*Author for correspondence JOURNAL OF NATURAL REMEDIES DOI: 10.18311/jnr/2021/26255 Article Received on: 09.10.2020 Accepted on: 25.01.2021 RESEARCH ARTICLE Revised on: 17.12.2020 In Vivo Phytotherapy in BALB/c Athymic Nude Mice: Hair Growth Promotion using Ficus religiosa L. and Morus alba L. Pramod S. Joshi 1* , Yogesh B. Patil 2 , Bhagyashri Nagarkar 3 , Tania S. Paul 3 and Kishori G. Apte 3 1 Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Pune, Maharashtra – 412115, India; pramodjoshi02@yahoo.com 2 Symbiosis Centre for Research and Innovation, Symbiosis International (Deemed University), Pune, Maharashtra – 412115, India 3 APT Research Foundation, Pune, Maharashtra – 411041, India Abstract 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. Keywords: Anagen Phase, Ficus religiosa L., Hair Follicles, Morus alba L., Nude Mouse 1. 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 3–5 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 . Tere appear to be no structural diferences 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 fve decades 8,9 . Phenotypically this mouse has hairless skin, however, its dermis has numerous active HFs that are anomalous with impaired keratinization 8,10 . Te compromised diferentiation 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 shafs 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, fbroblast growth receptor 2, insulin – like growth factor 1 receptor, and fbroblast growth factor 7