Journal of Ethnopharmacology 304 (2023) 116064 Available online 19 December 2022 0378-8741/© 2022 Elsevier B.V. All rights reserved. Ethnological validation of Ashwagandha (Withania somnifera L. Dunal) ghrita as ‘Vajikarana Rasayana’: In-silico, in-vitro and in-vivo approach Shailendra Gurav a, ** , Manish Wanjari b , Ritesh Bhole c , Nishikant Raut d , Satyendra Prasad d , Suprit Saoji e , Rupesh Chikhale f , Pukar Khanal g , Amit Pant h , Muniappan Ayyanar i , Nilambari Gurav j, * a Department of Pharmacognosy, Goa College of Pharmacy, Panaji, Goa University, Goa, India b Regional Ayurveda Research Institute, Pune, Maharashtra, India c Dr D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune, Maharashtra, India d Department of Pharmaceutical Sciences, R.T. M. University, Nagpur, Maharashtra, India e Formulation Development, Slayback Pharma India LLP, Hyderabad, Telangana, India f UCL School of Pharmacy, 2939 Brunswick Square, London, WC1N 1AX, United Kingdom g Department of Pharmacology and Toxicology, KLE College of Pharmacy Belagavi, KLE Academy of Higher Education and Research (KAHER), Belagavi, India h Department of Pharmacology and Neurosciences, Creighton University, Omaha, USA i Department of Botany, A.V.V.M. Sri Pushpam College (Autonomous), Poondi (Affliated to Bharathidasan University), Thanjavur, Tamil Nadu, India j Department of Pharmacognosy, PES’s Rajaram and Tarabai Bandekar College of Pharmacy, Ponda, Goa University, Goa, India A R T I C L E INFO Keywords: Aphrodisiac Indian ginseng Ayurveda Indian cow ghee PDE-5 ABSTRACT Ethnopharmacological relevance: Processing cow ghee (clarifed butterfat) with therapeutic herbs, i.e. ghrita, is recognized for augmenting the therapeutic effcacy of plant materials. Ashwagandha ghrita (AG) is an effective Ayurvedic formulation consisting of Indian ginseng, i.e., Withania somnifera (L.) Dunal, the main constituent used to treat infertility, weakness, gynaecological disorders, and general debility. Objectives: The present investigation was undertaken to corroborate the ethnopharmacological claim of AG as ‘Vajikarana Rasayana’ for its aphrodisiac potential using bioinformatics (in-silico) and experimental (in-vitro and in-vivo) approaches. Methods: AG was formulated as per the methods reported in Ayurved sarsangraha. AG was further subjected to HPLC, GCMS analysis, and biological (acute toxicity and aphrodisiac) assessment per the standard procedures. Thirty-eight bioactives of Indian ginseng were subjected to computational studies (molecular docking and network pharmacology) to confrm the plausible mechanism. Results: AG was found to be safe up to 2000 mg/kg body wt., and it showed dose-dependent upsurge (p < 0.01 and p < 0.05, wherever necessary) in mount and intromission frequency, genital grooming, and anogenital sniffng at 150 and 300 mg/kg body weight suggesting aphrodisiac activity. In-vitro studies demonstrated sig- nifcant relaxation of the Corpus Cavernosal Smooth Muscle at all concentrations in a dose-dependent manner. Furthermore, the results of molecular modelling studies were in agreement with the biological activity and showed interaction with phosphodiesterase-5 as a possible target. Conclusion: AG exhibited an aphrodisiac effect and substantiated the traditional claim of Indian ginseng-based ghrita formulation as ‘Vajikarana Rasayana’. 1. Introduction Withania somnifera (L.) Dunal, often known as Ashwagandha or In- dian ginseng, is a member of the nightshade (Solanaceae) family and, in Ayurveda (traditional Indian systems of medicine), it is revered as Rasayana. The effectiveness of Rasayana treatment has been praised for its ability to bestow superior Rasas and Dhatus on the body and to pro- mote optimal health (Gurav et al., 2020). The plant is widely used in various traditional systems of medicine to treat neurological disorders, * Corresponding author. Department of Pharmacognosy, PES’s Rajaram and Tarabai Bandekar College of Pharmacy, Ponda, Goa University, Goa, India. ** Corresponding author. Department of Pharmacognosy, Goa College of Pharmacy, Panaji, Goa University, Goa, India. E-mail addresses: shailendra.gurav@nic.in (S. Gurav), nilagurav@rediffmail.com (N. Gurav). Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm https://doi.org/10.1016/j.jep.2022.116064 Received 12 October 2022; Received in revised form 13 December 2022; Accepted 15 December 2022