Hair Growth Promoting Effect of Trichoxidil TM : A New Natural Compound for Hair Loss

Background: Androgenetic alopecia (AGA) is a most common condition of hair loss. Thus, the present study aimed to investigate the effect of Trichoxidil TM , a phytocomplex obtained from a blend of essential oils, in the treatment of hair loss caused by AGA. Methods: The CCD1072Sk cells were cultured for the 24-hour cell viability assessment and cytotoxicity of Trichoxidil TM . The expression of mRNA levels from KGF, IGF-1, and VEGF in fibroblasts was evaluated by RT-qPCR. Thirty-three volunteers, diagnosed with AGA, men and women, aged between 25 and 50 years, were divided into Control group, without treatment (n = 5); Trichosol TM vehicle group, without active (n = 5); Hydroalcoholic vehicle group, without active (n = 4); Trichosol TM vehicle group, with 2.5% minoxidil (n = 5); Hydroalcoholic vehicle group, with 2.5% minoxidil (n = 5); Trichosol TM group with 2.5% Trichoxidil TM (n = 5) and Hydroalcoholic vehicle group with 2.5% Trichoxidil TM (n = 4) to dermoscopic and histologic. Results: Fibroblasts exhibited higher proliferation when treated with higher concentrations of Trichoxidil TM . Trichoxidil TM significantly increased the expression of KGF, IGF-1, and VEGF mRNA in fibroblasts cells. Analysis of the capillary density showed that Trichoxidil TM associated with Trichosol TM vehicle, was the most effective association. In addition, it was observed an increased more effectively the percentage of anagen phase and reduction of the telogen when compared to other formulations. Conclusion: Trichoxidil TM promoted proliferative effects and positively modulated the expression of growth factors IGF-1, VEGF, and KGF, being a promising candidate for the treatment of hair loss caused by AGA.


Introduction
Androgenetic alopecia (AGA) is a most common condition of hair loss [1] [2]. It is characterized by a progressive loss of diameter, length, and pigmentation of the hair that causes serious psychological impacts [2] [3].
Clinical treatments have variable responses and require long-term care, a factor that reduces patient follow-up [2]. Classic clinical treatment is recommended and may or may not be associated with surgical treatments such as hair transplantation. Topical minoxidil and oral finasteride are among the most used medications [2] [4]. However, new clinical treatments are necessary [2]. The use of natural products treatment as an alternative or adjuvant therapy has been carried out for a long time [5].
Thus, the present study aimed to investigate the effect of Trichoxidil TM , a phytocomplex obtained from a blend of essential oils, on hair loss caused by AGA, analyzing the proportion of anagen and telogen follicles, as well as changes in other characteristics dermoscopic conditions observed in hair disorders, such as follicular units and capillary density. In addition, the study also quantified, in scalp fragments from volunteers, terminal follicles, vellus follicles and fibrous tract.

Culture and MTT Assay
The CCD1072Sk cell line was obtained from Rio de Janeiro Cell Bank (CCD1072Sk -ATCC CRL2088). The cells were cultured in a monolayer using IMDM (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 UI/mL penicillin/streptomycin, and 0.25 μg/mL Fungizone (Gibco) in a humidified atmosphere at 37˚C in 5% CO 2 . These cells were trypsinized three times per week using 0.25% trypsin/EDTA (Cultilab, Brazil). For the 24-hour cell viability assessment, the control and treated cells were centrifuged and resuspended in equal parts medium and trypan blue (0.05% solution) and counted using a hemocytometer. To evaluate the cytotoxicity of Trichoxidil TM , the essential oil was dissolved in the culture medium in appropriate concentrations. The cell viability of control and Trichoxidil TM (0.05% -5.0%)-treated fibroblasts cells were measured using a standard MTT assay. Briefly, 5 × 10 4 viable cells were seeded into clear 96-well flat-bottom plates (Corning) in IMDM medium supplemented with 10% fetal bovine serum (FBS) and incubated with different concentrations of the extract for 24 h. Then, 10 μL/well of MTT (5 mg/mL) was added and the cells were incubated for 4 h. Following incubation, 100 μL of 10% sodium dodecyl sulfate (SDS) solution in deionized water was added to each well and left overnight. The absorbance was measured at 595 nm using a FlexStation 3 Multi-Mode Benchtop Reader (Molecular Devices, Sunnyvale, CA, USA).

Reverse Transcription-Quantitative PCR (RT-qPCR)
The effect of Trichoxidil TM on the expression of mRNA of KGF; IGF-1; and VEGF in fibroblasts was evaluated by RT-qPCR. Cells were treated with Trichoxidil TM (2.5% and 5.0%) for 24 hours. Total RNA extracted from cells samples was converted to cDNA using a SuperScript® III RT kit (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. The concentration of RNA was detected using a NanoDrop 2000 (Thermo Fisher Scientific, Inc.). GAPDH was used as the internal control. The thermocycling conditions were as follows: 95˚C

In Vivo Studies: Volunteers and Study Groups
This study was carried out after approval by the local Ethics Committee and written consent was obtained from each subject (3.150.582 -approbation number). After obtaining informed written consent, all the patients were subjected to a detailed medical history followed by thorough general physical, dermatological and systemic examinations. Both the image acquisition by the Trichoscan equipment and the cylindrical fragments, 33 volunteers, diagnosed with androgenetic alopecia, men and women, aged between 25 and 50 years, were evaluated. Exclusion criteria were: 1) volunteers who were not diagnosed with AGA; and 2) volunteers who were not in the age range (25 and 50 years). The subjects were divided into four groups and assigned the drugs in a randomized manner.
Control group, without treatment (n = 5); Trichosol TM vehicle group, without active (n = 5); Hydroalcoholic vehicle group, without active (n = 4); Trichosol TM vehicle group, with 2.5% minoxidil (n = 5); Hydroalcoholic vehicle group, with 2.5% minoxidil (n = 5); Trichosol TM group with 2.5% Trichoxidil TM (n = 5) and Hydroalcoholic vehicle group with 2.5% Trichoxidil TM (n = 4). They were instructed to apply the solution to the balding area with a calibrated dropper twice daily at 12-hour intervals. The volunteers were evaluated in 2 phases throughout the study. Time zero (T = 0), the first evaluation and time ninety (T = 90), the second evaluation, after 90 days of treatment with the formulations developed for the study. In the evaluation, T = 0, data and images were obtained using the Trichoscan, followed by the collection of the cylindrical fragment for extraction of the total mRNA and histological analysis. The same procedure was repeated after 90 days of treatment (T = 90).

Dermoscopic Analysis (Trichoscan)
The FotoFinder Trichoscale softwear was used to evaluate the parameters of the hair growth phases, as the anagen and telogen phases. All patients were assessed and subjected to photographic records with a 10x magnification dermatoscope and a digital camera with 20x and 40x magnification on the small area of the shaved headscalp. The dermoscopy findings, for example, the numerical data report, as well as the photos generated by the equipment, were stored for statistical analysis and compared between the groups evaluated.

Scalp Fragments-Histologic Evaluation and RT-qPCR
Cylindrical scalp fragments were obtained on the vertex region. After trimming 1.0 cm 2 of hair, 4-mm punch biopsies were taken at T = 0 and T = 90. The punches were obtained parallel to the direction of hair growth. For follicular peribulbar evaluation and molecular biology analysis, scalp fragments were fixed for four h to 48 h, using 10% buffered formalin, and embedded in pure paraffin.
To histologic analysis, the scalp fragments were sectioned and stained with hematoxylin and eosin. The following parameters were evaluated: total follicles, vellus follicles, terminal follicle and fibrous tract. The presence of peribulbar changes (i.e. inflammation) were assessed by a dermatologist. Part of the scalp fragments were used to extract total RNA followed by the quantification of mRNA of KGF; IGF-1; and VEGF, as described above in the in vitro tests. All tissues were stored at −80˚C until the RNA extraction procedure.

Statistical Analysis
The obtained results were expressed as the mean ± standard error of mean (SEM) from at least three independent experiments, unless stated otherwise.
Paired data was evaluated by Student's t-test. One-way analysis of variance (ANOVA) was used for multiple comparisons. A p value of <0.05 was considered significant.

Trichoxidil TM Regulates KGF, IGF-1, and VEGF mRNA Levels in Fibroblasts Cells
To investigate whether Trichoxidil TM regulates KGF, IGF-1, and VEGF expression at transcriptional level, RT-PCR was performed. As shown in Figure 2,

Evaluation of Hair Growth of Volunteers by Dermoscopy (TrichoScan)
The results obtained at times T = 0 and T = 90 days are shown in Table 1. Anagen and telogen follicles, follicular units and capillary density were evaluated.
Both Trichoxidil TM and minoxidil increased all parameters evaluated. Analysis of the capillary density showed that Trichoxidil TM associated with Trichosol TM vehicle, was the most effective association (Table 1). Figure 3

Histological Evaluation of Transverse Scalp Sections
Total follicles, vellus follicles, terminal follicles and fibrous tract were evaluated, shown in Table 2. Figure 4(A) shows the percentage of total follicles in volunteers who used Trichoxidil TM or minoxidil formulations. Total follicles percentage was higher in volunteers treated with Trichoxidil TM associated with Trichosol TM ,    (Figure 4(B)). The percentage increase in terminal follicles was significant (*p < 0.05) in the volunteers who used the formulation of Trichoxidil TM in Trichosol TM (Figure 4(C)).

Figures 5(A)-(G) shows histological sections of anagen terminal follicles (capil-
lary channel diameter greater than the inner root sheath), without signs of apoptosis in the outer root sheath. Finally, the results described in Table 2 showed that the volunteers who received formulations with Trichoxidil TM , both in Trichosol TM vehicle and hydroalcoholic vehicle, showed a significant reduction (*p < 0.05) in the number of fibrous tracts.

Expression of Growth Factors at mRNA Levels in Scalp Fragments
We also investigated the effects of Trichoxidil

Discussion
Even though it is a prevalent condition, therapeutic options for AGA are limited and include finasteride and minoxidil, in addition to antiandrogens for female patients [6]. In this study, we introduced Trichoxidil TM , a phytocomplex developed from essential oils, that presents as an option for the treatment of hair growth. The in vitro results showed an absence of cytotoxicity by Trichoxidil TM and activation of fibroblast proliferation (CCD1072Sk cell line), besides to promoting upregulation in the expression of mRNAs levels of growth factors related to hair growth, such as insulin-like growth factor 1 (IGF-1) [7], keratinocyte growth factor (KGF) [8] [9] and vascular endothelial growth factor (VEGF) [10].
In the present work, hair growth was evaluated by dermoscopy, histological analysis and mRNA levels quantification. Dermoscopy, a non-invasive diagnostic method, which allows the visualization of microscopic details of skin lesions, besides revealing some structures of the skin below the surface that are not normally visible [11]. Thus, by dermoscopy, the results obtained showed that Tri-

Conclusion
Trichoxidil TM showed a proliferative effect in vitro and in vivo, possibly by positive modulation of growth factors, such as IGF-1, VEGF and, especially, KGF, revealing to be a promising candidate for treatment of hair loss caused by AGA.

Limitations of This Study
As limitations of the study, we can mention the lack of a control group without androgenetic alopecia, to obtain normal hair growth parameters.
ceutical TM LTDA. The authors would like to thank the Dermofit Compounding Pharmacy, São Paulo, SP, Brazil, for their support in preparing the formulations used in this work.

Statement of Data Availability
The authors declare that all raw data presented in this manuscript will be available upon request.

Conflicts of Interest
All authors disclose any influence of companies or manufacturers in the present study. In addition, all authors declare that the results of the study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation.