Hemp bark extract attenuates scopolamine-induced cognitive dysfunction through multi-target cholinesterase, MAO-B, and antioxidant mechanisms

Cognitive decline associated with Alzheimer's disease is driven by several interconnected mechanisms, including impaired cholinergic signaling, MAO-B-linked oxidative stress, and reduced neuronal antioxidant capacity. In this study, ethanol extracts prepared from hemp root, stem, bark, leaf, and seed were compared to identify hemp-derived materials with multi-target neuroprotective potential. Among the tested extracts, hemp bark extract (HBE) showed the most balanced activity profile, inhibiting acetylcholinesterase, butyrylcholinesterase, and MAO-B with IC 50 values of 0.56, 0.01, and 0.02 mg/mL, respectively. HBE at 0.2 μg/mL suppressed H 2 O 2 -induced intracellular reactive oxygen species formation and recovered superoxide dismutase activity in SH-SY5Y neuronal cells. In a scopolamine-induced mouse model, oral HBE administration at 100 and 200 mg/kg improved performance in the passive avoidance test and reduced escape latency in the water maze test, indicating protection against scopolamine-induced cognitive deficits. Chemical profiling by ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry revealed several cannabinoids in HBE, including cannabidiol, cannabidiolic acid, and cannabichromenic acid. Enzyme assays showed that cannabichromenic acid potently inhibited acetylcholinesterase and butyrylcholinesterase, with IC 50 values of 1.3 and 6.6 μM, respectively, whereas cannabidiol and cannabidiolic acid were the most active MAO-B inhibitors, with IC 50 values of 2.3 and 5.3 μM. Docking, GNINA rescoring, molecular dynamics simulations, and MM-GBSA/MM-PBSA analyses further supported stable interactions of these cannabinoids with the target enzymes, particularly cannabidiolic acid with acetylcholinesterase and MAO-B. These findings suggest that HBE protects against scopolamine-induced cognitive dysfunction through coordinated cholinesterase inhibition, MAO-B modulation, and antioxidant defense, with cannabidiol, cannabidiolic acid, and cannabichromenic acid acting as key multi-target constituents.