In Vivo Phytotherapy in BALB/c Athymic Nude Mice: Hair Growth Promotion using Ficus religiosa L. and Morus alba L

Ficus religiosa L. (FR) and Morus alba L. (ML) belonging to the family Moraceae have been tested as novel herbal agents for hair growth promotion and Hair Follicles (HFs) regeneration in BALB/c athymic nude mouse model. Current study tested different mixtures of 5% aqueous fractions: Test 1 (ML2+ML3+ML4+FR4), Test 2 (FR1+FR2+FR4), or Test 3 (ML2+ML3+ML4+FR1+FR2+FR4) from leaves of both plants including standard of care 2% minoxidil. Control mice were untreated. Animals were treated for 33 days by topical application on the back skin and changes in hair growth patterns were evaluated. Histology was performed to assess the HFs morphology, and modulation of hair cycle phases. Gene expression analysis was performed to understand potential mechanisms of action. All treatment groups had significantly higher anagen phase HFs compared with untreated control group based on histology analysis. Also, expressions of CD34, CD200R and Oct4 genes were upregulated in all treatment groups compared with untreated control. Present study demonstrated that a combinatorial therapy using either fractions of FR or FR and ML promoted hair growth and HFs regeneration through induction of anagen phase in conjunction with stem cells associated genes upregulation in BALB/c athymic nude mouse model of hair loss.


Introduction
Skin is considered as the largest organ in mammals that protects against external insults while, hair have evolved to protect skin and as a mode of communication via signals and cues. Epidermis is the outermost layer of the skin, which is extended into numerous HFs 1 . HFs are dynamic structures that post-birth undergo cycles of anagen, catagen and telogen phases in a repetitive manner 2 . C3H/HeJ mice and Dundee Experimental Bald Rat (DEBR) models are considered as the basis of developing new treatments for alopecia areata [3][4][5] and put rodents in forefront of preclinical research for human hair loss indication. Mouse has short hair cycle (3 weeks) with well delineated anagen and catagen phases divided into six and eight sub phases respectively 6 . There appear to be no structural differences between mouse and human hair cycles except, catagen phase in mouse exhibits hair bulb alterations in absence of retraction of vibrissae follicles 1 . Rat model has ability to transform human stem cells into dermal papilla followed by new HFs generation 7 .
Athymic nude mouse is devoid of fur and has been studied in biomedical research for more than five decades 8,9 . Phenotypically this mouse has hairless skin, however, its dermis has numerous active HFs that are anomalous with impaired keratinization 8,10 . The compromised differentiation of HFs in athymic nude mice is attributed to defects of the ectoderm that lead to structural abnormalities of cortex, hair cuticle and inner root sheath 11 . Since nude mice lack hair keratins, their hair shafts undergo twisting and coiling in the hair follicle infundibulum causing its dilation 12 . Studies on mutant mouse models of hair loss have implicated proteins such as bone morphogenic protein receptor, epidermal growth factor receptor, fibroblast growth receptor 2, insulin -like growth factor 1 receptor, and fibroblast growth factor 7 in hair growth pattern and overall hair cycle turnover 13 . Nude mouse model has been efficiently utilized to identify therapeutic targets such as Keratinocyte Growth Factor (KGF) 14 , tellurium immunomodulator AS101 15 , and cyclosporin A (CsA) 16 . Eclipta alba L. has been demonstrated to play a key role in facilitating nude mice follicular keratinocytes proliferation and delaying their terminal differentiation via TGF-β1 down regulation 17 . A study comparing C57BL/6 wild type (WT) and Foxn1-/-(B6.Cg-Foxn1nu/J) mice revealed that the very hairless phenotype in nude mice exists because of Foxn1−/− mutation influencing the downstream mechanisms of terminal keratinocyte differentiation and in addition, transcription factor Lhx2 (regulator of hair cycling and stem cells) appears to have a significant impact on mechanisms of stem cells regulation within the HFs bulge region 18 . Importantly, human and murine hair follicle stem cells share certain markers such as CD34 19,20 and CD200 [19][20][21] . HFs in the frontal parts of the scalp are deficient in CD34 in androgenetic alopecia (AGA), while, its expression is conserved in HFs of the occipital region 22 . Expression of CD200 is minimal in matrix cells in patchy alopecia, which may be an indication of compromised immune tolerance 22,23 therefore, transmission of immunoregulatory signal from CD200 through CD200 receptor (CD200R) is vital to reduce inflammatory reactions and facilitate immune tolerance 24 .
Ficus religiosa L. leaves containing carbohydrates, proteins, lipid, calcium, sodium, potassium, and phosphorus possess pharmacological activity against bacteria and fungi and they can also work as a wound healing agent 25 . A decade ago, an in vitro study was conducted using combination of extracts containing Aloe vera whole plant, Eucalyptus globulus leaves, Ficus infectoria bark, Ficus religiosa bark and Piper betel leaves to study antibacterial activity against resistant and sensitive strains isolated from infectious skin tissue (Staphylococcus, Klebsiella, Enterobacter, Escherichia and Micrococcus species). A mixture containing hot alcoholic extracts of Ficus infectoria, Ficus religiosa and Piper betel turned out to be the most efficacious against all bacteria including its bactericidal activity against resistant strains of Pseudomonas 26 . In more recent study, Ficus religiosa leaves extract was orally administered at doses up to 400 mg/kg for 55 days that significantly diminished the motor defects and provided brain protection from oxidative stress in rat models of Parkinson using 6 hydroxydopamine (6-OHDA) and catalepsy using haloperidol at test preparation doses of 200 and 400 mg/kg 27 . Ficus religiosa leaves extract also provided protection against isoniazidrifampicin and paracetamol induced liver toxicity in rats at doses of 100, 200 and 300 mg/kg 28 . In guinea pig model of asthma, administration of Ficus religiosa leaves extract (150 and 300 mg/kg, ip) generated significant inexpression effect to develop histamine & acetylcholine induced preconvulsive dyspnea 29 . Morus alba L. leaves predominantly containing flavonoids have activity as anti-microbial, anti-diabetic, anti-atherosclerotic, anti-obesity, and as a cardioprotective agent 30 . Abnormal overproduction of melanin in skin leads to dots, freckles, bruising keratosis, and hyperpigmentation. Compound 3 isolated from Morus alba leaves was demonstrated to impede melanin production via regulation of melanogenesis-related protein expression 31 . Till to date, there is little evidence generated that established efficacy of Ficus religiosa L. or Morus alba L. in skin or hair growth associated abnormalities. Considering scarcity of approved products for hair loss in humans and no evidence of hair growth promotion and HFs regenerative activities produced in rodent models using Ficus religiosa L. and Morus alba L. either as single agent or in combination, authors decided to test these two herbal agents in BALB/c athymic nude mouse model. 5% aqueous fractions from leaves of Ficus religiosa L. (FR1, FR2, FR3 and FR4) or Morus alba L. (ML1, ML2, ML3, and ML4) were tested for hair growth efficacy in pilot study using testosterone induced hair loss model in rats as described previously 32 . These fractions were topically applied on dorsal skin area for 28 days. Additional treatments included 5% aqueous FR leaves or ML leaves extract including a standard of care 2% minoxidil. In this study, 5% aqueous fractions ML2, ML3, ML4 of Morus alba L. leaves or 5% aqueous ML leaves extract yielded a biologically significant increase (based on % change) in hair weights compared to disease control (DC) and standard of care 2% minoxidil on day 21 of treatment in animals induced with alopecia, while Ficus religiosa L. leaves 5% aqueous fractions FR2, FR3, FR4 or 5% aqueous FR leaves extract generated overall lesser or comparable increase compared with 5% aqueous ML leaves fractions or 5% aqueous ML leaves extract including standard of care 2 % minoxidil (supplemental Figure 1). In pilot study using BALB/c athymic nude mice, 5% aqueous leaves extract of Ficus religiosa L. or Morus alba L. including standard of care 2% minoxidil were applied as topical preparations on dorsal skin of animals for 21 days. The genes expression analysis data of skin tissues collected at terminal sacrifice revealed that CD34, CD200R or Oct4 expressions were biologically upregulated in all treatments groups compared with vehicle control (supplemental Figure 2). These preliminary data indicated promising hair growth associated efficacious effects of fractions or extracts of both plants prompting authors to pursue current study, wherein same fractions as testosterone induced alopecia rat pilot study such as FR1, FR2, FR4 or ML2, ML3, ML4 in the form of different mixtures of 5% aqueous preparations were tested in BALB/c athymic nude mouse as a rodent model of choice to assess hair growth promotion and HFs regenerative activities and associated modulations of previously tested stem cells associated genes (CD34 and CD200R) and totipotency specific stemness transcriptional factor Oct4 after 33 days of topical application on dorsal skin area. Overall, this research opened the door of validating more therapeutic options for human hair loss indication and increased our understanding of roles of certain genes involved in stem cell biology of nude mouse HFs.
In the context of current study, a combinatorial therapy approach certainly is a viable option to promote hair growth and HFs regeneration.

Materials and Methods
The experiments were conducted as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines, India with approval from the Institutional Animal Ethics Committee (IAEC) of APT Testing Research Pvt. Ltd, Pune, Maharashtra, India. Study was assigned a project number RP50/1819.

Plants Samples Collection and Formulation
Dried leaves of Ficus religiosa L. and Morus alba L. were collected from the Pune region of the state of Maharashtra, India. The plants samples were identified by Botanical Survey of India (BSI), Western Regional Center, Pune, Maharashtra, India. 5% gel was prepared from aqueous fractions of Ficus religiosa L. and Morus Alba L. in combination according to modified protocol based on procedure described previously 33 . Briefly, the individual fractions were dissolved in 4 ml of distilled water followed by addition of 4% propylene glycol and 3% ethanol. In another beaker, 1% Carbopol was added to 4 ml of distilled water followed by addition of 0.2 % methyl paraben, 0.02 % propyl paraben and 0.03 % EDTA. The contents of both the beakers were mixed thoroughly using mortar and pestle, and a drop wise triethanolamine was added till gel formation was visible.

Animals and Treatment Protocol
BALB/c athymic nude mice at 7 weeks of age were purchased from Vivo Biotech, Hyderabad, India. Nude mice were maintained under aseptic conditions in a laminar-flow rack and 12 h light: dark periods at 24 ± 2 °C with relative humidity of 40-60 %. These conditions also included sterile food and bedding, reverse osmosis water, and sterilization of water bottles and cages. After at least 5 days of acclimatization to new environmental conditions, 30 mice were randomized into 5 groups of 6 mice (3 males and 3 females) each. Topical application of test preparations was performed when the synchronized telogen phase was observed in all the experimental groups. Ficus religiosa L. and Morus alba L. in the form of mixtures of 5% aqueous fractions from leaves such as Test1 (ML2+ML3+ML4+FR4), Test 2 (FR1+FR2+FR4), or Test 3 (ML2+ML3+ML4+FR1+FR2+FR4) including standard of care 2% minoxidil were topically applied daily at 0.1 ml/mouse on the back skin from suprascapular region to cover dorsal cervical region, dorsal thorax, and region caudal to the ribcage until completion of two hair growth generations (33 days) ( Table 1). Control mice were untreated.

Determination of Hair Length
Images of dorsal skin region from thoracic area to region caudal to ribcage area were taken using Nikon digital camera before (Day 0) and after Day 14 and 28 of initiating daily tests treatment. Care was taken to measure hair length consistently in same representative region in all groups i.e. dorsal thoracic area to region caudal to the ribcage. Microware 1000X 8 LED USB digital microscope was used to measure hair length.

Skin Sample Collection and Histology Analysis
The study animals were sacrificed by using CO 2 asphyxia and skin tissue samples including HFs of dorsal region from thoracic area to region caudal to the ribcage were collected at terminal sacrifice (Day 33) from different treatment groups including 2% minoxidil and untreated control groups in 10% NBF (neutral buffered formalin). After 72 hours, the fixed skin tissues were processed using Journal of Natural Remedies | ISSN: 2320-3358 http://www.informaticsjournals.com/index.php/jnr | Vol 21 (1) | January 2021 ascending series of graded ethanol, and xylene 34 . Tissues were embedded in paraffin blocks for micro-sectioning on automated tissue microtome (Leica, Germany) and tissue transverse sections of 5 µm were placed on clean grease free glass slides. Subsequently, tissue sections were stained with hematoxylin and eosin (H&E). These tissue sections were observed under binocular microscope with microphotography unit (Nikon, Japan). The morphology and the number of HFs in various phases (viz. Anagen, Catagen and Telogen) were evaluated microscopically in 5 fields per section at a magnification of 100×.

Gene Expression Analysis: CD34, CD200R, and Oct4
Total RNA was extracted from Day 33 skin samples using GeneJET RNA Purification Kit (Thermo Fisher Scientific). cDNA was then synthesized by reverse transcription using High-Capacity cDNA Reverse Transcription Kit with RNase inhibitor (Thermo Fisher Scientific). Quantitative PCR (RT-PCR) was performed with Quant Studio™ 5 Real-Time PCR System (Thermo Fisher Scientific). Genes expressions were evaluated using the primers as described previously for CD34 35 , CD200R 36 , and Oct4 37 . Relative gene expression was determined using the 2−ΔΔCt method and presented as the mean.

Statistical Analysis
The experimental data are expressed as the means. Oneway ANOVA was used for the assessment of significance between the different treatment groups. Statistical analysis was performed using Graphpad Prism Version 5. A value of p < 0.05 was considered to indicate a statistically significant difference.

Effects of Ficus religiosa L. and Morus alba L. on Hair Growth
The authors evaluated in-vivo hair growth promotion and HFs regenerative activities in BALB/c athymic nude mice after topically applying Ficus religiosa L. and Morus alba L. 5% aqueous fractions from leaves in the form of mixtures (Test 1, Test 2, or Test 3) or 2% minoxidil on dorsal surface of skin from suprascapular region to cover dorsal cervical region, dorsal thorax, and region caudal to the ribcage. Care was taken to measure hair length consistently in same representative region in all groups i.e. dorsal region from thoracic area to region caudal to the ribcage. The untreated control group of nude mice exhibited thin, transient, and variable hair growth during the entire treatment period of 33 days. Mean hair length did not differ among all groups (data not shown). Incidence of hair growth in mice on Day 0 (pre dose), Day 14, and Day 28 are shown in Table  2. Although, untreated control mice showed a variable background hair growth on Day 0, Day 14, and Day 28; Day 14 had greater hair growth and relatively higher incidence of animals showing hair growth in Test 1, Test 2, or Test 3 treated groups compared to 2% minoxidil and untreated control groups ( Figure 1, Table 2). Lack of hair growth on Day 28 of treatment in some of the same mice can be attributed to wave like pattern of hair growth 38 . Also, around 2 weeks of time frame of anagen phase 6,39 , and animal to animal variability noticed in mice may add to scarce hair growth.

Effects on Number and Development of HFs
Mixtures of 5% aqueous fractions from Ficus religiosa L. and Morus alba L. leaves stimulated HFs development that is evident from the photomicrographs obtained from the histology analysis ( Figure 2). Follicular development was determined according to the guideline previously proposed 40 . The quantitative analysis of the representative transverse skin tissue sections (from thoracic area to region caudal to the ribcage, Table 3, Figure 3) indicated that Test 1, Test 2, Test 3 or 2% minoxidil groups showed statistically and biologically higher number of HFs due to induction of anagen phase in the dermis and hypodermis region compared with untreated control. Also, telogen phase HFs were statistically significantly increased in Test 1, Test 2, Test 3 or 2% minoxidil groups compared with untreated control group. Telogen phase modulation appears to be treatments associated however, regular wave pattern of hair growth cycle phases in nude mice may be an additional factor contributing to modulation (Table  3). Overall, qualitative analysis under high power (100X) microscope field of skin tissue sections from thoracic area to region caudal to the ribcage revealed presence of different phases of hair cycle including anagen, catagen or telogen with anagen phase HFs significantly increased in Test 1, Test 2, Test 3 or 2% minoxidil groups as compared with untreated control group suggestive of initiation of HFs growth in these groups ( Figure 2). Although, Test 1, Test 2, or Test 3 showed biologically marginally higher anagen phase HFs than 2% minoxidil, it was Test 3 group that had relatively higher number of HFs in anagen phase suggestive of progressive HFs growth compared with Test 1 or Test 2 groups.

Expression of CD34, CD200R, and Oct4 during Follicular Morphogenesis
Current study compared the expressions of stem cells associated genes such as CD34, CD200R and a key transcriptional factor involved in induction of pluripotent stem cells (iPS); Oct4 using RT-PCR analysis of skin tissue collected at terminal sacrifice (Day 33, Figure 4). Mean relative gene expression (2−ΔΔCt) was statistically nonsignificant, however biologically higher in

Discussion
In this study, the researchers investigated the hair growth promotion and hair follicle regenerative activities of different mixtures of 5% aqueous fractions from leaves of Ficus religiosa L. and Morus alba L.  44 . Downregulation of TGF-β1 expression after treatment with Eclipta alba L. in nude mice was associated with stimulation of follicular keratinocytes proliferation and delay in keratinocytes terminal differentiation 17 . In nude mouse, a vital gene of epithelial cellness called LIM homeobox protein 2 (Lhx2) has been demonstrated to be constitutively upregulated along with significant changes in CD49f/CD34/CD200 cell subsets, that is indicative of changes in subpopulations of epithelial progeny 18 . The same research also indicated that Oct4+ stem cells population in dermal fractions of skin isolates from nude mice showed a greater expansion as compared with wild type counterpart. It has been demonstrated that defect in conversion of hair follicle stem cells to progenitor cell populations expressing CD200hiITGA6hi and CD34hi markers plays a pivotal role in the pathogenesis of AGA 45 .
Scientists have also demonstrated that CD200 expression on mouse follicular epithelium in skin region plays a critical role in decreasing inflammatory responses and potentially facilitates maintenance of immune tolerance to HFs associated autoantigens after active engagement with CD200R 24 . Current study findings are well aligned with previous data on Foxn1−/− mouse phenotype with respect to hair growth promotion 17,46 and robust modulatory effects of CD34, or Oct4 genes upregulation are indicative of beneficial effects of Test 1, Test 2 or Test 3 in HFs regenerative activity. Further, the authors speculate that, this phytotherapy may have downstream effects of stimulating progenitor cells population and facilitation of immune tolerance via upregulation of CD200R message. In addition, Test 3 results indicated overall advantage over rest of the treatments including 2% minoxidil in terms of marginally higher upregulation of all 3 genes. Despite animal to animal variability, overall histology and genes expression analysis data demonstrated hair growth promotion from thoracic region through caudal region to rib cage in Test 1, Test 2, Test 3 or 2% minoxidil treated groups compared with untreated control group. Future dose response studies in mutant rodent models using either same combinations of fractions used in current study or using different combinations and / or using different treatment preparations may enable more insight into pharmacology of both plants in hair loss indication.

Conclusion
Present study demonstrated that a combinatorial therapy using either fractions of FR or FR and ML promoted hair growth and HFs regeneration through induction of anagen phase in conjunction with stem cells associated genes upregulation in BALB/c athymic nude mouse model of hair loss.