The essential oil (EO) of Artemisia plants contains a large number of bioactive compounds that are widely used. The aim of this study was to analyse the chemical composition of EOs of six Arte-misia plants collected in Croatia and to test their cholinesterase inhibitory potential. GC-MS analysis of EO of A. absinthium showed that the dominant compounds are cis-sabinyl acetate and cis-epoxy-ocimene; in EO of A. abrotanum it is borneol; in EO of A. annua it is artemisia ketone, camphor, and 1,8-cineole; in EO of A. arborescens it is camphor and chamazulene; in EO of A. verlotiorum it is cis-thujone, 1,8-cineole, and trans-thujone; in EO of A. vulgaris, it is trans-thujone and trans-epoxy-ocimene. EO of the five studied Artemisia species from Croatia is rich in mono-terpenoid compounds (1,8-cineole, artemisia ketone, cis-thujone, trans-thujone, cis-epoxy-ocimene, camphor, borneol, and cis-sabinyl acetate). EO of A. arborescens is also rich in chamazulene. The results also showed that the tested EOs have moderate cholinesterase inhibi-tion potential, especially the EOs of A. annua, A. vulgaris, and A. abrotanum. This is the first anal-ysis of the chemical composition of the EOs of four Artemisia plants and the first analysis of cho-linesterase potential for plants collected in Croatia.

Affinity chromatography on procainamide-Sepharose has been an important step in the purification of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) since its introduction in 1978. The procainamide affinity gel has limitations. In the present report a new affinity gel called Hupresin^® was evaluated for its ability to purify truncated, partly-deglycosylated human butyrylcholinesterase (rHuBChE) expressed in a stably transfected CHO cell line. We present a detailed example of the purification of rHuBChE secreted into 3940 mL of serum-free culture medium. The starting material contained 13,163 units of BChE activity (20.9 mg). rHuBChE was purified to homogeneity in a single step by passage over 82 mL of Hupresin^® and elution with 0.1 M tetramethylammonium bromide in 20 mM TrisCl pH 7.5. The fraction with the highest specific activity of 630 units/mg contained 11 mg of BChE. Hupresin^® is superior to procainamide-Sepharose for purification of BChE, but is not suitable for purifying native AChE because Hupresin^® binds AChE so tightly that AChE is desorbed with denaturing solvents such as 50% acetonitrile or 1% trifluoroacetic acid. Procainamide-Sepharose will continue to be useful for purification of AChE.

Cholinesterase (ChE) and secretase (BACE) inhibitors, and fibril- and β-amyloid-suppressing medicines are used to treat Alzheimer's disease (AD) symptomatically. The prevalence and complex nature of AD have increased the urgent need for multi-targeted directed ligands (MTDLs). This is because MTDLs can prevent potential drug-drug interactions during poly-therapy and have a better therapeutic profile than single targeted agents. Using piperazine linker or spacer and two primary scaffolds, benzofuran and 8-hydroxyquinoline, a unique class of multi-targeted medicines was recently reported by our group. These compounds showed re-markable effectiveness in inhibiting Aβ1-42 aggregation and acting as iron chelators.The results prompted us to synthesize an additional series of compounds as multimodal anti-AD agents and investigate its inhibitor activities to ChE (AChE/BChE) and BACE1 enzymes. The resulting in-hibitory effects suggested that the compounds under study might be used to improve cognitive function. The docking analysis' findings revealed that the compounds bind to AChE and BChE by forming H-bond interactions with amino acid residues at binding sites and µ-stacking inter-actions with aromatic residues, whereas the binding to BACE1 only revealed H-bond interac-tions with amino acid residues at binding sites.

Effective therapeutics for Alzheimer's disease (AD) are in great demand worldwide. In our previous work, we responded to this need by synthesizing novel drug candidates consisting of 4-amino-2,3-polymethylenequinolines conjugated with butylated hydroxytoluene via fixed-length alkylimine or alkylamine linkers (spacers) and studying their bioactivities pertaining to AD treatment. Here, we report significant extensions of these studies, including the use of variable-length spacers and more detailed biological characterizations. Conjugates were potent inhibitors of acetylcholinesterase (AChE, minimum IC50 15.1±0.2 nM) and butyrylcholinesterase (BChE, minimum IC50 5.96±0.58 nM), with weak inhibition of off-target carboxylesterase. Conjugates with alkylamine spacers were more effective cholinesterase inhibitors than alkylimine analogs. Optimal inhibition for AChE was exhibited by cyclohexaquinoline and for BChE by cycloheptaquinoline. Increasing spacer length elevated potency against both cholinesterases. Structure-activity relationships agreed with docking results. Mixed-type reversible AChE inhibition, dual docking to catalytic and peripheral anionic sites, and propidium iodide displacement suggested the potential of hybrids to block AChE-induced β-amyloid (Aβ) aggregation. Hybrids also exhibited inhibition of Aβ self-aggregation in the thioflavin test; those with a hexaquinoline ring and C8 spacer were the most active. Conjugates demonstrated high antioxidant activity in ABTS and FRAP assays as well as inhibition of luminol chemiluminescence and lipid peroxidation in mouse brain homogenates. Quantum-chemical calculations explained antioxidant results. Computed ADMET profiles indicated favorable blood-brain barrier permeability suggesting CNS activity potential. Thus, the conjugates could be considered promising multifunctional agents for potential treatment of AD.