Identification and Verification of the Anti‐Insomnia Compounds From Herbal Pair Albiziae Cortex and Polygoni Multiflori Caulis Using Immobilized 5‐HT 1A Receptor Chromatography

ABSTRACT This study employed an affinity chromatography approach to investigate the potential molecular mechanism of the Albiziae Cortex and Polygoni Multiflori Caulis herbal pair, which is commonly used for treating insomnia. Briefly, the haloalkane dehalogenase and its substrate were fused separately onto the serotonin 1A receptor and amino microspheres. Receptors were bound onto chromatographic stationary phases specifically through biological orthogonal interactions. Subsequently, catechin, methyl‐O‐digalloyl‐hexose, 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside (THSG), and julibroside J 35 were identified as active components in the herbal pair extract targeting the receptor. A mice model of insomnia was used to validate the anti‐insomnia effects. The herbal‐pair extract, catechin, and THSG significantly reduced sleep latency and increased sleep duration. Furthermore, on‐column analysis and molecular docking revealed the binding characteristic. Adsorption isotherm analysis indicated a homogeneous binding process, which was further supported by injection amount‐dependent analysis. The latter also provided binding constants of (5.70 ± 0.61) × 10 5 M −1 for catechin and (4.05 ± 0.20) × 10 5 M −1 for THSG. Molecular docking revealed critical binding residues (Asp116, Val117, Thr121, Lys191, Thr196, and Ser199), primarily via van der Waals forces and hydrogen bonds. Collectively, immobilizing the serotonin 1A receptor enables the screening of biologically active compounds from complex systems effectively and shows a degree of general applicability.