A novel method for efficient screening of hyaluronidase inhibitors based on the integration of paper-based sensors and immobilized affinity chromatography

Hyaluronidase (HAase) degrades hyaluronic acid (HA), a key glycosaminoglycan of the extracellular matrix involved in tissue hydration, cell motility, and inflammation. Excessive HAase activity is associated with diseases, including cancer metastasis and inflammation, making HAase inhibitors vital for treatment strategies. Meanwhile, efficiently analyzing active ingredients in complex herbal medicines is key for drug discovery, but developing a simple and accurate method remains challenging. Here, an integrated approach combining paper-based sensing and column-free affinity chromatography to efficiently screen potential HAase inhibitors from herbal sources was established. The paper-based sensing module uses HA's rheological properties to quickly assess the inhibitory effects of herbal extracts on HAase. Herbals with inhibitory activity are then analyzed using affinity chromatography to capture, separate, and identify bioactive components interacting with HAase. The feasibility of both biosensing and affinity chromatography was validated using negative and positive control drugs, and Galla chinensis was successfully identified from 40 herbal samples as a potent HAase inhibitor, with tannic acid (TA) being the active compound (IC 50  = 51.22 μM). Molecular docking and dynamics simulations revealed a strong binding affinity between TA and HAase. TA's anti-inflammatory effects were further confirmed by its significant inhibition of LPS-induced IL-4, IL-5, IL-6, and TNF-α in cell experiments. In summary, this integrated strategy presents a promising and efficient approach for the discovery and development of novel HAase inhibitors, as well as other enzyme inhibitors derived from natural medicinal sources.