Phytochemical Analysis, Antioxidant Activity and Bioassay-Guided Isolation of Acetylcholinesterase and Butyrylcholinesterase Inhibitors from <i>Horsfieldia polyspherula</i> Bark (Myristicaceae)

Alzheimer's disease (AD) is a neurodegenerative condition brought on by aging and characterised by progressive decline in cognitive function and abnormalities in the central cholnergic system. <i>β</i>-amyloid deposits, neurofibril tangle aggregation, oxidative stress or reduced level of acetylcholine are a few causes that have been linked to AD. In this study, the bioassay-guided isolation from ethyl acetate (EtOAc) extract of <i>Horsfieldia polyspherula</i> bark led to the isolation of nine compounds namely, 16-phenylhexadecanoic acid (<b>1</b>), undecylbenzene (<b>2</b>), 3,4-dihydroxybenzoic acid (<b>3</b>), dodecanoic acid (<b>4</b>), tetradecanoic acid (<b>5</b>), pentadecanoic acid (<b>6</b>), 1-tridecene (<b>7</b>), stigmasterol (<b>8</b>) and trimyristin (<b>9</b>). Phytochemical analysis revealed the presence of flavonoids, steroids, lignin, alkaloids, phytosterol and triterpenoids. The DPPH scavenging activity of EtOAc extract was related to the phenolic content (116.67 ± 16.98 GAE mg/g) and other non-phenolics such as lower fatty acids. Meanwhile, the DPPH scavenging activity was found to be concentration-dependent and correlated with both flavonoid and phenolic content. Furthermore, EtOAc and methanol (MeOH) extracts of <i>H. polyspherula</i> bark showed significant inhibitory activity at 100 μg/mL on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), with EtOAc extract showing 77.2% and 64.1% inhibition and MeOH extract showing 37.5% and 39.2% inhibition, respectively. Additionally, the IC₅₀ for BuChE and AChE of the EtOAc extract were found to be effective, with 15.41 ± 0.78 μg/mL and 7.67 ± 0.13 μg/mL, respectively. Compound <b>1</b> exhibited dual inhibition of 40.99 ± 1.99 μM (BuChE) and 46.83 ± 2.44 μM (AChE), while compounds <b>2</b> and <b>3</b> showed IC₅₀ values above 200 μM. This study revealed that this plant shows a significant potential as anti-cholinesterase focusing on acetylcholinesterase (AchE) and butyrylcholinesterase (BuChE). This is the first report on <i>Horsfieldia polyspherula</i> and their biological activity.