Efficacy and Anti-Inflammatory Activity of Ashwagandha Sustained-Release Formulation on Depression and Anxiety Induced by Chronic Unpredictable Stress: in vivo and in vitro Studies

Abstract

Background

Stress is the psychological, physiological, and behavioral response of an individual’s body when they perceive a lack of equilibrium between the demands placed upon them and their ability to meet those demands. Adaptogens are herbs that help with stress management, and Ashwagandha is one such safe and effective adaptogen.

Objective

We evaluated the anti-neuroinflammatory potential of Ashwagandha sustained-release formulation (AshwaSR) by estimating the in vitro expression of pro-inflammatory cytokines, and its efficacy on anxiety and depression in an in vivo study.

Methods

Our in vitro study investigated the anti-inflammatory potential of AshwaSR by estimating the expression of tumour necrosis factor [TNF]-α and interleukin [IL]-1β levels in LPS-induced THP-1 human monocytes, and the antioxidant effects by its potential to inhibit the superoxide [SO] generation in PMA-induced HL-60 human monocytic cells. The in vivo study assessed the efficacy of AshwaSR on chronic unpredictable stress (CUS)-induced comorbid anxiety and depression in Sprague Dawley rats. Antidepressant and anxiolytic effects of AshwaSR were evaluated by open field test (OFT), elevated plus maze (EPM), forced swim test (FST), and Morris water maze (MWM) test.

Results

AshwaSR inhibited TNF-α, IL-1β and superoxide production in a dose-dependent manner in the in vitro study. The in vivo CUS model induced depression-like and anxiety-like behaviour. Treatments with AshwaSR and escitalopram showed improvement in the EPM and MWM models compared to the CUS-group.

Conclusion

In vitro study demonstrated that AshwaSR inhibits expressions of pro-inflammatory cytokines, IL-1β and TNF-α, and superoxide production. Further, the in vivo study confirmed its anxiolytic and stress-relieving effects in the CUS model that confirmed AshwaSR’s potential in managing stress and stress-related symptoms.

Keywords:

• Withania somnifera
• antidepressant
• anxiolytic stress
• CUS model
• anti-inflammatory
• antioxidant

Abbreviations

AshwaSR, Ashwagandha sustained-release formulation; CL, control; CNS, central nervous system; CUS, chronic unpredictable stress; EPM, elevated plus maze; ESC, Escitalopram; FBS, Fetal Bovine Serum; FST, forced swim test; HBSS, Hank’s Balanced Salt Solution; HPA, hypothalamic-pituitary-adrenal; IAEC, Institutional Animal Ethics Committee; IL, interleukin; LPS, lipopolysaccharide; MWM, Morris water maze; OFT, open field test; PMA, phorbol 12-myristate 13-acetate; SR, sustained-release; TNF, tumor necrosis factor; WHO, World Health Organization.

Data Sharing Statement

We will not be sharing the raw data.

Animals and Ethics

We considered 32 male rats, Rattus norvegicus (Sprague Dawley), aged 8–12 weeks and weighed between 214.4 to 275.8 g. 3–4 animals per cage were housed at 22±3°C temperature and 30–70% relative humidity and 12 h light–dark cycle except on the day of inversion of the light/dark cycle for induction of CUS. All the animals were housed in the solid-bottom autoclaved polypropylene cages with a stainless-steel top grill with separate provisions for animal feed. They received rodent feed pellets (Lab meal-M^®, VRK nutritional solutions Pvt. Ltd., Pune) regularly, and mineral water for drinking in polycarbonate bottle ad libitum. Corncob was used as bedding material which was changed twice a week.

The study plan was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of the Laila Impex R&D center and was documented in the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (CPCSEA registration No. of the Test Facility: 204/PO/Rc/S/2000/CPCSEA) specified “Form-B” plan (IAEC number:LI/IAEC/LI190106). This study was conducted with the strict adherence to the recommendations of animal welfare guidelines regarding animal care, and all animals were handled humanely with due regard for their welfare in compliance with CPCSEA regulations. All the procedures were implemented in this study were in match with the accepted practices to minimize/avoid risk of causing pain, distress or discomfort to the animals.

Consent for Publication

All authors confirm that the details of images, videos, recordings, etc can be published, and that all authors provide their consent for the same.

Acknowledgments

The study was sponsored by Nutriventia Limited, and Laila Nutraceuticals, India. The authors would like to acknowledge Ms. Arohi Sarang, Ms. Neelanjana Bhattacharyya and Ms. Komal Dobariya from the CBCC Global Research team for their writing and editorial support in the development of this manuscript.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

Venkateswarlu Somepalli and AV KrishnaRaju are employees of Laila Nutraceuticals, India. Rajat Shah and Shefali Thanawala are employees of Nutriventia Limited, India. Rajat Shah has a patent pending, International (PCT) application WO 2023/021434.

Additional information

Funding

This study was funded by Nutriventia Limited and Laila Nutraceuticals.