Identification of aldose reductase inhibitors from the flowers of Tussilago farfara using PCN-222 metal-organic framework-based immobilization-free affinity selection coupled with high-performance liquid chromatography

Abstract

Inhibition of aldose reductase (AR) activity is promising for mitigating diabetic complications. This study proposed a novel screening approach combining PCN-222 metal-organic framework (MOF)-based immobilization-free affinity selection with high-performance liquid chromatography to identify AR inhibitors (ARIs) from natural product extracts. As a proof-of-concept, the AR inhibitory activity of the flowers of Tussilago farfara L. (FTF) was evaluated, and ARIs in FTF were identified using the proposed approach. To validate the screening results, major constituents of FTF were isolated for functional assays, inhibitory kinetics, and interaction mechanisms. The results demonstrated significant inhibitory activity of FTF extracts against AR (IC₅₀ = 3.49 µg/mL), with chlorogenic acid (IC₅₀ = 0.97 µM, K_i = 1.21 µM, K_a = 4.88 × 10⁴ L/mol), isochlorogenic acid A (IC₅₀ = 0.76 µM, K_i = 0.61 µM, K_a = 5.25 × 10⁴ L/mol), isochlorogenic acid C (IC₅₀ = 1.83 µM, K_i = 1.24 µM, K_a = 4.25 × 10⁵ L/mol), and isochlorogenic acid B (IC₅₀ = 0.95 µM, K_i = 0.40 µM, K_a = 3.64 × 10⁴ L/mol) identified using the proposed method, where all exhibited high binding affinity to AR. In contrast, rutin (IC₅₀ = 3.69 µM, K_i = 5.60 µM, K_a = 2.93 × 10⁴ L/mol), despite being confirmed as an ARI, exhibited weaker binding affinity to AR and was not identified by this approach. These findings demonstrate the effectiveness of the proposed method for efficiently identifying enzyme inhibitors with high binding affinities, and represent the first application of MOFs in immobilization-free affinity selection screening methods.