Abstract
Background:
Growing interest in natural veterinary products has highlighted Cymbopogon nardus (citronella) and Azadirachta indica (neem) as promising botanical ingredients due to their antimicrobial, anti-inflammatory, repellent, and hair-supportive properties. These characteristics render them suitable for topical shampoo formulations designed to enhance skin and coat health, prevent ectoparasite infestation, and minimize hair shedding in companion animals. However, combined herbal formulations necessitate thorough in vivo safety and efficacy validation before practical implementation.

Aim:
This study aimed to formulate shampoos with combined C. nardus and A. indica extracts at 0.5%, 1.0%, and 2.0% concentrations, to evaluate their safety profile, and efficacy in enhancing hair growth parameters, follicular density and reducing shedding in animal models

Methods:
LC–MS/MS analysis identified 18 phytochemical compounds (6 additional signals classified as analytical artifacts). Three shampoo formulations were subsequently assessed for organoleptic properties, homogeneity, pH, viscosity, foam height, and physicochemical stability. Acute dermal irritation tests were performed on four adult female New Zealand White rabbits, while ocular irritation tests were conducted on six rabbits, with both evaluated using the Draize scoring system at 1, 24, 48, and 72 hours post-application. Efficacy was assessed using 108 female DDY mice divided into 18 experimental groups (n = 6 per group) over 28 days. Parameters included weekly hair length measurements (weeks 1–4), a pull test to assess hair fragility, and histopathological quantification of follicular density at week 4. Data were analyzed using two-way ANOVA followed by Tukey's HSD post-hoc test (p < 0.05).

Results:
All formulations satisfied quality standards (pH 7; viscosity 950 ± 5 to 1430 ± 8 cP; homogeneous consistency). Irritation indices ranged from negligible to slight. Hair growth parameters improved significantly in a dose and frequency dependent manner, with the 2% formulation producing the greatest increases in hair length (5.57 ± 0.25 mm) and follicular density (156.67 ± 2.52 follicles/cm²), alongside near complete shedding reduction (p < 0.05).

Conclusion:
Shampoos combining C. nardus and A. indica shampoos (0.5–2%) are topically safe and effectively enhance hair growth and follicular density, with the 2% formulation demonstrating optimal performance.

Key words: Azadirachta indica; Cymbopogon nardus; Hair growth; Irritation test; Veterinary shampoo.