Design space optimization for the extraction of anti-thrombin-rich phenolics and flavonoids from Justicia adhatoda L. using response surface methodology and in silico validation of their anti-thrombin activity

The study focused on optimizing the extraction of anti-thrombin phenolics and flavonoids from the stem and flower of Justicia adhatoda (JA) using response surface methodology (RSM). Key factors utilized for the optimization included HCl concentration (0.1–1 N), extract concentration (50–150 mg/mL), methanol proportion (0–100%), and incubation temperature (32 ± 2 °C for hot and 6 ± 2 °C for cold). Fluorescence microscopy identified plant parts enriched with pharmacologically active compounds. The optimized extracts demonstrated substantial levels of phenolics, flavonoids and thrombin inhibitory activity across all samples. Antioxidant activity was measured using the DPPH and ABTS radical scavenging assays. Furthermore, multivariate optimization enhanced the antioxidant properties of the extracts. LC–MS analysis of the optimized extracts from JA (stem and flower) identified the presence of 22 compounds, with anti-thrombin polyphenols and flavonoids predominating, constituting approximately 30% and 35%, respectively. In silico studies, including molecular docking and dynamic simulations, revealed that the flavonoid Naringenin (−4.868 kcal mol⁻¹) had a higher inhibitory potential against the thrombin protein compared to the reference drug Dabigatran (−4.269 kcal mol⁻¹). This study demonstrated that flavonoids with significant anti-thrombin activity can be effectively extracted from J. adhatoda stem and flower using this optimized extraction procedure.