Design and Preclinical Evaluation of a Novel Anxiety Chamber for Assessing Aromatherapy-Mediated Anxiolytic Effects in Rodents

Background: Anxiety disorders are increasingly prevalent, often linked to chronic social defeat stress and everyday social challenges such as hesitation and fear in routine interactions. While pharmacological agents like benzodiazepines remain standard therapy, their side effects necessitate safer alternatives. Essential oils contain bioactive phytochemicals that may serve as potential anxiolytic agents. Objective: The present study aimed to design and evaluate a novel Anxiety Chamber for behavioural screening of anxiolytic activity and to investigate the role of volatile oils (Eucalyptus, Rosemary, and Lemongrass), with supporting FTIR analysis of their chemical constituents. Methods: A square wooden chamber was divided into two compartments: a dark chamber and an anxiety-inducing chamber (fitted with bright LEDs and a buzzer). Mice (Swiss albino) were allowed to explore both chambers through a semi-arched tunnel. Anxiety was induced in the illuminated chamber, and the time spent in each chamber was recorded at 5, 15, 30, 60, and 120 minutes. Animals were divided into six groups: Control, Negative Control, Standard (Diazepam 1 mg/kg), and three Test groups exposed to aromatherapy with volatile oils in varying ratios (50:25:25, 25:50:25, 25:25:50). FTIR analysis identified phytoconstituents previously reported to possess anxiolytic and CNS-modulating activity, thereby supporting the observed behavioural outcomes. Results: The negative control group exhibited reduced exploration of the anxiety chamber (38 sec at 30 min). Diazepam-treated animals showed progressive increases in chamber exploration (173 sec at 120 min). Test groups exposed to volatile oils demonstrated anxiolytic activity, with the 25:50:25 ratio (Rosemary-dominant) producing the greatest effect (188 sec at 120 min). Time spent in the chamber increased significantly (p < 0.05) from 38 sec in control to 173 sec with diazepam and 188 sec with the rosemary-rich formulation. Conclusion: The novel Anxiety Chamber demonstrated cost-effectiveness and reliability as a behavioural model for anxiolytic screening in rodents. Validation with diazepam and volatile oils confirmed its applicability, with increased time spent in the chamber indicating reduced anxiety levels. Among the tested formulations, the rosemary-enriched blend (25:50:25) exhibited superior anxiolytic efficacy. FTIR analysis identified phytoconstituents with established CNS-modulating properties, supporting the pharmacological relevance of these essential oils. Overall, the findings suggest that optimized aromatherapy formulations may serve as a natural and effective alternative for anxiety management.