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 IC₅₀ values of 0.56, 0.01, and 0.02 mg/mL, respectively. HBE at 0.2 μg/mL suppressed H₂O₂-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 IC₅₀ values of 1.3 and 6.6 μM, respectively, whereas cannabidiol and cannabidiolic acid were the most active MAO-B inhibitors, with IC₅₀ 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.