1. Introduction

Interleukin (IL-1) was shown to be a potent inhibitor of hair growth. Furthermore, it was reported to be a significant factor in alopecia areata (AA) which is a disease of the anagen stage hair follicles, considered to be autoimmune in origin [1, 2]. In isolated hair follicles IL-1β decreased hair growth approximately 60-80%. This hair growth inhibition was mediated by cAMP ². IL-1α and IL-1β share common receptors. Rückert, et al. [3] reported that high dose proinflammatory cytokines such as IL-1β induced apoptosis of hair bulb keratinocytes in vivo. The hair loss that is seen in transgenic mice overexpressing IL-1α in the epidermis and outer root sheath was reported and compatible with the view of inflammatory cytokines may induce apoptosis within the hair follicle epithelium [4]. In AA a more serious disease was encountered in patients who show gene polymorphism and have insufficient amount of IL-1 receptor antagonists [2]. There were many publications on the mechanism of action of minoxidil. Topical 5% minoxidil solution twice a day on 47 patients was reported to be effective in AA [5]. Minoxidil’s effect on stimulating hair growth does not depend on either a hormonal factor or on the inhibitor action on 5α-reductase. It was also reported that minoxidil increased the mRNA expression for the VEGF at the dermal papilla level [6]. In cell cultures, minoxidil extend the survival and slowed the aging of human keratinocytes and it acts on follicular matrix cells extending the anagen phase [7, 8]. When considering the well documented effects of minoxidil on hair growth it is logical to use it as a reference for comparison purposes of any other formulation to promote hair growth. We developed a botanical extract and carried out a clinical study to show its hair growth promoting effects [9]. Chamomilla recutita was reported to contain apigenin as an active ingredient [10] whereas Urtica urens and Urtica dioica were a source of kaempferol, myricetin, linoleic acid [11], oleic acid [12], iron [13] and beta-sitosterol [14]. Equisetum arvense was reported to contain apigenin, kaempferol and beta-sitosterol [15]. Together with apigenin and beta-sitosterol, Achillea millefolium contained folic acid and several minerals [16] as active ingredients. Ceratonia siliqua, on the other hand, contained, tocopherol, thiamine, riboflavin, pantothenic acid, folic acid, zinc, copper, iron as some of the ingredients [17]. Among these active ingredients apigenin, kaempferol, myricetin, linoleic acid, oleic acid, iron and zinc were reported to stimulate hair growth [18]. In this study, we compared minoxidil and the experimental extract in the human keratinocyte cell line (HaCaT).

2. Materials and Methods

2.1. Cell Culture

The human keratinocyte cell line (HaCaT) was cultured in Dulbecco’s Modified Eagle’s medium (DMEM) with high glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2mM l-glutamine and 100U/ml gentamicin. Cells were maintained at 37°C in a humidified atmosphere at 5% CO₂ in Newbrunswick incubator. All supplements and media were purchased from Sigma Aldrich.

2.2. Preparation of Minoxidil Solution

522.5 mg minoxidil was dissolved in 25 ml distilled water - 25 ml ethanol mixture to get 5mM minoxidil solution. This solution is used as 100% sample and other concentrations (10%, 5%, 3%, 1% and 0.2%) of the solution were prepared by dilution with distilled water.

2.3. Preparation of Plant Extract

The plants were obtained from a commercial plant supplier (Martin Bauer Group, Germany). Chamomilla recutita (Chamomile flowers), Urtica urens (Small nettle leaves), Urtica dioica (nettle leaves), Equisetum arvense (Horsetail leaves), Achillea millefolium (Common yarrow flowers), Ceratonia siliqua (Carob fruit) were used as the plants for extract. Dried plants were fine-cut. Forty grams of plant mixture was extracted with 500 mL distilled water for 3 hours at 100°C using a soxhlet extractor. The extract was filtered through a 0.22 µm filter.

2.4. XTT Cell Proliferation and Cytotoxicity Assay

The Cell Proliferation Kit II (Roche XTT) was used for cytotoxicity assay. This assay based on the cleavage of XTT by metabolic active cell, resulting in an orange formazan dye. The amount of orange formazan dye is quantitated using microplate reader. Briefly, HaCaT cells were seeded into 96-well plates (1x10⁴ cells/well) and were subjected to different concentrations (100%, 10%, 5%, 3%, 1% and 0,2%) an of minoxidil solution and botanical extract. After 72 hour incubation period, 50 µl XTT and activator reagents were added to the each plates according to the manufacturer’s instructions. Then, cells were incubated at 37°C for 4 hours in order that XTT reagent was reduced to formazan compound. The absorbance was read in BIO-RAD microplate reader (Japan) at 450 nm subtracting the background measurement of 620 nm.

2.5. RNA Isolation

HaCaT cells were incubated with 1% concentration of both minoxidil solution and the botanical extract before total RNA isolation. Total RNA was extracted by using TRI-reagent according to manufacturer’s instructions (Sigma Aldrich). The concentration and purity of isolated RNA samples were determined by measuring optical densities at 260 nm and 280 nm using BioSpec- nano (Japan).

2.6. Reverse Transcription

Roche Transcriptor First Strand cDNA Synthesis Kit was used for reverse transcription. cDNA synthesis was performed with 500 ng total RNA, 2 µM final concentration of gene specific primers of VEGF, KGF, IL-1α, SRD5α type 2 and GAPDH (Integrated DNA Technologies), 10 U of Transcriptor Reverse Transcriptase, 20 U of Protector RNase Inhibitor, 1mM each of dNTP mix and Transcriptor Reverse Transcription Buffer (5X) according to the manufacturer’s instructions (Roche).

2.7. Real-Time Quantitative Polymerase Chain Reaction

Real-time PCR (RT-qPCR) reaction was carried out in Light Cycler 96 (Roche) and Fast Start DNA Green Master Kit (Roche) was used. Briefly, total volume of reaction mix was 20 µl containing 10 µl SYBR Green Master Mix (2X), 0.5 µM of reverse and forward primers (Table 1), 2.5 ng cDNA and appropriate amount of nuclease free water. All samples were run as triplicates in each run including a non-template control and four standards (1:10, 1:100 and 1:1000). The PCR parameters were determined separately for each target according to melting and annealing temperatures of primers. Each parameter includes a pre-incubation step for 10 min at 95°C and followed by 45 cycles of 3-step amplification and melting step. Melting curve analysis was performed to verify specificity. Three repeats of gene expression analysis were done (n=3). For quantitation of RT-qPCR results, ΔΔC_t method was used (2^-ΔΔCt).

Table 1. Primer sequences for selected genes (5’ to 3’)

2.8. Statistical Analysis

All data are representative of three experiments and expressed as mean ± standard error of the means (SEM). Statistical evaluation was performed by Unpaired t-test using Graph Pad Prism 5 Software (USA) and the results with p value less than 0.05 were accepted as significant.

3. Results and Discussion

3.1. Cytotoxicity Analysis

Minoxidil solution and the botanical extract showed cytotoxic effect for HaCaT cells at high concentrations. For the subsequent analysis, the possible highest concentration was determined to be 1% (Figure 1).

Figure 1. Cytotoxicity analysis results of minoxidil solution and the plant extract (Dashed bars represent the concentrations chosen for incubation.)

Gene Expression Analysis (Real Time qPCR)

Results of gene expression analysis via RT-qPCR showed that both the botanical extract and minoxidil caused a statistically significant downregulation of IL-1α gene expression compared to untreated control cells. The botanical extract resulted in 0.19 fold change in the IL-1α gene expression level whereas minoxidil treatment resulted in 0.34 fold change (Figure 2). SRD5α type 2 gene expression was not significantly affected by the botanical extract treatment. However, it was downregulated upon minoxidil treatment (Figure 2). VEGF and KGF gene expression were significantly affected by minoxidil and botanical extract treatments in a similar way. VEGF was downregulated upon both treatments: Its expression was reduced to one-seventh of the untreated control cells by the botanical extract while minoxidil reduced it to a one-fourth of the controls (Figure 2). Both minoxidil and the botanical extract treatment reduced KGF gene expression to a one-sixth of the controls.

In a recent study [19] it was shown that twice a day minoxidil spray was effective promoting hair density in frontotemporal and vertex regions in men. On the other hand, our clinical study [9] showed that concomitant use of a hair serum and the shampoo formulated with the botanical extract decreased hair loss in subjects suffering from the TE and AGA. Therefore, in this study, minoxidil was compared to our botanical extract at the cellular level. According to the results of our cell culture studies, minoxidil suppressed SRD5α2, VEGF, and KGF expression and the botanical extract suppressed VEGF and KGF in a similar way as minoxidil (Table 2), however, it did not show a significant effect on SRD5α2 expression. Based on these results, clinically approved effects of minoxidil and our botanical extract on the reduction of hair loss cannot be linked to the changes in VEGF and KGF gene expression since both of them was unexpectedly downregulated by both minoxidil and the botanical extract (Figure 2). There can be various mechanisms by which minoxidil and our botanical extract perform their hair-loss-reducing action. Inhibition of 5α-reductase II may be one of those mechanisms for minoxidil but not for our botanical extract (Figure 2). Among the factors that we tested, IL-1α was the only one which was affected by minoxidil and by the botanical extract in the same manner and also the one which was expectedly downregulated by both. The botanical extract reduced IL-1α gene expression to a one-fifth of the control similar to the reduction to a one-third of the control upon minoxidil treatment (Figure 2). Since IL-1α was shown to inhibit human hair follicle growth and the hair fiber production in whole-organ cultures, downregulation of this factor may explain the effect of our botanical extract and of minoxidil on hair loss [20].

Table 2. IL-1a /GAPDH, SRD5a /GAPDH, VEGF /GAPDH and KGF /GAPDH fold change values for plant extract or minoxidil-treated HaCaT cells

Figure 2. Gene expression levels of IL-1a, SRD5a2, VEGF, and KGF upon plant extract or minoxidil treatment

4. Conclusions

By taking all those into consideration, it was our hypothesis that botanical extracts consisting of different plants can be effective against hair loss. However, their effectiveness must be evaluated by placebo-controlled clinical studies as well as further cell, tissue and organ culture studies.