In Vitro Enzyme Kinetics of Ashwagandha Extract on Plant Amylase and Protease Activity

Withania 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 α-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 α-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, V_max 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 α-amylase (affinity loss at preserved capacity) and non-competitively inhibited a cysteine protease with low-micromolar potency.