AbstractWithania somnifera (Ashwagandha) contains withanolides and alkaloids that may modulate enzymes. However, standardized kinetic evidence on plant hydrolases is limited. This study attempts to quantify inhibitory or stimulatory effects of a marker-standardized Ashwagandha extract on plant a-amylase and a cysteine protease, define mechanism and estimate potency. Cell-free spectrophotometric assays were run at 30°C with controlled pH, seven substrate levels, and four inhibitor levels, each in technical triplicate. Initial rates were obtained from blank-corrected 0–60s traces and converted using pathlength-adjusted extinction coefficients. Across 168 conditions, 94. 0% of traces met linearity (R2 = 0. 98). For a-amylase, Vmax changed modestly (119. 87 ± 2. 28 to 128. 56 ± 3. 63 µM min–1) while Km increased (148. 58 ± 9. 57 to 511. 65 ± 34. 56 µM), and LB slopes increased with preserved intercepts, supporting competitive inhibition; Ki was 4. 06 ± 0. 16 µM (95% CI 3. 75–4. 38). For protease, Vₘax declined (89. 85 ± 1. 60 to 24. 92 ± 0. 54 µM min–1) at near-constant Km (˜ 100 µM), with rising LB intercepts, indicating non-competitive inhibition; Ki was 3. 78 ± 0. 14 µM (95% CI 3. 50–4. 06). Model adequacy was high for primary fits (amylase R2 0. 988–0. 996; protease R2 0. 975–0. 984). Under tightly controlled in-vitro conditions, Ashwagandha extract competitively inhibited plant a-amylase (affinity loss at preserved capacity) and non-competitively inhibited a cysteine protease with low-micromolar potency.
Kumar et al. (2025) studied this question.