Exogenous applications of humic acid and salicylic acid differentially affect biomass accumulation and production of rare pharmacologically active withanolides in in vitro cultures of Withania coagulans

Abstract

Ashwagandha(Withania coagulans) is a medicinal plant valued for its withanolide content, known for anti-cancer, anti-microbial, immunomodulatory, and hepatoprotective properties.However, due to habitat losss and overharvesting, it is now considered endangered. This study aimed to enhance biomass and withanolide production in W. coagulans callus cultures using humic acid (HA) and salicylic acid (SA) as elicitors. HA at 10 µM significantly boosted biomass (fresh weight (FW): 3.6 g/L, dry weight (DW): 267 mg/L), antioxidant enzymatic activity (peroxidase (POD) and superoxidase dismutase (SOD)) and non-enzymatic antioxidant potential, including DPPH (80.2%), ABTS (287.1 µM of Trolox equivalent antioxidant capacity (TEAC) and FRAP (548.2 µM TEAC). In contrast, SA at 60 µM negatively affected biomass and phytochemical content. HPLC analysis confirmed enhanced accumulation of withanolides and coagulanolide under HA treatment. Overall, low concentrations of HA and SA proved more effective and economically viable than high concentrations, enhancing both growth and secondary metabolite production. These findings support optimized in vitro elicitation strategies for conservation and sustainable use of W. coagulans, offering potential for commerical-scale cultivation and pharmaceutical applications. Future research may focus on understanding the molecular mechanisms behind elicitor responses and scaling up production systems to meet industrial demands.