Metabolomic profiling of Ashwagandha (Withania somnifera): linking bioactive compounds to functional therapeutic roles.

BACKGROUND: Withania somnifera (Ashwagandha) is a widely used medicinal plant known for its neuroprotective, hepatoprotective, fertility-enhancing, and anti-inflammatory properties. Although it has a long history of use in traditional medicine, a detailed understanding of its phytochemical constituents and associated biological activities remains limited. AIM: To perform comprehensive metabolomic profiling of Withania somnifera and identify its major bioactive secondary metabolites, with a focus on compounds contributing to neuroprotection, liver health, reproductive support, and inflammation modulation. METHODS: Root extracts of Withania somnifera (Ashwagandha) were subjected to methanol-based organic phase extraction followed by LC-MS/MS analysis, resulting in the characterization of 1,020 metabolites. Among these, 1,010 compounds were classified as putatively annotated (MSI Level 2/3 annotations) metabolites based on MS1 data, while 10 highly abundant metabolites were further confirmed by MS/MS fragmentation and assigned MSI Level 2 annotations. Relative abundance analysis was performed to determine the predominant phytochemical constituents of the extract. Key metabolites identified through this approach were subsequently correlated with the known pharmacological properties of Ashwagandha, including its immunomodulatory, anti-inflammatory, antioxidant, adaptogenic, and neuroprotective effects. RESULTS: The analysis identified a diverse array of secondary metabolites, many present at low individual abundance ( < 0.30 % ). Notable compounds included the alkaloids Gentianaine ( 8.44 % ) and Sauroxine ( 2.94 % ), phenolics such as Mulberrofuran A ( 14.58 % ) and Sulphuretin ( 9.61 % ), and terpenoids including Pinocarvone and Ergosterol. (% of total annotated metabolites) These compounds were associated with anti-oxidative, neuro-regenerative, hepatoprotective, and anti-inflammatory effects. Additional metabolites, including Gomphrenin-I, Sitosterol, Pfaffic acid, and β -Carotene, were linked to reproductive health. Embelin and Kievitone were noted for their potential roles in enhancing oxidative stress responses. Despite the low abundance of individual compounds, the overall metabolite profile suggests substantial synergistic bioactivity. CONCLUSION: Metabolomic profiling of Withania somnifera reveals a rich composition of bioactive secondary metabolites with potential roles in neuroprotection, hepatoprotection, antioxidant defense, and reproductive support. These findings support its traditional use and highlight its potential for modern therapeutic applications in promoting stress resilience, vitality, and overall wellbeing.