Herbal allies in antibiotic warfare: Unveiling potent β-lactamase inhibitors with innovative cellulose-based colorimetric sensors

β-Lactamases (β-LS) are a significant threat to public health due to their role in conferring antibiotic resistance against nearly all clinically used β-lactam antibiotics. Consequently, the search for β-lactamase inhibitors (β-LSI), which can reversibly bind to and inactivate β-LS, has intensified. Here, a sensitive and rapid cellulose-based colorimetric sensor integrated with affinity identification model was designed for high-throughput screening of β-LSI. The innovative concept of a nitrocefin-based colorimetric paper sensor strategy was effective in rapidly detecting the inhibitory effects of herbals on β-LS. Then, potential β-LSI present in the screened herbals were captured, separated, and identified utilizing affinity identification model. The efficacy of the developed method was validated with both negative and positive drugs and was subsequently applied to the screening of β-LSI in 7 different herbal samples. Notably, Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou exhibited significant β-LS inhibitory activity. Further affinity separation and LC-MS analyses identified 2 bioactive compounds, spinosin, and magnoflorine, as the agents responsible for this inhibitory effect, with IC 50 values of 143.53 μM and 294.66 μM, respectively. The inhibitory effects and mechanisms were validated through antibacterial activity testing, molecular docking, and dynamic simulation. Notably, spinosin combined with aztreonam showed greater antibacterial effectiveness than avibactam. This study not only establishes a platform for the effective preliminary screening of β-LSI as a potential antimicrobial resistance-reversing agent for future clinical applications, but also inspires the further development of other cellulose-based colorimetric sensors for the rapid and high-throughput discovery of various enzyme inhibitors in complex natural medicines.