Anxiety-related behavioural responses to light-dark transitions in zebrafish larvae treated with a Withaniasomnifera (L.) Dunal water extract and selected withanolides.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal (Solanaceae) is used traditionally for anxiety; however, the activity of the distilled-water root extract and its major withanolide constituents remains incompletely characterised in larval zebrafish.

AIM OF THE STUDY: To evaluate the anxiolytic-like effects of a distilled-water root extract and selected withanolides in a larval zebrafish model, and to explore predicted pharmacokinetic properties and putative pharmacodynamic targets of withanolide A using in silico methods.

MATERIALS AND

METHODS

The root powder was extracted with distilled-water and marker compounds were quantified by UPLC-PDA/MS. Anxiolytic-like effects were evaluated in 5 dpf zebrafish larvae using continuous-light and light-dark transition assays, with locomotor activity and reverse-thigmotaxis as behavioural endpoints. Physicochemical and pharmacokinetic properties of withanolide A, withanone and withaferin A were predicted using SwissADME and PreADMET, and network pharmacology analysis was performed for withanolide A.

RESULTS

Withaferin A and withanolide A were detected in the extract; withanone was not detected. The extract showed anxiolytic-like activity at 250 μg/mL by reducing dark-phase locomotor activity and increasing central-zone exploration, with effects comparable to 10 μM diazepam. Withanolide A was the most active pure compound, particularly at 100 μM, where it reduced thigmotaxis without suppressing locomotor activity. In silico profiling predicted favourable drug-likeness and intestinal absorption, but limited blood-brain barrier penetration. Network analysis identified 33 anxiety-related targets, including CRHR1, CYP3A4, NR3C1, GRM5, AR, MAOB and OPRM1.

CONCLUSIONS

The findings support the ethnomedicinal use of W. somnifera for anxiety-related conditions and suggest that withanolide A may contribute to these anxiolytic-like effects through putative multi-target mechanisms.