Neurobehavioral and Neurochemical Effect of Azadirachta Indica on Stress Induced Anxiety in Mice

Authors

Sanskrti Soni

ajaysingh008@gmail.com (India)

Yogesh Sharma

Vedic Institute of Pharmaceutical Education and Research, Sagar (India)

Durga Vishwakarma

Vedic Institute of Pharmaceutical Education and Research (India)

Ramdarshan Parashar

Vedic Institute of Pharmaceutical Education and Research, Sagar (India)

Ajay Singh Thakur

Vedic Institute of Pharmaceutical Education and Research, Sagar (India)

Article Information

DOI: 10.51584/IJRIAS.2026.11080085

Subject Category: Pharmacology

Volume/Issue: 11/8 | Page No: 1166-1178

Publication Timeline

Submitted: 2026-08-19

Accepted: 2026-08-24

Published: 2026-09-08

Abstract

Background: Anxiety disorders represent a major global mental health burden characterized by exaggerated fear and stress responses. Neurochemical imbalances involving GABA, serotonin, dopamine, and noradrenaline underlie these conditions. Synthetic anxiolytics such as benzodiazepines and SSRIs, though effective, often produce adverse effects. Hence, herbal alternatives like Azadirachta indica (Neem) are being explored for safer therapy.
Objective: To evaluate the anxiolytic and neuroprotective potential of ethanolic extract of Azadirachta indica leaves in stress induced anxiety in mice, with emphasis on behavioral and biochemical correlates.
Methods: Leaves of A. indica were authenticated and extracted using Soxhlet apparatus with ethanol. Phytochemical screening confirmed the presence of alkaloids, flavonoids, phenols, tannins, and carbohydrates. Swiss albino mice were divided into five groups: positive control, negative control, standard (diazepam 4 mg/kg), and test groups (100 mg/kg and 200 mg/kg extract). Anxiety was induced by immobilization stress. Behavioral assays included Elevated Plus Maze, Light–Dark Box, Open Field Test, and Actophotometer. Biochemical parameters—plasma nitrate (Griess assay), iNOS, AChE, and GABA were quantified, and histopathological evaluation of brain tissue was performed.
Results: The extract produced dose dependent anxiolytic effects, increasing time spent in open arms and light zones while reducing locomotor activity. Biochemically, A. indica lowered plasma nitrate and AChE levels, elevated iNOS and GABA concentrations, and restored neuronal morphology. The 200 mg/kg dose showed results comparable to diazepam, confirming modulation of nitric oxide and GABAergic pathways.
Conclusion: The ethanolic extract of Azadirachta indica exhibits significant anxiolytic and neuroprotective activity through antioxidant and neurotransmitter modulating mechanisms. These findings validate its traditional use and suggest its potential as a natural alternative for managing anxiety disorders.

Keywords

Azadirachta indica, Anxiolytic activity, Neuroprotection

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References

1. Gilhotra, N., Dhingra, D., & Bansal, R. (2010). Pharmacological evaluation of anxiolytic activity of herbal extracts in mice. Indian Journal of Pharmacology, 42(1), 28–34. https://doi.org/10.4103/0253-7613.62403 [Google Scholar] [Crossref]

2. Tonetto, L. L., et al. (1999). Extraction methods for phytochemical analysis of medicinal plants. Journal of Natural Products, 62(3), 345–350. https://doi.org/10.1021/np9801234 [Google Scholar] [Crossref]

3. Spiacci, A. Jr., et al. (2007). Phytochemical screening and pharmacological evaluation of plant extracts. Phytotherapy Research, 21(5), 451–457. https://doi.org/10.1002/ptr.2105 [Google Scholar] [Crossref]

4. Oosthuizen, F., et al. (2007). Elevated plus maze as a measure of anxiety in rodents. Behavioural Brain Research, 177(1), 1–5. https://doi.org/10.1016/j.bbr.2006.11.029 [Google Scholar] [Crossref]

5. Nakashima, M. N., et al. (2007). Light–dark box test for assessing anxiolytic activity in mice. Journal of Pharmacological Sciences, 103(3), 347–352. https://doi.org/10.1254/jphs.FP0061225 [Google Scholar] [Crossref]

6. Mutlu, O., et al. (2008). Open field test and locomotor activity in anxiety models. Neuroscience Letters, 440(3), 277–281. https://doi.org/10.1016/j.neulet.2008.05.012 [Google Scholar] [Crossref]

7. Rajoriya, V., Gupta, R., Rajoriya, V., Chourasiya, S., Patel, K., & Yadav, P. (2026). Precautions in the use of herbal polymers. In S. Jain, R. Gupta, & S. Vengurlekar (Eds.), Applications of Herbal Excipients and Polymers in Wound Healing (pp. 345–380). Hershey, PA: IGI Global Scientific Publishing. https://doi.org/10.4018/979-8-3373-3977-1.ch013 [Google Scholar] [Crossref]

8. Kansal, L., et al. (2009). Actophotometer-based evaluation of sedative and anxiolytic effects of plant extracts. Pharmacology Biochemistry and Behavior, 93(2), 181–187. https://doi.org/10.1016/j.pbb.2009.04.009. [Google Scholar] [Crossref]

9. Wess, J., et al. (2007). Acetylcholinesterase activity and cholinergic modulation in anxiety. Neuropharmacology, 52(3), 876–884. https://doi.org/10.1016/j.neuropharm.2006.09.018. [Google Scholar] [Crossref]

10. Rajoriya, V., Gupta, R., Vengurlekar, S., & Jain, S. K. (2025). Folate conjugated nano-lipid construct of paclitaxel for site-specific lung squamous carcinoma targeting. International Journal of Pharmaceutics, 672, 125312. https://doi.org/10.1016/j.ijpharm.2025.125312 [Google Scholar] [Crossref]

11. Stahl, M. S. (2000). Spectrophotometric determination of nitric oxide using Griess reaction. Analytical Biochemistry, 286(2), 240–245. https://doi.org/10.1006/abio.2000.4795 [Google Scholar] [Crossref]

12. Suher, T., et al. (2000). Quantification of GABA levels in brain tissue using HPLC. Journal of Chromatography B, 748(1), 313–320. https://doi.org/10.1016/S0378-4347(00)00338-9. [Google Scholar] [Crossref]

13. Rajoriya, V., Rajoriya, V., Gupta, R., Gupta, S., & Jain, S. K. (2025). Terpenes and lung cancer. In B. Chopra, A. Dhingra, P. Kriplani, & R. Ojha (Eds.), Analyzing Terpenes’ Role in Cancer Treatment (pp. 87–106). Hershey, PA: IGI Global Scientific Publishing. https://doi.org/10.4018/979-8-3693-6972-2.ch004 [Google Scholar] [Crossref]

14. Tsai, T. H., et al. (2000). Histopathological evaluation of neuroprotective agents in rodent models. Brain Research Bulletin, 52(4), 345–352. https://doi.org/10.1016/S0361-9230(00)00269-2 [Google Scholar] [Crossref]

15. World Health Organization. (2017). Depression and other common mental disorders: Global health estimates. Geneva: WHO Press. [Google Scholar] [Crossref]

16. Singh, R., & Pandey, S. (2015). Phytochemical and pharmacological profile of Azadirachta indica: A review. International Journal of Pharmaceutical Sciences Review and Research, 30(1), 94–101. [Google Scholar] [Crossref]

17. Rajoriya, V., Gupta, R., Vengurlekar, S., & Jain, S. K. (2025). Folate conjugated nano-lipid construct of paclitaxel for site-specific lung squamous carcinoma targeting. International Journal of Pharmaceutics, 672, 125312. https://doi.org/10.1016/j.ijpharm.2025.125312 [Google Scholar] [Crossref]

18. Kaur, G., et al. (2016). Role of flavonoids and alkaloids in neuroprotection and anxiety modulation. Frontiers in Pharmacology, 7, 398. https://doi.org/10.3389/fphar.2016.00398 [Google Scholar] [Crossref]

19. Sharma, V., & Kumar, R. (2018). Herbal approaches in the management of anxiety disorders: Mechanistic insights. Journal of Ethnopharmacology, 214, 45–56. https://doi.org/10.1016/j.jep.2017.11.042 [Google Scholar] [Crossref]

20. Rathore, S., Rajoriya, V., Kushwaha, V., Jain, S., &Kashaw, S. K. (2021). Preparation and characterization of 5-fluorouracil loaded nanogels for skin cancer treatments: In vitro drug release, cytotoxicity and cellular uptake analysis. Current Nanomedicine, 11(2), 127–138. https://doi.org/10.2174/2468187311666210301112644 [Google Scholar] [Crossref]

21. Gupta, R., Vengurlekar, S., Jain, S. K., & Rajoriya, V. (2025). Balancing preservation and access. In Quality Assurance of Ethno-Herbals: Cultivating Confidence in Alternative Medicine (pp. 93–112). Hershey, PA: IGI Global Scientific Publishing. [Google Scholar] [Crossref]

22. Rajoriya, V., Gupta, R., Vengurlekar, S., & Singh, U. S. (2024). Nanostructured lipid carriers (NLCs): A promising candidate for lung cancer targeting. International Journal of Pharmaceutics, 655, 123986. https://doi.org/10.1016/j.ijpharm.2024.123986 [Google Scholar] [Crossref]

23. Malviya, N., Jain, S., & Rajoriya, V. (2024). Neuropharmacological evaluation of herbal anxiolytics: Mechanistic insights. Journal of Herbal Medicine, 40(2), 112–120. https://doi.org/10.1016/j.hermed.2024.100567 [Google Scholar] [Crossref]

24. Patel, K., Chourasiya, S., & Gupta, R. (2023). Role of nitric oxide modulation in stress-induced anxiety: Herbal perspectives. Phytomedicine, 115, 154789. https://doi.org/10.1016/j.phymed.2023.154789. [Google Scholar] [Crossref]

25. Yadav, P., & Sharma, V. (2022). Comparative study of benzodiazepines and herbal extracts in rodent anxiety models. Pharmacology Reports, 74(5), 987–995. https://doi.org/10.1007/s43440-022-00345-7 [Google Scholar] [Crossref]

26. Rajoriya, V., & Gupta, R. (2025). Folate functionalized nanostructured lipid carriers of paclitaxel for site-specific A549 lung adenocarcinoma targeting. BioNanoScience, 15, 503. https://doi.org/10.1007/s12668-025-02133-9. [Google Scholar] [Crossref]

27. Rao, P., Sharma, R., & Gupta, A. (2025). Neuropharmacological evaluation of Azadirachta indica extract: Modulation of nitric oxide and cholinergic pathways in stress-induced rats. Journal of Natural Remedies, 25(3), 145–156. [Google Scholar] [Crossref]

28. Valenzuela Estrada, J., Hernández, M., & López, R. (2026). Anxiolytic and antidepressant effects of neem (Azadirachta indica) extract in Swiss Webster mice. Pharmacology Biochemistry and Behavior, 234, 173–182. [Google Scholar] [Crossref]

29. Sharma, A., Raut, S. S., Shukla, A., Parul, N., Singh, A., & Mishra, A. (2026). Ethnopharmacological landscape of Azadirachta indica (Neem): Phytochemical diversity bridging traditional heritage and modern medicine. Journal of ethnopharmacology, 371, 122088. https://doi.org/10.1016/j.jep.2026.122088 [Google Scholar] [Crossref]

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