Design, synthesis and biological evaluation of bakuchiol derivatives as multi-target agents for the treatment of Alzheimer's disease.

The concept of multi-target-directed ligands offers fresh perspectives for the creation of brand-new Alzheimer's disease medications. To explore their potential as multi-targeted anti-Alzheimer's drugs, eighteen new bakuchiol derivatives were designed, synthesized, and evaluated. The structures of the new compounds were elucidated by IR, NMR, and HRMS. Eighteen compounds were assayed for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in vitro using Ellman's method. It was shown that most of the compounds inhibited AChE and BuChE to varying degrees, but the inhibitory effect on AChE was relatively strong, with fourteen compounds showing inhibition of >50% at the concentration of 200 µM. Among them, compound 3g (IC50 = 32.07 ± 2.00 µM) and compound 3n (IC50 = 34.78 ± 0.34 µM) showed potent AChE inhibitory activities. Molecular docking studies and molecular dynamics simulation showed that compound 3g interacts with key amino acids at the catalytically active site (CAS) and peripheral anionic site (PAS) of acetylcholinesterase and binds stably to acetylcholinesterase. On the other hand, compounds 3n and 3q significantly reduced the pro-inflammatory cytokines TNF-α and IL-6 released from LPS-induced RAW 264.7 macrophages. Compound 3n possessed both anti-acetylcholinesterase activity and anti-inflammatory properties. Therefore, an in-depth study of compound 3n is expected to be a multi-targeted anti-AD drug.

Amaryllidaceae alkaloids from the bulbs of Crinum latifolium L. and their cholinesterase inhibitory activities.

Eleven previously undescribed Amaryllidaceae alkaloids, crinalatifolines A-K (1-11), and two first naturally occurring alkaloids, dihydroambelline (12) and N-demethyldihydrogalanthamine (13), were isolated from the bulbs of Crinum latifolium L. Additionally, thirty-seven known alkaloids and one alkaloid artifact were also isolated from this plant species. Their structures and absolute configurations were elucidated using extensive spectroscopic techniques, including IR, NMR, MS, and ECD. Evaluations of the cholinesterase inhibitory activities of most of these compounds were conducted. Among the tested compounds, ungeremine exhibited the highest potency against acetylcholinesterase and butyrylcholinesterase, with the IC50 values of 0.10 and 1.21 µM, respectively. These values were 9.4- and 2.4-fold more potent than the reference drug galanthamine.

N-Methyl Costaricine and Costaricine, Two Potent Butyrylcholinesterase Inhibitors from Alseodaphne pendulifolia Gamb.

Studies have been conducted over the last decade to identify secondary metabolites from plants, in particular those from the class of alkaloids, for the development of new anti-Alzheimer's disease (AD) drugs. The genus Alseodaphne, comprising a wide range of alkaloids, is a promising source for the discovery of new cholinesterase inhibitors, the first-line treatment for AD. With regard to this, a phytochemical investigation of the dichloromethane extract of the bark of A. pendulifolia Gamb. was conducted. Repeated column chromatography and preparative thin-layer chromatography led to the isolation of a new bisbenzylisoquinoline alkaloid, N-methyl costaricine (1), together with costaricine (2), hernagine (3), N-methyl hernagine (4), corydine (5), and oxohernagine (6). Their structures were elucidated by the 1D- and 2D-NMR techniques and LCMS-IT-TOF analysis. Compounds 1 and 2 were more-potent BChE inhibitors than galantamine with IC50 values of 3.51 ± 0.80 µM and 2.90 ± 0.56 µM, respectively. The Lineweaver-Burk plots of compounds 1 and 2 indicated they were mixed-mode inhibitors. Compounds 1 and 2 have the potential to be employed as lead compounds for the development of new drugs or medicinal supplements to treat AD.

Identification of potential inhibitors of cholinergic and ß-secretase enzymes from phytochemicals derived from Gongronema latifolium Benth leaf: an integrated computational analysis.

Neurodegenerative disorders (NDDs) are associated with increased activities of the brain acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and ß-secretase enzyme (BACE1). Inhibition of these enzymes affords therapeutic option for managing NDDs such as Alzheimer's disease (AD) and Parkinson's disease (PD). Although, Gongronema latifolium Benth (GL) has been widely documented in ethnopharmacological and scientific reports for the management of NDDs, there is paucity of information on its underlying mechanism and neurotherapeutic constituents. Herein, 152 previously reported Gongronema latifolium derived-phytochemicals (GLDP) were screened against hAChE, hBChE and hBACE-1 using molecular docking, molecular dynamics (MD) simulations, free energy of binding calculations and cluster analysis. The result of the computational analysis identified silymarin, alpha-amyrin and teraxeron with the highest binding energies (-12.3, -11.2, -10.5 Kcal/mol) for hAChE, hBChE and hBACE-1 respectively as compared with those of the reference inhibitors (-12.3, -9.8 and - 9.4 for donepezil, propidium and aminoquinoline compound respectively). These best docked phytochemicals were found to be orientated in the hydrophobic gorge where they interacted with the choline-binding pocket in the A-site and P-site of the cholinesterase and subsites S1, S3, S3' and flip (67-75) residues of the pocket of the BACE-1. The best docked phytochemicals complexed with the target proteins were stable in a 100 ns molecular dynamic simulation. The interactions with the catalytic residues were preserved during the simulation as observed from the MMGBSA decomposition and cluster analyses. The presence of these phytocompounds most notably silymarin, which demonstrated dual high binding tendencies to both cholinesterases, were identified as potential neurotherapeutics subject to further investigation.

A retrospective screening method for carbamate toxicant exposure based on butyrylcholinesterase adducts in human plasma with ultra-high performance liquid chromatography-tandem mass spectrometry.

Carbamate pesticides are extensively used in agriculture for their inhibition to acetylcholinesterase and damages to the insects' neural systems. Because of their toxicity, human poisoning incidents caused by carbamate pesticide exposure have occurred from time to time. What's more, some lethally toxic carbamate toxicants known as carbamate nerve agents (CMNAs) have been supplemented in Schedule 1 of the Annex on Chemicals in the Chemical Weapons Convention (CWC) by Organisation of the Prohibition of Chemical Weapons (OPCW) from 2020. And some other carbamates, like physostigmine, have been used in clinical treatment as anticholinergic drugs and their misuse may also cause damages to the body. Similar to organophosphorus toxicants, carbamate toxicants would react with butyrylcholinesterase (BChE) in plasma when entering the human body, resulting in the BChE adducts, based on which the exposure of carbamate toxicants could be detected retrospectively. In this study, methylcarbamyl nonapeptide and dimethylcarbamyl nonapeptide from pepsin digestion of BChE adducts were identified with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in product ion scan mode. Carbofuran was chosen as the target to establish the detection method of carbamate toxicant exposure based on methylcarbamyl nonapeptide digested from methylcarbamyl BChE. Procainamide-gel affinity purification, pepsin digestion and UHPLC-MS/MS analysis in multiple reaction monitoring (MRM) mode were applied. Under the optimized conditions of sample preparation and UHPLC-MS/MS MRM analysis, the limits of detection (LODs) reached 10.0 ng/mL of plasma exposed to carbofuran with satisfactory specificity. The quantitation approach was established with d3-carbofuran-exposed plasma as the internal standard (IS) and the linearity range was 30.0-1.00 × 103 nmol/L (R2 >0.998) with the accuracy of 95.6%-107% and precision of ≤9% relative standard deviation (RSD). The applicability was also evaluated by N,N-dimethyl-carbamates with the LODs of 30.0 nmol/L for pirimicarb-exposed plasma based on dimethylcarbamyl nonapeptide. Because most of carbamate toxicants has methylcarbamyl or dimethylcarbamyl groups, this approach could be applied on the retrospective screening of carbamate toxicant exposure including CMNAs, carbamate pesticides or carbamate drugs. This study could provide an effective means in the fields of CWC verification, toxicological mechanism investigation and down-selection of potential treatment options.

Amaryllidaceae Alkaloids from Clivia miniata (Lindl.) Bosse (Amaryllidaceae): Isolation, Structural Elucidation, and Biological Activity.

Clivia miniata (Amaryllidaceae) is an herbaceous evergreen flowering plant that is endemic to South Africa and Swaziland and belongs to one of the top-10 traded medicinal plants in informal medicine markets in South Africa. The species has been reported as the most important component of a traditional healer's pallet of healing plants. Eighteen known Amaryllidaceae alkaloids (AAs) of various structural types, and one undescribed alkaloid of homolycorine-type, named clivimine B (3), were isolated from Clivia miniata. The chemical structures of the isolated alkaloids were elucidated by a combination of MS, HRMS, 1D and 2D NMR techniques and by comparison with literature data. Compounds isolated in a sufficient quantity, and not tested previously, were evaluated for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BuChE; E.C. 3.1.1.8) inhibition activities.

Kinetic and structural studies on the inhibition of acetylcholinesterase and butyrylcholinesterase by a series of multitarget-directed galantamine-peptide derivatives.

Complex neurological disorders, including Alzheimer's disease, are one of the major therapeutic areas to which multitarget drug discovery strategies have been applied in the last twenty years. Due to the complex multifactorial etiopathogenesis of Alzheimer's disease, it has been proposed that to be successful the pharmaceutical agents should act on multiple targets in order to restore the complex disease network and to provide disease modifying effects. Here we report on the synthesis and the anticholinergic activity profiles of seven multitarget anti-Alzheimer compounds designed by combining galantamine, a well-known acetylcholinesterase inhibitor, with different peptide fragments endowed with inhibitory activity against BACE-1. A complementary approach based on molecular docking simulations of the galantamine-peptide derivatives in the active sites of acetylcholinesterase and of the related butyrylcholinesterase, as well as on inhibition kinetics, by global fitting of the reaction progress curves, allowed to gain insights into the enzyme-inhibitor mechanism of interaction. The resulting structure-activity relationships pave the way towards the design of more effective pharmacodynamic/pharmacokinetic multitarget inhibitors.

In Vitro and In Silico Evaluation of Cholinesterase Inhibition by Alkaloids Obtained from Branches of Abuta panurensis Eichler.

Alkaloids are natural products known as ethnobotanicals that have attracted increasing attention due to a wide range of their pharmacological properties. In this study, cholinesterase inhibitors were obtained from branches of Abuta panurensis Eichler (Menispermaceae), an endemic species from the Amazonian rainforest. Five alkaloids were isolated, and their structure was elucidated by a combination of 1D and 2D 1H and 13C NMR spectroscopy, HPLC-MS, and high-resolution MS: Lindoldhamine isomer m/z 569.2674 (1), stepharine m/z 298.1461 (2), palmatine m/z 352.1616 (3), 5-N-methylmaytenine m/z 420.2669 (4) and the N-trans-feruloyltyramine m/z 314.1404 (5). The compounds 1, 3, and 5 were isolated from A. panurensis for the first time. Interaction of the above-mentioned alkaloids with acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes was investigated in silico by molecular docking and molecular dynamics. The molecules under investigation were able to bind effectively with the active sites of the AChE and BChE enzymes. The compounds 1-4 demonstrated in vitro an inhibitory effect on acetylcholinesterase with IC50 values in the range of 19.55 µM to 61.24 µM. The data obtained in silico corroborate the results of AChE enzyme inhibition.

Monoterpene indole alkaloids from Vinca minor L. (Apocynaceae): Identification of new structural scaffold for treatment of Alzheimer's disease.

One undescribed indole alkaloid together with twenty-two known compounds have been isolated from aerial parts of Vinca minor L. (Apocynaceae). The chemical structures of the isolated alkaloids were determined by a combination of MS, HRMS, 1D, and 2D NMR techniques, and by comparison with literature data. The NMR data of several alkaloids have been revised, corrected, and missing data have been supplemented. Alkaloids isolated in sufficient quantity were screened for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BuChE; E.C. 3.1.1.8) inhibitory activity. Selected compounds were also evaluated for prolyl oligopeptidase (POP; E.C. 3.4.21.26), and glycogen synthase 3ß-kinase (GSK-3ß; E.C. 2.7.11.26) inhibition potential. Significant hBuChE inhibition activity has been shown by (-)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole with an IC50 value of 0.65 ± 0.16 µM. This compound was further studied by enzyme kinetics, along with in silico techniques, to reveal the mode of inhibition. This compound is also predicted to cross the blood-brain barrier (BBB) through passive diffusion.

Anti-aging and antioxidant of four traditional malaysian plants using simplex centroid mixture design approach.

Aging is a naturally biological process with adverse effects. The continuous accumulation of reactive oxygen species (ROS) trigger cellular and tissue damage by activating several aging enzymes. The antioxidant properties of traditional medicinal plants used by Jakun aborigine's community are a promising approach to alleviate aging process and prevent Alzheimer. The aim of the current investigation was to optimize a novel anti-aging formulation from traditional plants (Cnestis palala stem, Urceola micrantha stem, Marantodes pumilum stem and Microporus xanthopus fruiting bodies) using simplex centroid mixture design (SCMD). After selecting the optimal formulations based on desirability function of antioxidant activity (DPPH, ABTS ˙ + and FRAP), they were further examined against the activity of aging-related-enzymes (collagenase, tyrosinase, acetyl- and butyrylcholinesterase). The single extracts of C. palala, U. micrantha and the binary mixture of C. palala and U. micrantha were the optimal formulations with high antioxidant activities. Single extract of U. micrantha showed the highest inhibition towards matrix metalloproteinase-1 (49.44 ± 4.11 %), while C. palala water extract showed highest inhibitions towards tyrosinase (14.06 ± 0.31%), acetylcholinesterase (32.92 ± 2.13%) and butyrylcholinesterase (34.89 ± 2.84%) enzymes. The single extracts of C. palala and U. micrantha displayed better activity as compared to the binary mixture formulation. In conclusion, these findings could be a baseline for further exploration of novel anti-aging agents from natural resources.

Possible Role of Butyrylcholinesterase in Fat Loss and Decreases in Inflammatory Levels in Patients with Multiple Sclerosis after Treatment with Epigallocatechin Gallate and Coconut Oil: A Pilot Study.

(1) Background. Multiple sclerosis (MS) is characterised by the loss of muscle throughout the course of the disease, which in many cases is accompanied by obesity and related to inflammation. Nonetheless, consuming epigallocatechin gallate (EGCG) and ketone bodies (especially ß-hydroxybutyrate (ßHB)) produced after metabolising coconut oil, have exhibited anti-inflammatory effects and a decrease in body fat. In addition, butyrylcholinesterase (BuChE), seems to be related to the pathogenesis of the disease associated with inflammation, and serum concentrations have been related to lipid metabolism. Objective. The aim of the study was to determine the role of BuChE in the changes caused after treatment with EGCG and ketone bodies on the levels of body fat and inflammation state in MS patients. (2) Methods. A pilot study was conducted for 4 months with 51 MS patients who were randomly divided into an intervention group and a control group. The intervention group received 800 mg of EGCG and 60 mL of coconut oil, and the control group was prescribed a placebo. Fat percentage and concentrations of the butyrylcholinesterase enzyme (BuChE), paraoxonase 1 (PON1) activity, triglycerides, interleukin 6 (IL-6), albumin and ßHB in serum were measured. (3) Results. The intervention group exhibited significant decreases in IL-6 and fat percentage and significant increases in BuChE, ßHB, PON1, albumin and functional capacity (determined by the Expanded Disability Status Scale (EDSS)). On the other hand, the control group only exhibited a decrease in IL-6. After the intervention, BuChE was positively correlated with the activity of PON1, fat percentage and triglycerides in the intervention group, whereas these correlations were not observed in the control group (4). Conclusions. BuChE seems to have an important role in lipolytic activity and the inflammation state in MS patients, evidenced after administering EGCG and coconut oil as a ßHB source.

Effect of fish paste products, fish balls 'tsumire', intake in Sprague-Dawley rats.

The fish paste product, fish balls 'tsumire', is a traditional type of Japanese food made from minced fish as well as imitation crab, kamaboko and hanpen. Although tsumire is known as a high-protein and low-fat food, there is a lack of scientific evidence on its health benefits. Hence, we aimed to investigate the effects of tsumire intake on organ weight and biomarker levels in Sprague-Dawley rats for 84 d as a preliminary study. Six-week-old male Sprague-Dawley rats were divided into two groups: group I, fed normal diets, and group II, fed normal diets with 5 % dried tsumire. Throughout the administration period, we monitored their body weight and food intake; at the end of this period, we measured their organ weight and analysed their blood biochemistry. No significant differences were observed with respect to body weight, food intake, organ weight and many biochemical parameters between the two groups. It was found that inorganic phosphorus and glucose levels were higher in group II rats than in group I rats. On the other hand, sodium, calcium, amylase and cholinesterase levels were significantly lower in group II than in group I. Interestingly, we found that the levels of aspartate aminotransferase, alanine transaminase, lactate dehydrogenase and leucine aminopeptidase in group II were significantly lower than in group I, and that other liver function parameters of group II tended to be lower than in group I. In conclusion, we consider that the Japanese traditional food, 'tsumire,' may be effective as a functional food for human health management worldwide.

Pharmacophore-based drug design of AChE and BChE dual inhibitors as potential anti-Alzheimer's disease agents.

For the Alzheimer's disease (AD) with complex pathogenesis, single target drugs represent one of the most effective therapeutic strategies in clinical. However, the traditional concept of "a disease, a target" is difficult to find very effective drugs, and multi-target drugs have already become new hot spot in drug development for this disease. In our present study, our efforts toward discovering new cholinesterase (ChE) inhibitors aided by computational methods will provide useful information as anti-AD agents in the future. The best 3D-QSAR acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors pharmacophore hypotheses Hypo1 A and Hypo1 B were generated and validated by HypoGen program in Discovery Studio 2016 based on the training set of flavonoids, and then they were used as 3D query for screening the ZINC database. Next, the hit molecules were then subjected to the ADMET and molecular docking study to prioritize the compounds. Finally, 6 compounds showed good estimated activities and promising ADMET properties. The result of best compound ZINC08751495 with AChE estimate activity (0.028), BChE estimate activity (1.55), AChE fit value (9.369), BChE fit value (8.415), AChE -CDOCKER ENERGY (30.22), BChE -CDOCKER ENERGY (33.13) has the potential for further development as a supplement to treat Alzheimer's disease.

In silico, theoretical biointerface analysis and in vitro kinetic analysis of amine compounds interaction with acetylcholinesterase and butyrylcholinesterase.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are considered important target for drug design against Alzheimer's disease. In the present study in silico analysis; theoretical analysis of biointerface between ligand and interacting amino acid residues of proteins; and in vitro analysis of enzyme inhibition kinetics were carried out to delineate the inhibitory property of amine compounds against AChE/BChE. High throughput virtual screening of amine compounds identified three compounds (2-aminoquinoline, 2-aminobenzimidazole and 2-amino-1-methylbenzimidazole) that best interacted with AChE/BChE. Molecular docking analysis revealed the interaction of these compounds in the active site gorge of AChE/BChE, in particular with amino acid residues present in the peripheral anionic site. Molecular dynamics simulation confirmed the stable binding of these compounds with AChE/BChE. Binding energy calculated through MMGBSA method identified the non-covalent interactions (electrostatic and Van der Waals interactions) have contributed to the stable binding of the amine compounds with the AChE/BChE. Biointerface between amine compounds and AChE/BChE were visualized through Hirshfeld surface analysis. The inter-fragment interaction energies for the possible contacts between amine compounds and amino acid residues were carried out for the first time. All the amine compounds showed mixed-type of inhibition with moderate Ki value in in vitro analysis.

Amygdalus spinosissima root extract enhanced scopolamine-induced learning and memory impairment in mice.

The Amygdalus spinosissima (Rosaceae) plant has been used in the Iranian folk medicine as a remedy for the burn wound. Hence, in this study, we aimed to determine the possible medicinal potential of the plant focusing on the root part. The bioactive phenolic and flavonoid compounds present in the root extract of the Amygdalus spinosissima plant as well as its antioxidant and anti-inflammatory properties were determined. Moreover, the effects of root extract on learning and memory in mice were evaluated. The results revealed that the root methanolic extract contained phenolic and flavonoid compounds including apigenin, quercetin, rutin, kaempferol, gallic acid, syringic acid, ferulic acid, and ellagic acid. The extract possessed antioxidant, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibitory activities in vitro. These biological activities were attributed to the presence of phenolics and flavonoids. The A. spinosissima root extract improved learning and memory function in scopolamine-induced memory dysfunction in mice as determined using the Morris water maze task. The extract modulated the AChE, BChE, and inflammatory genes and enhanced the expression of the antioxidant enzymes in the brain. Consequently, A. spinosissima root extract could be considered as a promising source of potent bioactive compounds in the retarding the development of neurodegenerative diseases such as Alzheimer's disease.

UPLC-PDA-ESI-QTOF-MS/MS fingerprint of purified flavonoid enriched fraction of Bryophyllum pinnatum; antioxidant properties, anticholinesterase activity and in silico studies.

CONTEXT: Bryophyllum pinnatum (Lam.) Oken (Crassulaceae) is used traditionally to treat many ailments. OBJECTIVES: This study characterizes the constituents of B. pinnatum flavonoid-rich fraction (BPFRF) and investigates their antioxidant and anticholinesterase activity using in vitro and in silico approaches. MATERIALS AND METHODS: Methanol extract of B. pinnatum leaves was partitioned to yield the ethyl acetate fraction. BPFRF was isolated from the ethyl acetate fraction and purified. The constituent flavonoids were structurally characterized using UPLC-PDA-MS2. Antioxidant activity (DPPH), Fe2+-induced lipid peroxidation (LP) and anticholinesterase activity (Ellman's method) of the BPFRF and standards (ascorbic acid and rivastigmine) across a concentration range of 3.125-100 µg/mL were evaluated in vitro for 4 months. Molecular docking was performed to give insight into the binding potentials of BPFRF constituents against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). RESULTS: UPLC-PDA-MS2 analysis of BPFRF identified carlinoside, quercetin (most dominant), luteolin, isorhamnetin, luteolin-7-glucoside. Carlinoside was first reported in this plant. BPFRF significantly inhibited DPPH radical (IC50 = 7.382 ± 0.79 µg/mL) and LP (IC50 = 7.182 ± 0.60 µg/mL) better than quercetin and ascorbic acid. Also, BPFRF exhibited potent inhibition against AChE and BuChE with IC50 values of 22.283 ± 0.27 µg/mL and 33.437 ± 1.46 µg/mL, respectively compared to quercetin and rivastigmine. Docking studies revealed that luteolin-7-glucoside, carlinoside and quercetin interact effectively with crucial amino acid residues of AChE and BuChE through hydrogen bonds. DISCUSSION AND CONCLUSIONS: BPFRF possesses an excellent natural source of cholinesterase inhibitor and antioxidant. The material could be further explored for the potential treatment of oxidative damage and cholinergic dysfunction in Alzheimer's disease.

Design and synthesis of garlic-related unsymmetrical thiosulfonates as potential Alzheimer's disease therapeutics: In vitro and in silico study.

Garlic contains a wide range of organosulfur compounds, which exhibit a broad spectrum of biological activities. Amongst the sulfur-containing compounds in garlic, the thiosulfonates are considerably popular in various fields. In light of this, we decided to investigate the enzyme inhibition ability of thiosulfonates. In this paper, the synthesis and biological activity of a small library of unsymmetrical thiosulfonates as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are described. The activity evaluation revealed nanomolar IC50 and Ki values against both enzymes tested. Furthermore, molecular docking studies allowed for the determination of possible binding interactions between the thiosulfonates and AChE.

Phenotype and genotype characteristics of 58 patients showing a prolonged effect of succinylcholine: A four-year experience.

INTRODUCTION: This study sought to describe the phenotype and genotype characteristics of patients referred to our laboratory to undergo further assessment due to a suspicion of a prolonged effect of suxamethonium attributed to BChE deficiency. METHODS: All patients referred to our laboratory from January 2016 to December 2019 due to the suspicion of a prolonged effect of suxamethonium were included in this study. The determination of BChE activity and genotyping using complete nucleotide sequencing of the entire complementary DNA-coding region with flanking intron-exon boundaries were completed. RESULTS: During this four-year period, 58 patients were referred to our laboratory for the investigation of prolonged neuromuscular block due to BChE deficiency. Among them, 52 showed a BChE deficiency related to BCHE gene mutations. The most commonly detected genotype was compound homozygous atypical variant (p.Asp98Gly)/homozygous Kalow variant (p.Ala569Thr) (p.[Asp98Gly;Ala567Thr];[p.Asp98Gly;Ala567Thr]). Further, we recorded four new BCHE variants, which seem to be associated with prolonged post suxamethonium apnoea: p.(Trp205Cys), p.(Leu222His), p.(Glu469Gln), and p.(Lys276Ter). CONCLUSION: During a four-year period, among the 58 patients referred to our laboratory, we have found four new BCHE variants, which seem to be associated with prolonged post suxamethonium apnoea (p.(Trp205Cys), p.(Leu22His), p.(Glu469Gln), and p.(Lys276Ter)).

Comparative effects of berberine and piperine on the neuroprotective potential of neostigmine.

OBJECTIVES: This study examined effect of berberine and piperine on neuroprotective potential of neostigmine in the management of neurological disorders. METHODS: Berberine and neostigmine were weighed (30 g), dissolved in distilled water (30 mL) separately, while, 30 mg piperine was dissolved in ethanol (0.45 mL), made up to 30 mL with distilled water. Antioxidant activities in 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH), 2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonate) radical (ABTS), Fe-chelation, ferric reducing properties (FRAP), nitric oxide (NO) and hydroxyl (OH) radical scavenging abilities and Fe2+, cisplatin and sodium nitroprusside (SNP) induced lipid peroxidation (LPO), and acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and monoamine oxidase (MAO) activities were assessed in vitro. RESULTS: The result revealed that tested compounds inhibited enzymes activities dose-dependently. However, berberine (IC50=0.17 mg/mL) had slight higher AChE inhibitory effect than piperine and neostigmine (p<0.05). Also, berberine had the highest BChE inhibitory effect (IC50=0.16 mg/mL) while piperine exhibited the highest MAO inhibitory effect (IC50=0.21 mg/mL). Berberine, piperine and neostigmine exhibited high antioxidant properties and inhibited Fe2+, cisplatin and SNP induced LPO. CONCLUSIONS: Both alkaloids demonstrated antiradical scavenging ability comparable to neostigmine action against Alzheimer's disease (AD). The modulatory and antioxidant berberine and piperine properties on these enzymes (AChE, BChE and MAO) could be possible underlying mechanisms in employing these compounds as a complementary therapy in neurodegenerative diseases (NDDs) management.

Molecular Modeling Studies on the Multistep Reactivation Process of Organophosphate-Inhibited Acetylcholinesterase and Butyrylcholinesterase.

Poisoning with organophosphorus compounds used as pesticides or misused as chemical weapons remains a serious threat to human health and life. Their toxic effects result from irreversible blockade of the enzymes acetylcholinesterase and butyrylcholinesterase, which causes overstimulation of the cholinergic system and often leads to serious injury or death. Treatment of organophosphorus poisoning involves, among other strategies, the administration of oxime compounds. Oximes reactivate cholinesterases by breaking the covalent bond between the serine residue from the enzyme active site and the phosphorus atom of the organophosphorus compound. Although the general mechanism of reactivation has been known for years, the exact molecular aspects determining the efficiency and selectivity of individual oximes are still not clear. This hinders the development of new active compounds. In our research, using relatively simple and widely available molecular docking methods, we investigated the reactivation of acetyl- and butyrylcholinesterase blocked by sarin and tabun. For the selected oximes, their binding modes at each step of the reactivation process were identified. Amino acids essential for effective reactivation and those responsible for the selectivity of individual oximes against inhibited acetyl- and butyrylcholinesterase were identified. This research broadens the knowledge about cholinesterase reactivation and demonstrates the usefulness of molecular docking in the study of this process. The presented observations and methods can be used in the future to support the search for new effective reactivators.