEM) under incubation at 37 °C and 5% CO2.
2.3. Cell Viability
The MTT assay was used to determine the cytotoxicity of biotin, dexpanthenol, L-menthol, zinc pyrithione, and sulforaphane, which are substances that relieve hair loss, and to test the cytotoxicity of sulforaphane, which is expected to be effective in relieving hair loss symptoms. RAW264.7 macrophage cells and Hepa1c1c7 cells were seeded into 96-well plates at a concentration of 5 × 104 cells per well and were treated with different concentrations (0 μg/mL to 50 μg/mL) of biotin, dexpanthenol, l-menthol, and zinc pyrithione for 24 h. Subsequently, an MTT solution of 5 mg/mL (Quanti-Max™, Biomax, Korea) was added to each well and then maintained in an incubator under the conditions of 37 °C and 5% CO2 for 4 h. Finally, a UV MAX kinetic microplate reader (Synergy™ microplate reader, BioTek, Winwooski, USA) was used to measure the absorbance at 450 nm. The experiment was independently repeated three times under the same experimental conditions.
2.4. Isolation of Total RNA and Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated with an RNeasy Mini Kit (RNeasy Mini kit, Qiagen, Germantown, MD, USA). RNA was reverse-transcribed with an oligo (dT) primer using the SuperScript First-strand Synthesis System (Intron Power cDNA Synthesis Kit, Intron, Korea). RT-PCR was performed using recombinant Taq DNA polymerase according to the manufacturer’s instructions (2xPCR master mix solution, Intron, Korea). The GAPDH mRNA level was used as a control. The following primers were used: Dhrs9: Forward 5′-GGAAACTTAGCAGCCAGAAC-3′ and Reverse 5′-AACACGCCAAGAACACCAG-3′; and Akr1c2: Forward 5′-AATGGCCCTGAAACCAGGAG-3′ and Reverse 5′-GACCACAATCCCACGCTGTA-3′; GAPDH: Forward 5′ACCACAGTCCATGCCATCAC-3′ and Reverse 5′-CACCACCCTGTTGCTGTAGCC-3′.The conditions for PCR were 30 cycles of 94 °C denaturation for 30 s, 60 °C annealing for 30 s, and 72 °C extension for 30 s. The PCR products were analyzed by electrophoresis on 2% agarose gels stained with RedSafe staining solution (Intron, Korea).
2.5. Western Blotting
Cell extracts were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane and immunoblotted with anti-rabbit DHRS9 polyclonal (MBS768606, MyBioSource, San Diego, CA, USA), anti-rabbit polyclonal AKR1C2 (PA5-36572, Invitrogen, Waltham, MA, USA), or anti-β-Actin rabbit polyclonal antibodies(Ab8227, Abcam, Cambridge, MA, USA), followed by incubation with secondary anti-goat antibody (Ab6721, Abcam, Cambridge, MA, USA). Reactive bands were detected by chemiluminescence using a western lightning reagent (Clarity™ Western ECL Substrate, Bio-Rad, Hercule, CA, USA).
2.6. Prototype Preparation of a Functional Cosmetic Formulation to Relieve Hair Loss
A prototype formulation was prepared for the clinical application test to promote the relief of hair loss symptoms. Besides the active ingredients, including dexpanthenol, biotin, l-menthol, and sulforaphane, which are functional cosmetic materials that relieve hair loss, according to the MFDS in Korea. Most the other ingredients listed in the formula ingredients statement were purchased from Sigma-Aldrich (St. Louis, MO, USA). A prototype formulation was manufactured at a small scale at a pilot plant in Interkos, Inc. (Inchon, Korea).
2.7. Clinical Application Test for Hair Loss
2.7.1. Purpose of the Trial
This test will be used to evaluate the capability of the prototype formulation to relieve hair loss symptoms after 18 weeks of use. The trial target formulation was tentatively designated as The Fact/BHCG (Biodegradable Hair Control Gel).
2.7.2. Description for the Trial Target
Men and women from 18 to 54 years old were diagnosed by a basic and specific (BASP) classification. The basic type was grouped from male and female with an androgenic alopecia that was classified autonomously as M1 or higher, C1 or higher, or U1 or higher. The specific type was also grouped from male and female, with androgenic alopecia classified autonomously as V1 or higher, or F1 or higher. Men and women were diagnosed as 2 or 2A or higher by Norwood-Hamilton classification, and as more than 1 by Ludwig classification.
2.7.3. Evaluation Methods
All measurements and evaluations were carried out in a space without air movement or direct sunlight. Based on the skin stability of the subjects under constant temperature and humidity conditions (22 ± 2 °C and 50 ± 5%), the evaluation was performed before and after use of the test product implemented in the trial.
2.7.4. Visual Evaluation for the Crown of the Head and Forehead Line
In this test, a high-resolution digital camera (DSLR, Canon Inc., Tokyo, Japan) was used to take pictures of the crown of the head at a 90-degree angle and the forehead line at a 45-degree angle under the same conditions. After 6, 12, and 18 weeks of treatments, the test product was implemented for the trial, and the structure and location with relief of hair loss were observed from the crown of the head and forehead line [
23].
2.7.5. Quantitative Evaluation Using Phototricogram
In this test, a folliscope (LeadM, Seoul, Korea) was used to evaluate hair loss and growth rates by magnifying areas of scalp or hair before and after 6, 12, and 18 weeks treatments of the test product that was implemented for the trial. After cutting the hair on areas with hair loss, a small dot tattoo with a diameter of 1 mm was applied to evaluate the center of the tattoo each time using a phototricogram. It was evaluated statistically whether the test product group showed a trend of an increase in hair growth after using the product from 6 weeks to 18 weeks.
2.7.6. Statistical Analysis
To verify the statistical significance before and after the test product was used, a statistical analysis was performed using Embedded on SPSS Statistics 26 (IBM, Armonk, NY, USA), and the significance was confirmed when the significance probability was p < 0.05 in the 95% confidence interval. The resulting value, which was calculated through device evaluation, was expressed by obtaining the mean and standard deviation as a continuous variable, and the resulting value of the questionnaire evaluation was expressed as a frequency and percentage as categorical variables.
The normality of the data was verified using the SPSS Shapiro–Wilk test. In the case of repeating measured data with a measurement point of three or more times, if normality was satisfied, the test was performed using repeated measures analysis of variance, which is a parametric method, followed by Bonferroni correction, and the normality was not satisfied [
24]. If not, it was tested with Friedman test, which is a nonparametric method, followed by Bonferroni correction, and post-tested with the Wilcoxon signed rank test [
25].
4. Discussion
In this present study, we determined the hair loss-relieving efficacy of a mixture of sulforaphane, dexpanthenol, and l-menthol. To measure its efficacy, we performed a clinical application test using the prototype formulation. The results after 18 weeks of using the prototype formulation revealed a significant difference in the hair loss-relieving efficacy when compared to that before using the product. To further confirm this effect at the molecular and cellular level, we then tested the expression levels of Ak1c21 and Dhrs9 in the in vitro cell culture experiment. Hepa1c1c7 cells were treated with sulforaphane or a mixture of biotin, dexpanthenol, and l-menthol. This study showed that sulforaphane alone achieved a hair loss-relieving effect in our experimental cell culture conditions. Although the mixture of biotin, dexpanthenol, and l-menthol did not show any difference under a time- or dose-dependent manner in our in vitro experiments, we speculated the possibility that these mixtures might have a hair loss-relieving effect in the clinical trial test to relieve hair loss, because the mechanisms of action of these mixtures are still not fully understood yet. Actually, the MFDS, a cosmetics regulatory authority in Korea, approved dexpanthenol, biotin, l-menthol, and zinc pyrithione as functional cosmetic materials that can help to relieve hair loss symptoms. We excluded biotin and zinc pyrithione from our in vitro cell culture experiments since biotin and zinc pyrithione are widely used in cosmetic products and show cytotoxicity at certain temperature ranges. Though the use of biotin or zinc pyrithione as a hair growth supplement is prevalent, research demonstrating the efficacy of biotin [
27] or zinc pyrithione is limited [
28].
As one of the main ingredients promoting the relief of hair loss symptoms in our experiments, topical dexpanthenol has emerged as a frequently used formulation in the field of dermatology and skin care. Various studies have confirmed that dexpanthenol offers moisturizing [
29] and skin barrier enhancing potential [
30]. Other human studies also demonstrated an increased concentration of pantothenic acid in the hair and hair roots after topical administration [
31]. Although discovered decades ago, the action mechanisms of dexpanthenol have not been fully clarified. Recently, various trends in the applications of new technologies focused on dexpanthenol’s mode of action at the molecular level.
Regarding the effect of l-menthol on the relief of hair loss in previous in vivo experiments, the transcriptome analysis of skin from the dorsal side of a mouse treated with l-menthol compared to that of the controls revealed significant changes in keratin, keratin-associated protein, forkhead box, sonic hedgehog, fibroblast growth factor 10, desmoglein 4, deoxyribonuclease-like 2, and cadherin 3, which are known to play roles in promoting hair growth [
32].
Our finding that sulforaphane induces Akr1c21 is consistent with previous studies [
33]. It was also shown that sulforaphane not only induced Akr1c21 expression in cultured murine Hepa1c1c7 hepatocytes but also alleviated hair loss symptoms in a clinical application test for 23 people with AGA. Sulforaphane treatment induced the expression of another isoform of 3α-HSD, Dhrs9, in an in vitro cell culture experiment. Dhrs9 has several NE-F2 binding sites in the promoter region that bind to Nrf2, which are known to be induced by sulforaphane [
18,
20]. Thus, it is highly likely that sulforaphane might enhance the degradation of DHT, not only via the induction of degrading enzymes 3α-HSD, but also by functional activation of these enzymes. Further studies remain to test this possibility soon.
The synergistic effect of sulforaphane, dexpanthenol, and l-menthol in promoting the relief of hair loss also remains to be further investigated. Notably, our synergistic effects would be drawn from the potential effects of dexpanthenol and l-menthol at the molecular levels, even though the action mechanisms of the two substances have not been fully elucidated.
Our present results suggest that sulforaphane may enhance efficacy to relieve hair loss with the two substances described above, possibly synergistically. Although the mechanism of action of sulforaphane, dexpanthenol, and l-menthol is not fully understood, we believe that these potential bioenhancers may be applied to develop functional cosmetics applications for the relief of hair loss. Actually, based on the clinical application results obtained from this test formulation, we expect that we may be able to launch new excellent functional cosmetics in the global market.