Fluorescence paper sensor meets magnetic affinity chromatography: discovering potent neuraminidase inhibitors in herbal medicines

Abstract

Given the inherent complexity of natural medicines, finding a straightforward and efficient method for identifying active ingredients remains a significant challenge, yet it is of paramount importance. Influenza virus neuraminidase (NA), a primary target for anti-influenza drug development, plays a crucial role in the infection process, making it essential to develop rapid and facile methods for screening NA inhibitors. Herein, we developed a novel and efficient analytical technique for the identification of NA inhibitors from complex herbal medicines by integrating dual sensing with affinity chromatography. This approach simplifies the experimental process and highlights the benefits of being quicker, more sensitive, and cost-effective. Regarding the biosensing section, the innovative concept of a 4-methylumbelliferyl-N-acetylneuraminic acid-NA-based fluorescence paper sensor strategy enables the rapid detection of NA inhibitors in complex herbal samples. In affinity chromatography, bioactive compounds were precisely captured, separated, and identified. The efficacy and reliability of the developed method were confirmed using both negative and positive controls. Then, the method was applied to screen for NA inhibitors in 20 different herbal medicines. The results revealed that Bupleurum chinense DC. exhibited the most pronounced inhibitory effect on NA. Subsequent analysis utilizing affinity chromatography identified three bioactive compounds, namely saikosaponin a, saikosaponin d, and baicalin, as the active agents responsible for this inhibitory effect, with IC₅₀ values of 177.3 μM, 262.9 μM, and 241.4 μM, respectively. Molecular docking studies further indicated that these three bioactive compounds exhibit a strong binding affinity with NA. This research provides novel insights into the screening of enzyme inhibitors within herbal medicines.

Keywords: 4-Methylumbelliferyl-N-acetylneuraminic acid; Affinity chromatography; Biosensing; Enzyme inhibitor; Neuraminidase.