DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF WITHAFERIN A AND WITHANOLIDE A IN METHANOLIC EXTRACT OF WITHANIA SOMNIFERA ROOTS AND CORRELATION WITH IN VITRO ANTIOXIDANTA ANTI-INFLAMMATORY ACTIVITIES

Abstract

Background

Withania somnifera (Ashwagandha) is a medicinal plant rich in withanolides, primarily Withaferin A and Withanolide A, known for their antioxidant and anti-inflammatory properties. Accurate quantification of these bioactive compounds is essential for quality control and therapeutic standardization of herbal formulations.

Objective

To develop and validate a simple, robust, and stability-indicating RP-HPLC method for the simultaneous quantification of Withaferin A and Withanolide A in W. somnifera root extract and to correlate their content with in vitro antioxidant and anti-inflammatory activities.

Methods

A methanolic extract of W. somnifera roots was prepared and analyzed using an optimized RP-HPLC method employing a C18 column, acetonitrile–acidified water mobile phase, and PDA detection at 227 nm. The method was validated per ICH Q2(R1) guidelines. Forced degradation studies were performed under acidic, basic, oxidative, thermal, and photolytic conditions. Antioxidant assays (DPPH, ABTS, FRAP, TAC) and anti-inflammatory assays (protein denaturation, HRBC membrane stabilization, lipoxygenase inhibition) were conducted. Pearson correlation analysis was used to determine relationships between withanolide content and biological activities.

Results

The method produced well-resolved peaks with retention times of 6.42 min (Withaferin A) and 8.73 min (Withanolide A), and demonstrated excellent linearity (R² > 0.999), accuracy (98–102%), precision (%RSD < 2), and sensitivity (LOD 0.18–0.22 µg/mL). Forced degradation studies confirmed the method's stability-indicating capability, with major degradation observed under oxidative and alkaline conditions. Antioxidant IC₅₀ values of the extract ranged from 54.77–63.42 µg/mL, while anti-inflammatory IC₅₀ values ranged from 68.94–81.62 µg/mL. Strong inverse correlations (r > –0.80) were observed between withanolide content and biological activities, confirming their contribution to extract potency.

Conclusion

A validated, stability-indicating RP-HPLC method was successfully developed for simultaneous estimation of Withaferin A and Withanolide A in W. somnifera extract. The method is suitable for routine quality control and standardization. The strong correlation between withanolide content and antioxidant and anti-inflammatory activities highlights their therapeutic significance and supports further pharmacological and formulation-based research.