Determination of Enzyme Inhibition Potential and Anticancer Effects of Pistacia khinjuk Stocks Raised in In Vitro and In Vivo Conditions

In this study, antihypertensive, anticholinesterase, antiurease, antityrosinase and antielastase enzyme inhibition and anticancer activities of in vivo (male and female) and in vitro samples (root, stem and leaf parts) of the Pistacia khinjuk Stocks were investigated comparatively. In this context, in vitro shoot cultures were obtained from germinated mature seeds. Then, the juvenile shoots were proliferated in Murashige and Skoog (MS) medium supplemented with 1 mg/L 6-Benzylaminopurine (BAP). In terms of anticancer activity, the whole of the samples studied was found to have apoptotic effects against MCF-7 (breast cancer) and HT-29 (colon cancer) cell lines. The extracts obtained from in vivo female root parts showed better cytotoxicity than all the other tested extracts on MCF-7 (IC50: 31.86 ± 1.40 µg/mL) and HT-29 cell series (IC50: 59.60 ± 0.69 µg/mL). Even though all the samples showed a strong butyrylcholinesterase enzyme inhibition (BChE) activity, it was detected that none of the samples had shown acetylcholinesterase enzyme inhibition (AChE). It was also determined that in vivo leaf samples of female trees had the highest BChE activity (Inhibition%: 75.20 ± 1.50). All the samples showed a low-moderate level of urease and tyrosinase enzyme activity, while in vivo samples showed a significant level of the elastase enzyme activities (Inhibition%: 58.72 for female root extracts; 58.25 for female leaf extracts, at 50 µg/mL concentration), and they were more active than the standard oleanolic acid (Inhibition%: 39.46 ± 0.52). The antihypertensive activities as the inhibition of angiotensin I-converting enzyme (ACE) of in vivo samples (Inhibition%: 95.88 for female stem extracts; 95.18 for female root extracts) were detected as close to the standard (Inhibition%: 96.64 ± 1.85) used. In general, it can be stated that in vivo samples had higher biological activities compared to in vitro ones. Consequently, according to our results, it was concluded that in vitro stem parts of khinjuk pistachio could also be evaluated as an alternative new antihypertensive, antielastase and anticancer agent source.

A Bioorthogonally Synthesized and Disulfide-Containing Fluorescence Turn-On Chemical Probe for Measurements of Butyrylcholinesterase Activity and Inhibition in the Presence of Physiological Glutathione

Butyrylcholinesterase (BChE) is a biomarker in human blood. Aberrant BChE activity has been associated with human diseases. Here we developed a fluorescence resonance energy transfer (FRET) chemical probe to specifically quantify BChE activity in serum, while simultaneously discriminating against glutathione (GSH). The FRET chemical probe 11 was synthesized from a key trifunctional bicyclononyne exo-6 and derivatives of 5-(2-aminoethylamino)-1-naphthalenesulfonic acid (EDANS) and 4-[4-(dimethylamino)phenylazo]benzoic acid (DABCYL). EDANS fluorescence visualization and kinetic analysis of 11 in the presence of diverse compounds confirmed the outstanding reactivity and specificity of 11 with thiols. The thiol-dependent fluorescence turn-on property of 11 was attributed to a general base-catalyzed SN2 nucleophilic substitution mechanism and independent of metal ions. Moreover, all thiols, except GSH, reacted swiftly with 11. Kinetic studies of 11 in the presence of covalently modified GSH derivatives corroborated that the steric hindrance of 11 imposing on GSH was the likely cause of the distinguished reactivity. Since GSH commonly interferes in assays measuring BChE activity in blood samples, the 11-based fluorescent assay was employed to directly quantify BChE activity without GSH interference, and delivered a linear range of 4.3–182.2 U L−1 for BChE activity with detection limit of 4.3 U L−1, and accurately quantified serum BChE activity in the presence of 10 μM GSH. Finally, the 11-based assay was exploited to determine Ki of 5 nM for tacrine inhibition on BChE catalysis. We are harnessing the modulated characteristics of 6 to synthesize advanced chemical probes able to more sensitively screen for BChE inhibitors and quantify BChE activity in serum.

Decreased Activity of Circulating Butyrylcholinesterase in Blood Is an Independent Prognostic Marker in Pancreatic Cancer Patients

Introduction: The activity of butyrylcholinesterase (BChE) in blood reflects liver function and has recently been associated with systemic inflammatory response and tumor cachexia. As these conditions have been previously linked with pancreatic cancer (PC), the purpose of the present study was to evaluate the prognostic impact of plasma BChE in PC. Methods: Data from 574 consecutive PC patients, treated between 2004 and 2018 at a single academic center, was evaluated. The primary endpoint was cancer-specific survival (CSS), analyzed by Kaplan–Meier curve, and both univariate and multivariate Cox proportional models. Results: BChE activity negatively correlated with other liver parameters (bilirubin, gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and C-reactive protein (CRP)), and positively correlated with albumin levels, respectively (p < 0.01). In univariate analysis, a low plasma BChE activity was a factor of poor CSS (hazard ratio: 1.4, 95% confidence interval: 1.129–1.754, p = 0.002). In multivariate analysis, tumor stage, tumor grade, administration of chemotherapy, bilirubin levels and a low BChE activity (hazard ratio: 1.42, 95% confidence interval: 1.10–1.82; p = 0.006) were identified as independent prognostic factors. Conclusion: Decreased activity of BChE in blood plasma predicts shorter survival time in PC patients. Therefore, BChE might be helpful in additional stratification of patients into different prognostic risk groups.

Early diagnosis of nerve agent exposure with a mobile test kit and implications for medical countermeasures: a trigger to react

Recent uses of nerve agents underline the need of early diagnosis as trigger to react (initiating medical countermeasures, avoiding cross-contamination). As organophosphorus (OP) pesticide poisoning exerts the same pathomechanism, that is, inhibition of the pivotal enzyme acetylcholinesterase (AChE), a portable cholinesterase (ChE) test kit was applied in an emergency room for rapid diagnosis of OP poisoning. OP nerve agents or pesticides result in the inhibition of AChE. As AChE is also expressed on erythrocytes, patient samples are easily available. However, in most clinics only determination of plasma butyrylcholinesterase (BChE) is established which lacks a pathophysiological correlate, shows higher variability in the population and behaves different regarding inhibition by OP and reactivation by oximes. The ChE test kit helped to diagnose atypical cases of OP poisoning, for example, missing of typical muscarinic symptoms, and resulted in administration of pralidoxime, the oxime used in Serbia. The ChE test kit also allows an initial assessment whether an oxime therapy is successful. In one case report, AChE activity increased after oxime administration indicating therapeutic success whereas BChE activity did not. With only BChE at hand, this therapeutic effect would have been missed. As inhibition of AChE or BChE activity is determined, the CE-certified device is a global diagnostic tool for all ChE inhibitors including carbamates which might also be misused as chemical weapon. The ChE test kit is a helpful point-of-care device for the diagnosis of ChE inhibitor poisoning. Its small size and easy menu-driven use advocate procurement where nerve agent and OP pesticide exposure are possible.

Combining Chalcones with Donepezil to Inhibit Both Cholinesterases and Aβ Fibril Assembly

The fact that the number of people with Alzheimer’s disease is increasing, combined with the limited availability of drugs for its treatment, emphasize the need for the development of novel effective therapeutics for treating this brain disorder. Herein, we focus on generating 12 chalcone-donepezil hybrids, with the goal of simultaneously targeting amyloid-β (Aβ) peptides as well as cholinesterases (i.e., acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)). We present the design, synthesis, and biochemical evaluation of these two series of novel 1,3-chalcone-donepezil (15a–15f) or 1,4-chalcone-donepezil (16a–16f) hybrids. We evaluate the relationship between their structures and their ability to inhibit AChE/BChE activity as well as their ability to bind Aβ peptides. We show that several of these novel chalcone-donepezil hybrids can successfully inhibit AChE/BChE as well as the assembly of N-biotinylated Aβ(1–42) oligomers. We also demonstrate that the Aβ binding site of these hybrids differs from that of Pittsburgh Compound B (PIB).

Terpenes and Phenylpropanoids as Acetyl- and Butyrylcholinesterase Inhibitors: A Comparative Study

Background: Cholinesterase inhibitors are routinely applied in the treatment of Alzheimer’s disease, and seeking new cholinesterase inhibitors is a priority. Objective: Twenty seven compounds were compared, including ones not previously tested. An attempt was undertaken to precisely describe the role of alcohol in the inhibitory activity. This paper underlines the role of a “false positive” blank sample in the routine analysis. Methods: The inhibition of cholinesterase was measured using Ellman’s colorimetric method with a few modifications designed by the authors (including the “false-positive” effect). The inhibitory role of ethanol and methanol was also carefully evaluated. The present and past results were compared taking the source of enzyme and alcohol content into consideration. Results: For the first time, new inhibitors were identified, namely: methyl jasmonate, 1R-(−)-nopol ((anti-acetyl-(AChE) and butyrylcholinesterase (BChE) activity)) and 1,4-cineole, allo-aromadendrene, nerolidol, β-ionone, and (R)-(+)-pulegone (anti-BChE activity). Oleanolic acid and (+)-β-citronellene (not previously studied) proved to be inefficient inhibitors. For a number of well-known inhibitors (such as nerol, (−)-menthol, (+)-menthol, isoborneol, (−)-bornyl acetate, limonene, α-pinene, β-pinene, α- ionone, and eugenol) some serious discrepancies were observed between our findings and the results of previous studies. Ethanol and methanol showed no anti-AChE activity up to 0.29% (v/v) and 0.23% (v/v), respectively. Similarly, ethanol up to 0.33% (v/v) and methanol up to 0.29% (v/v) did not inhibit the activity of BChE. Conclusion: It can be stated that the impact of alcohol should be precisely determined and that blank “false-positive” samples should be processed together with test samples. Furthermore, the effect of the enzyme origin on the result of this test must be taken into consideration.

A randomized crossover trial assessing the effects of acute exercise on appetite, circulating ghrelin concentrations, and butyrylcholinesterase activity in normal-weight males with variants of the obesity-linked FTO rs9939609 polymorphism

ABSTRACT Background The fat mass and obesity-associated gene (FTO) rs9939609 A-allele is associated with higher acyl-ghrelin (AG) concentrations, higher energy intake, and obesity, although exercise may mitigate rs9939609 A-allele–linked obesity risk. Butyrylcholinesterase (BChE) hydrolyzes AG to des-acyl-ghrelin (DAG), potentially decreasing appetite. However, the effects of the FTO rs9939609 genotype and exercise on BChE activity, AG, DAG, and energy intake are unknown. Objective We hypothesized that individuals homozygous for the obesity-risk A-allele (AAs) would exhibit higher postprandial AG and energy intake than individuals homozygous for the low obesity-risk T-allele (TTs), but that exercise would increase BChE activity and diminish these differences. Methods Twelve AA and 12 TT normal-weight males completed a control (8 h rest) and an exercise (1 h of exercise at 70% peak oxygen uptake, 7 h rest) trial in a randomized crossover design. A fixed meal was consumed at 1.5 h and an ad libitum buffet meal at 6.5 h. Appetite, appetite-related hormones, BChE activity, and energy intake were assessed. Results AAs displayed lower baseline BChE activity, higher baseline AG:DAG ratio, attenuated AG suppression after a fixed meal, and higher ad libitum energy intake compared with TTs [effect sizes (ESs) ≥ 0.72, P ≤ 0.049]. Exercise increased Δ BChE activity in both genotypes (ESs = 0.37, P = 0.004); however, exercise lowered AG and the AG:DAG ratio to a greater extent in AAs (P ≤ 0.023), offsetting the higher AG profile observed in AAs during the control trial (ESs ≥ 1.25, P ≤ 0.048). Exercise did not elevate energy intake in either genotype (P = 0.282). Conclusions Exercise increases BChE activity, suppresses AG and the AG:DAG ratio, and corrects the higher AG profile observed in obesity-risk AA individuals. These findings suggest that exercise or other methods targeting BChE activity may offer a preventative and/or therapeutic strategy for AA individuals. This trial was registered at clinicaltrials.gov as NCT03025347.

Drop of Butyrylcholinesterase Activity after Cyclophosphamide Conditioning as a Predictive Marker of Liver Transplant-Related Complications and Its Correlation with Transplant-Related Mortality in Pediatric Hematopoietic Stem Cell Recipients

Transplant-related liver complications are a potentially fatal condition of hematopoietic stem cell transplantation (HSCT) in pediatric patients, actually representing one of the main factors involved in transplant-related mortality (TRM). The search for a specific marker capable of predicting the development of this condition is a relevant clinical issue. We have observed a variable reduction in serum butyrylcholinesterase (BChE) activity after a cyclophosphamide-containing conditioning regimen. This study aims to determine the cutoff of BChE activity reduction that might be a specific prognostic marker for liver complications after HSCT. Our results show that the reduction of BChE values below 2000 U/L the day before the transplantation is an indicator strongly associated with the transplant-related liver complications (p < 0.0001). The incidence of overall survival at 1 year was significantly higher in the BChE > 2000 U/L group compared to the BChE < 2000 U/L group (84.7% versus 58.5%, p < 0.001), while the TRM rate was significantly lower (8.1% versus 23.1%, p < 0.05). None of the patients undergoing prophylaxis with defibrotide developed severe liver complications. Starting defibrotide treatment at the first signs of hepatic dysfunction in patients with particularly low BChE activity levels reduces severe liver transplant-related complications.

In Vivo Effects of Neostigmine and Physostigmine on Neutrophil Functions and Evaluation of Acetylcholinesterase and Butyrylcholinesterase as Inflammatory Markers during Experimental Sepsis in Rats

Introduction. Recent studies have shown that acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) may serve as important diagnostic and therapeutic targets in sepsis. Since polymorphonuclear neutrophils (PMNs) play a pivotal role in the early phase of sepsis, we evaluated the potential therapeutic effects of cholinesterase inhibitors on PMN functions during cecal ligation and puncture- (CLP-) induced sepsis and investigated the roles of AChE and BChE as inflammatory markers under standardized experimental conditions.Methods. Sham surgery or CLP was performed in male Wistar rats (n=60). Animals were randomized into four groups: physostigmine, 100 μg/kg; neostigmine, 75 μg/kg; 0.9% saline (control group); and sham group, each applied four times over 24 h. The levels of reactive oxygen species (ROS) production and CD11b/CD62l expression were quantified by flow cytometry att=0, 6, 15, 20, and 24 h. Blood gas analysis as well as AChE and BChE activity levels was measured by validated point-of-care measurements. Clinical scores and survival times were determined.Results. CLP induced a significant increase in ROS production and CD11b upregulation by rat PMNs. Treatment with physostigmine or neostigmine significantly reduced ROS production and CD11b upregulation by PMNs 20 h after CLP induction. In physostigmine-treated animals, survival times were significantly improved compared to the control animals, but not in neostigmine-treated animals. While AChE activity significantly decreased in the control animals att>6 h, AChE activity did not change in the sham group. BChE activity decreased att>20 hin the control animals.Conclusion. While AChE activity may serve as an acute inflammatory marker, BChE activity shows a delayed decrease. Administration of centrally acting physostigmine in CLP-induced sepsis in rats has protective effects on PMN functions and improves survival times, which may be of interest in clinical practice.

Thiol–ene click reaction-induced fluorescence enhancement by altering the radiative rate for assaying butyrylcholinesterase activity

A fluorescent molecular probe for assaying BChE activity based on thiol-triggered fluorescence enhancement via a thiol–ene click reaction was developed.