Acetylcholinesterase (AChE) inhibitors remain the primary therapeutic drug that can alleviate Alzheimer’s disease’s (AD) symptoms. Several Cassia species have been shown to exert significant anti-AChE activity, which can be an alternative remedy for AD. Cassia timoriensis and Cassia grandis are potential plants with anti-AChE activity, but their phytochemical investigation is yet to be further conducted. The aims of this study were to identify the phytoconstituents of C. timoriensis and C. grandis and evaluate their inhibitory activity against AChE and butyrylcholinesterase (BChE). Two compounds were isolated for the first time from C. timoriensis: arachidyl arachidate (1) and luteolin (2). Five compounds were identified from C. grandis: β-sitosterol (3), stigmasterol (4), cinnamic acid (5), 4-hydroxycinnamic acid (6), and hydroxymethylfurfural (7). Compound 2 showed significant inhibition towards AChE (IC₅₀: 20.47 ± 1.10 µM) and BChE (IC₅₀: 46.15 ± 2.20 µM), followed by 5 (IC_50: 40.5 ± 1.28 and 373.1 ± 16.4 µM) and 6 (IC₅₀: 43.4 ± 0.61 and 409.17 ± 14.80 µM) against AChE and BChE, respectively. The other compounds exhibited poor to slightly moderate AChE inhibitory activity. Molecular docking revealed that 2 showed good binding affinity towards TcAChE (PDB ID: 1W6R) and HsBChE (PDB ID: 4BDS). It formed a hydrogen bond with TYR121 at the peripheral anionic site (PAS, 2.04 Å), along with hydrophobic interactions with the anionic site and PAS (TRP84 and TYR121, respectively). Additionally, 2 formed three H-bonds with the binding site residues: one bond with catalytic triad, HIS438 at distance 2.05 Å, and the other two H-bonds with GLY115 and GLU197 at distances of 2.74 Å and 2.19 Å, respectively. The evidence of molecular interactions of 2 may justify the relevance of C. timoriensis as a cholinesterase inhibitor, having more promising activity than C. grandis.

Seventeen methanol extracts from different plant parts of five different Cassia species, including C. timorensis, C. grandis, C. fistula, C. spectabilis, and C. alata were screened against acetylcholinesterase (AChE). C. timorensis extracts were found to exhibit the highest inhibition towards AChE whereby the leaf, stem, and flower methanol extracts showed 94–97% inhibition. As far as we are aware, C. timorensis is one of the least explored Cassia spp. for bioactivity. Further fractionation led to the identification of six compounds, isolated for the first time from C. timorensis: 3-methoxyquercetin (1), benzenepropanoic acid (2), 9,12,15-octadecatrienoic acid (3), β-sitosterol (4), stigmasterol (5), and 1-octadecanol (6). Compound 1 showed moderate inhibition towards AChE (IC₅₀: 83.71 μM), while the other compounds exhibited poor to slightly moderate AChE inhibitory activity. Molecular docking revealed that the methoxy substitution of 1 formed a hydrogen bond with TYR121 at the peripheral anionic site (PAS) and the hydroxyl group at C5 formed a covalent hydrogen bond with ASP72. Additionally, the OH group at the C3′ position formed an interaction with the protein at the acyl pocket (PHE288). This possibly explains the activity of 1 in blocking the entry of acetylcholine (ACh, the neurotransmitter), thus impeding the hydrolysis of ACh.

Despite being widely used traditionally as a general tonic, especially in South East Asia, scientific research on Cassia timoriensis, remains scarce. In this study, the aim was to evaluate the in vitro activities for acetylcholinesterase (AChE) inhibitory potential, radical scavenging ability, and the anti-inflammatory properties of different extracts of C. timoriensis flowers using Ellman’s assay, a DPPH assay, and an albumin denaturation assay, respectively. With the exception of the acetylcholinesterase activity, to the best of our knowledge, these activities were reported for the first time for C. timoriensis flowers. The phytochemical analysis confirmed the existence of tannins, flavonoids, saponins, terpenoids, and steroids in the C. timoriensis flower extracts. The ethyl acetate extract possessed the highest phenolic and flavonoid contents (527.43 ± 5.83 mg GAE/g DW and 851.83 ± 10.08 mg QE/g DW, respectively) as compared to the other extracts. In addition, the ethyl acetate and methanol extracts exhibited the highest antioxidant (IC₅₀ 20.12 ± 0.12 and 34.48 ± 0.07 µg/mL, respectively), anti-inflammatory (92.50 ± 1.38 and 92.22 ± 1.09, respectively), and anti-AChE (IC₅₀ 6.91 ± 0.38 and 6.40 ± 0.27 µg/mL, respectively) activities. These results suggest that ethyl acetate and methanol extracts may contain bioactive compounds that can control neurodegenerative disorders, including Alzheimer’s disease, through high antioxidant, anti-inflammatory, and anti-AChE activities.