Modulation of Hair Growth Promoting Effect by Natural Products

A large number of people suffer from alopecia or hair loss worldwide. Drug-based therapies using minoxidil and finasteride for the treatment of alopecia are available, but they have shown various side effects in patients. Thus, the use of new therapeutic approaches using bioactive products to reduce the risk of anti-hair-loss medications has been emphasized. Natural products have been used since ancient times and have been proven safe, with few side effects. Several studies have demonstrated the use of plants and their extracts to promote hair growth. Moreover, commercial products based on these natural ingredients have been developed for the treatment of alopecia. Several clinical, animal, and cell-based studies have been conducted to determine the anti-alopecia effects of plant-derived biochemicals. This review is a collective study of phytochemicals with anti-alopecia effects, focusing mainly on the mechanisms underlying their hair-growth-promoting effects.


Introduction
Hair affects the impacts of thermoregulation, physical protection, and social interaction [1]. Hair disorders, which include alopecia, anagen effluvium, telogen effluvium, hirsutism, hypertrichosis, and miniaturization, negatively affect health [2]. Alopecia is defined as a decrease in hair density and can be considered a common symptom of several pathologies, such as inflammation and infection [3]. Alopecia can be a sign of a systemic disease, including thyroid dysfunction, systemic lupus erythematosus, trichotillomania, or infection [3]. Although alopecia is a non-life-threatening condition, it can affect the esthetics, social activities, and quality of life of individuals [4]. Hair growth is the result of the growth and differentiation of hair follicles (HFs) comprising dermal papilla cells (DPCs) and epithelial cells. The formation of HF involves four cycles: anagen (growth), catagen (regression), telogen (rest), and exogen (shedding). The hair cycle transition is controlled by several growth stimulatory or inhibitory factors [5]. Alopecia can be categorized into several classes. Androgenetic alopecia, the most common form of alopecia, is a male or female pattern hair loss that is characterized by a progressive loss of hair diameter, length, and pigmentation. It is caused by genetic factors and inadequate androgen signaling [6]. Androgenetic alopecia also worsens conditions such as drug side effects, acute stressors, and weight loss [6]. Alopecia areata is an autoimmune disorder (similar disorders include thyroid disease, celiac disease, vitiligo, and atopy) caused by lymphocytes that attack the bulb of HFs in the anagen phase [7]. This disease affects up to 2% of the population and commonly occurs during childhood or adolescence [7]. Patients with alopecia areata may experience sudden hair loss [8]. Telogen effluvium refers to the loss of telogen hair due to abnormal hair circulation, and generally 100-200 telogen hairs are lost every day [9]. The causes of acute telogen effluvium (in which hair loss lasts less than six months) includes systemic disease, drugs, fever, psychological/emotional stress, weight loss, childbirth, iron and vitamin D deficiency, inflammatory scalp disorders, interruption of oral contraceptives, Table 1. Bioactive components from plants with hair-growth-promoting therapeutic effects.

Botanical Name Parts Used Bioactive Components Type of Extract Ref
Citrus limon L., Fragaria ananassa L., Secale cereale L.

Wnt/β-Catenin
Wnt/β-catenin signaling is broadly utilized and plays a crucial role in HF morphogenesis [144]. In canonical Wnt/β-catenin signaling, Wnt proteins bind to Frizzled (FZD) receptors and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors. Once activated, the β-catenin complex with APC and Axin is phosphorylated by casein kinase Iα (CKIα) and glycogen synthase kinase-3β (GSK-3β). Therefore, β-catenin accumulates in the cytoplasm and binds to the transcription factor T-cell factor/lymphoid enhancing factor (TCF/LEF) in the nucleus [145]. The study reported that Wnt/β-catenin activation promotes hair regeneration by inhibiting GSK-3β in hDPCs [146]. Sinapic acid treatment increased the protein level of β-catenin by upregulating phosphorylated GSK-3β and Akt [16]. Oleuropein elevated nuclear β-catenin protein expression and increased LEF1 and cyclin D1 mRNA expression in DPCs [19]. Oleuropein administration increased the mRNA levels of Wnt10B, LRP5, and FZDR1, and the protein level of β-catenin in mice compared to minoxidil-treated C57BL/6 mice [19]. RGO remarkably increased the protein expression of β-catenin and LEF1 induced by testosterone and RGO co-treatment in dorsal skin tissues compared to treatment with testosterone alone [22]. RGO also enhanced β-catenin and phosphorylated GSK-3β protein expression in C57BL/6 mouse skin compared to that in the minoxidil-treated group [38]. Thuja orientalis (TO) is used to treat dermatitis, gout, and chronic tracheitis [147]. TO (5.05 mg/cm 2 /day) administration promoted hair growth and the early anagen phase, and prolonged the mature anagen phase in mice [39]. Immunohistochemical analysis showed increased β-catenin expression after TO treatment [39]. 3,4,5-Tri-O-caffeoylquinic acid (TCQA) is a caffeoylquinic acid derivative that acts as a neuroprotective agent and protects against amyloid-β (Aβ)-induced cell death [148,149]. Topical treatment with 1% TCQA prolonged anagen phase induction in C3H mice for 30 days [40]. It was also confirmed that β-catenin expression increased in the skin of TCQA-treated mice and HFDPCs [40]. Gene expression profile data revealed an increase in the expression of the canonical Wnt-associated genes, Ctnnb1, Wls, Wnt2b, and Wnt4 after TCQA treatment [40]. Ishige sinicola (IS) is a brown alga that exhibits various activities, including osteoblastic bone formation [150] and anti-inflammatory effects [151]. IS treatment for 3 weeks increased hair-fiber length in rat vibrissa follicles and induced anagen progression of the hair shaft [41]. Protein expression of β-catenin and phosphorylation of GSK3β were increased after treatment with IS in cultured DPCs [41]. Prunus mira Koehne (PK) is a wild peach species that contains various nutrients and fatty acids [152]. Zhou et al., showed the hair-promoting effect of nut oil from PK [42]. They demonstrated that nut oil from PK increased hair length in mice and upregulated Wnt10B, β-catenin, and GSK-3β expression in mice [42]. Costunolide, a constituent of Saussurea lappa, has been used as an antioxidant, anti-inflammatory, and anticancer agent [153][154][155]. Costunolide increased cell proliferation and β-catenin expression in hHFDPCs [43]. Morroniside is the main component of Cornus officinalis and possesses neuroprotective, anti-apoptotic, and antioxidant effects [156][157][158][159]. Morroniside increased Wnt/β-catenin signaling by upregulating Wnt10B, β-catenin, and LEF1 in cultured human ORS cells [44]. In a mouse model, morroniside promoted the anagen phase and delayed the catagen phase of HF, which was partly related to an increase in β-catenin expression [44]. 3-DSC inhibited the phosphorylation of β-catenin protein but promoted the transcriptional activity of TCF/LEF [36]. Timosaponin BII extracted from Anemarrhena asphodeloides rhizome is known to have antioxidant [160], anti-inflammatory [161,162], and anticancer [163] properties. A clinical study showed that application of timosaponin BII (0.5%) containing scalp care solution for 28 days resulted in improvement in hair and scalp conditions, particularly hair luster, scalp hydration, hair fall number, and scalp redness level [164]. A recent study showed that timosaponin BII (0.5%) increased the hair regrowth area and HF number in mice [45]. Moreover, timosaponin BII treatment was shown to upregulate β-catenin and Wnt10B expression in the dorsal skin of mice [45]. The application of 20 mg/mL liposomal honokiol also increased Wnt3a and β-catenin expression [34]. Malva verticillata (MV) is a traditional herb native to Mongolia that contains flavan-3-ols, flavonoids, and fatty acids [119]. MV is a therapeutic candidate for diabetes [121], bone disease [165], and cancer [166]. MV leaves, stems, and seeds have been shown to be a rich source of phenolic compounds. Microbial fermentation has been used to increase the extraction yield of bioactive compounds from natural products [167]. Bacillus subtilis fermentation of MV leaves exhibited an antioxidant and osteogenic effect [168]. The seeds of MV (50 µg/mL) activated β-catenin protein expression in cultured DPCs [32]. Myristoleic acid, an active compound of MV, upregulates Wnt reporter activity [32]. Another study showed that LA in MV seeds increased cell proliferation and phosphorylation of GSK-3 and β-catenin in DPCs [33]. Salvia plebeia (SP) belongs to the family Labiaceae and is used for its antioxidant [169], anti-inflammatory [170,171], and anti-influenza [172] effects. It is reported to contain flavonoids, phenolic acids, and other nutrients [169]. SP extract enhanced the proliferation of hHFDPC and increased the TCF/LEF-luciferase activity as well as the level of β-catenin protein expression [46]. The use of hair tonics, including Broussonetia papyrifera (BP) extract, showed an increase in total hair count for 12 weeks in a clinical study. In addition, BP treatment increased TCF/LEF-luciferase activity and β-catenin protein levels in vitro [37]. Undariopsis peterseniana (UP), an edible brown seaweed, is a rich source of nutrients and acts as an antioxidant and anti-inflammatory agent [173][174][175][176]. UP extract was shown to induce hair growth in ex vivo organ cultures [47]. UP extract upregulated the phosphorylation levels of β-catenin and GSK-3β compared with the control in DPCs [47].

5α-Reductase Inhibitory Effect
Testosterone is converted to DHT by 5α-reductase, which is known to cause androgenic alopecia [177]. Androgenic alopecia affects over 50% of men over the age of 50 [178], compared to only 25% of women by the age of 49 and 41% of women by the age of 69 years [179]. Finasteride, a 5α-reductase inhibitor, has shown improvement in androgenic alopecia in clinical trials [13]. However, the use of 5-α reductase inhibitors has revealed that it has sexual and reproductive side effects [180]. DHT binds to androgen receptors in DPCs, leading to the onset of the telogen phase [177]. Sophora flavescens or Undariopsis peterseniana treatment showed potent 5α-reductase inhibitory effects [24,47]. Octaphlorethol A, a constituent of Ishige sinicola, inhibited 5α-reductase activity compared to that of finasteride [41]. Costunolide treatment downregulated testosterone-induced 5αreductase mRNA expression in hDPCs [43]. Puerariae Flos (PF) has been used as a medicinal herb for its antioxidant, antidiabetic, and protective effects against ethanol-induced injury [181][182][183]. PF extract showed inhibitory effects on 5α-reductase and hair-growthpromoting effects in mice [48]. Cacumen platycladi (CP) is a Chinese medicine containing organic acids, flavonoids, and phenylpropanoids [184]. Treatment with CP decreased DHT levels and 5α-reductase expression while promoting hair growth in vivo [49]. Another study showed that CP volatile oil treatment increased the proliferation of hDPCs and shortened the time of hair regrowth [185]. Ginseng rhizomes have been used in medical remedies [186,187]. Ginsenoside Ro, a major ginsenoside constituent in the ginseng rhizome, has anti-inflammatory [188,189], antioxidant [190], and anti-obesity [191] effects. Murata et al., revealed that red ginseng rhizome extract, ginsenoside Ro, and ginsenoside Rg3 showed inhibitory effects on 5α-reductase activity [50]. The study also showed that topical administration of red ginseng rhizomes (2 mg/mouse) and ginsenoside Ro (0.2 mg/mouse) induced hair regrowth in testosterone-treated mice [50]. Physcion, a component of Polygonum multiforum (PM), has anti-inflammatory, antioxidant, and anticancer effects [192,193]. Treatment with physcion exhibited hair growth-promoting activity in testosterone-treated C57BL/6 mice [51]. In addition, physcion inhibits 5α-reductase expression in vivo [51]. Rosmarinus officinalis belongs to the Lamiaceae family and has been widely studied for its antibacterial, antioxidant, and anticancer activities [194][195][196]. Murata et al. showed that Rosmarinus officinalis leaf extract improved hair regrowth in mice, as well as 5α-reductase inhibitory activity [52]. Avicennia marina is used in traditional medicine for the treatment of skin diseases, rheumatism, ulcers, and smallpox [197]. Avicequinone C, isolated from Avicennia marina, attenuated 5α-reductase inhibitory activity (IC 50 of 38.8 ± 1.29 µM) [53].

Sonic Hedgehog (Shh) Signaling
The hedgehog pathway is one of the most important signaling pathways in tissue development, homeostasis, and repair [201]. Hedgehog signaling is triggered by Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Shh signaling plays an important role in HF development [202]. Hedgehog signaling occurs through the binding of hedgehog ligands to the receptor Patched (PTCH) 1, which suppresses the activation of Smoothened (SMO). Inhibition of SMO results in the translocation of the glioma-associated (GLI) gene to the nucleus [202]. Shh plays an essential role in the cell-cell interactions involved in the morphogenesis of hair follicles [203]. Hair follicle development results from complex signaling between epithelial and mesenchymal cells. Various signaling pathways are involved in hair follicle development, such as Wnt, bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), Notch, and ectodysplasin, and they exhibit signaling crosstalk with the hedgehog pathway [201]. RGO treatment upregulated Shh signaling-related expression of Shh, SMO, and GLI1 in testosterone-induced C57BL/6 mice [22] and in mouse skin [38]. TO treatment resulted in an increase in Shh expression [39]. Costunolide elevated GLI1 mRNA and protein expression in hDPCs [43]. Epigallocatechin-3-gallate (EGCG) is a major bioactive molecule in green tea that has been shown to act on multiple molecular targets to ameliorate various human diseases [204]. Green tea leaf extracts have polyphenolic components that exhibit anti-inflammatory and stress-inhibitory effects, which may influence mouse hair growth [205]. EGCG treatment promoted the growth of mink hair follicles and the proliferation of DPCs and outer root sheath cells (ORSCs) [55]. EGCG has also been shown to increase the protein levels of Shh, PTCH, Smo, and Gli1 in hair follicles [55].

Apoptosis
Apoptosis plays an important role in morphological development and is accompanied by a number of characteristic morphological changes, including cell shrinkage, nuclear condensation, and cellular fragmentation [206]. There are two major apoptosis signaling pathways: the death receptor (extrinsic) pathway and the mitochondria-mediated pathway [207]. The extrinsic pathway is initiated by cell-surface-expressed death receptors of the tumor necrosis factor superfamily. Once the receptor is activated, caspase-8 is activated and initiates apoptosis by direct cleavage of downstream effector caspases [207]. The intrinsic pathway is initiated by intracellular stresses, and it induces permeabilization of the outer mitochondrial membrane and activates the mitochondrial pathway. Once mitochondrial permeabilization occurs, cytochrome c is released into the cytosol and associates with caspase-9 and Apaf-1 to form apoptosomes, which can activate caspase-3 or caspase-7, causing apoptosis [207]. Apoptosis signaling is attenuated by a particular group of proteins (Bcl-2, Bcl-xL, and Mcl-1) [208]. Another group of proteins (Bax, Bak, Bok, Bim, Bad, Bcl-xS, and Bid) act as apoptotic agonists that promote apoptosis [208]. In the anagen phase, hair follicular cells undergo dynamic cell proliferation and differentiation to form the hair shaft [209]. During the catagen phase, growth factors expressed by DPCs cause a decrease in the proliferation and differentiation of hair matrix keratinocytes, leading to apoptosis [210,211]. The anagen-catagen transition plays a clinically important role in human hair growth; the abnormal termination of the anagen phase has been shown to cause gradual hair thinning [211]. Therefore, extension of the anagen phase is a key strategy for the prevention of hair loss. Caffeine administration inhibits apoptosis and necrosis in human ORS keratinocytes [20]. The decrease in the protein level of Bcl-2 in testosterone-treated mice was reversed after treatment with RGO, LA, and SITOS [22]. Treatment with RGO after UVC exposure inhibited cleaved caspase-3, cleaved caspase-9, and cleaved poly-ADP ribose polymerase (PARP) expression in mouse skin tissues [38]. In addition, RGO treatment resulted in an increased Bax/Bcl-2 ratio in the UVC-treated group compared to that in the control group [38]. Quercitrin administration resulted in a decrease in mRNA expression of Bad, although the mRNA and protein expression of Bcl-2 increased after treatment of cultured hDPCs with 10 nM and 100 nM quercitrin [23]. PM extract also showed an increase in Bcl-2 mRNA expression and a decrease in Bad expression in hDPCs [30]. Panax ginseng extract inhibited apoptosis in Dickkopf-1 (DKK-1)-induced ORS keratinocytes as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining [56]. Moreover, Panax ginseng extract downregulated the mRNA expression of Bcl-2 and upregulated the mRNA expression of Bax in DKK-1induced ORS keratinocytes and HF [56]. AS treatment increased protein expression of Bcl-2 but led to a decrease in the protein levels of Bax, PARP, and caspase-3 in HFDPCs [31]. SP extract elevated the protein expression of Bcl-2 and decreased Bax expression compared with the negative or positive control (10 µM minoxidil) in hDPCs [46]. The extract from the berries Serenoa repens (SR), commonly known as saw palmetto, showed therapeutic effects as a 5α-reductase inhibitor [212]. SR and DHT co-treatment stimulated hair growth compared to that induced by DHT in vivo [57]. Moreover, SR treatment led to a decrease in the protein expression of TGF-β2, cleaved caspase-3, and Bax, but increased Bcl-2 expression compared to DHT treatment [57].

Cell Cycle
Cell proliferation is controlled by factors that regulate the transition between different cell cycle stages in mammalian cells [213]. Cell cycle progression also plays a major role in HF biology [214]. The cell cycle consists of four phases: gap phase 1 (G1), DNA synthesis (S), gap phase 2 (G2), and mitosis (M) [214]. Cyclins and cyclin-dependent kinases (CDKs) function as regulators of the G1/S or G2/M phases [215]. Cyclin D1 binds to CDK4 and CDK6 and drives cell cycle progression into the G1 phase [213]. Cyclin E associates with CDK2 to promote G1-S phase transition [214]. Sinapic acid treatment was accompanied by an increase in cyclin D1 and the distribution of cells in the G0/G1 phase, as well as a decreased distribution in the S and G2/M phases [16]. Cyclin D1 is a direct target for transactivation by the β-catenin/LEF-1 pathway through an LEF-1 binding site in the cyclin D1 promoter and is a direct downstream molecule in the β-catenin pathway [213]. Woo et al. suggested that sinapic acid treatment increased cell growth and cell cycle progression through an increase in cyclin D1 expression [16]. Administration of RGO increased the protein expression of cyclin D1 and cyclin E in testosterone-treated mice [22] and C57BL/6 mouse models [38]. UP treatment upregulated cyclin D1, phospho(ser780)-pRB, cyclin E, phospho-CDK2, and CDK2 protein expression in DPCs [47]. Treatment with IS upregulated cyclin E and CDK2 expression in cultured DPCs [41]. LA in MV seeds increased the mRNA levels of cyclin D1 and CDK2 in vitro [33]. EGCG treatment increased the number of cells in S phase, and upregulated the protein levels of cyclin D1 and cyclin B1 [55].

Conclusions
This study reviewed the beneficial effects of herbs and their bioactive compounds on hair growth, and their underlying mechanisms of action (growth factors and cytokines, Wnt/β-catenin, 5α-reductase inhibitory effect, sonic hedgehog signaling, apoptosis, and cell cycle progression). The herbs and their constituents investigated in this study act via multiple signaling mechanisms in the prevention of alopecia. Therefore, they have the potential to be more effective than minoxidil and finasteride, which are conventionally used to treat hair loss. In this review, we have attempted to provide a database of phytochemicals for hair-growth-promoting effects. This information will serve as a basis for developing more effective therapeutic agents for the treatment of alopecia and improving our understanding of their mechanisms of action.

Conflicts of Interest:
The authors declare no conflict of interest.