A quantitative screening strategy based on activity-content ratio for prioritizing bioactive compounds in medicinal...

Accurately evaluating the intrinsic bioactivity of individual constituents, independent of their abundance, remains a major challenge in natural product screening. To address this, we proposed the activity-content ratio (ACR) as a novel index for direct intrinsic inhibitory potency characterization of constituents. Utilizing an at-line nanofractionation platform, an ACR-based screening method for acetylcholinesterase inhibitors was established. The method was validated using both mixed standards and a complex extract of Corydalis rhizoma alkaloids. Results demonstrated a strong linear correlation (R ² > 0.93) between ACR% and IC₅₀ for 16 active constituents. High-ACR% compounds, such as berberine, palmatine, and dehydrocorydalin, exhibited significant inhibitory activities at nanomolar concentrations. The model's broad applicability was further confirmed by screening Citri reticulatae pericarpium flavonoids for α-glucosidase inhibitors, showing excellent linearity (R ² = 0.96) for the five identified active constituents. Notably, rutin displayed the highest ACR% and an IC₅₀ of 15.21 ± 1.23 μM. The consistent performance of the ACR model across two distinct enzymatic targets and chemical families (alkaloids and flavonoids) demonstrates its reliability and broad applicability. This study provides a reliable and efficient quantitative tool for bioactivity evaluation, which is expected to greatly accelerate the discovery and quality control of bioactive medicinal plants.