A precision inhibitor screening strategy from complex herbal medicine for hyaluronidase based on carbon quantum dot sensing and targeted capture technology

Abstract

Hyaluronidase (HAase), an enzyme responsible for hyaluronic acid (HA) degradation, compromises the structural integrity of HA by cleaving anti-inflammatory high-molecular-weight HA into pro-inflammatory lower molecular weight forms. Consequently, developing simple and efficient strategies for screening HAase inhibitors is of critical importance. Herein, a novel biosensing-based targeted affinity screening method was established for the identification of HAase inhibitors from complex herbal medicines. In the biosensing system, carbon dots served as fluorescent probes, while HA-functionalized gold nanoparticles acted as quenchers, and the addition of HAase induced a marked fluorescence recovery. Under optimized conditions, HAase exhibited a good linear response over the concentration range of 5.3 to 343.8 U mL⁻¹. This method enables rapid and efficient detection of HAase inhibitory activity in complex herbal extracts. Subsequently, active components were selectively captured, separated, and identified using immobilized HAase affinity chromatography. Following validation with both negative and positive control drugs, the screening model successfully identified Rhodiola rosea L. among 43 herbal medicines as a potent HAase. Gallic acid (GA) was identified as the active constituent, exhibiting an IC₅₀ value of 111.7 μM. Results from molecular docking and molecular dynamics simulations demonstrated a strong binding affinity between GA and HAase. Additionally, cellular experiments confirmed the anti-inflammatory activity of GA, as evidenced by its significant inhibition of LPS-induced inflammatory markers IL-4, IL-5, IL-6, and TNF-α. Collectively, the integrated biosensing and affinity-based screening strategy established in this study provides a precise and efficient platform for the discovery of bioactive compounds from complex natural products.