Reversible inhibition of acetylcholinesterase by eseroline,an opioid agonist structurally related to physostigmine (eserine) and morphine

The action of eseroline—(3aS, 8aR)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo[2,3-b] indol-5-ol—salicylate was tested on preparations of ChE from different sources and on the longitudinal muscle of guinea-pig ileum. While eseroline is extremely weak-acting on horse serum BuChE (K_i = 208 ± 42 μM), it is a rather strong competitive inhibitor of AChE's, its K_i being 0.15 ± 0.08 μM, 0.22 ± 0.10 μM and 0.61 ± 0.12 μM in electric eel, human RBC and rat brain, respectively. Eseroline inhibitory action on AChE is independent of the duration of pre-incubation and appears fully developed in less than 15 sec. This action is also rapidly reversible; after pre-incubation followed by dilution, maximum enzymic activity is regained within 15 sec. The electrically-evoked contractions of the longitudinal strip were inhibited by concentrations of eseroline in the range 0.2–15 μM, while they were increased by concentrations over 20 μM. In the same preparation, without electrical stimulation, but in the presence of naloxone, eseroline induced contractions at concentrations higher than 5 μM. This effect was antagonized by atropine. The inhibitory activity of eseroline parallels, as regards selectivity, potency and kinetics, that of the phenolic anticurare agent edrophonium, while it differs markedly from that of physostigmine.     

In vitro assays of the antibacterial and antioxidant activity of aqueous leaf extracts from different Prunus salicina Lindl. cultivars

The growing interest in the substitution of synthetic food antioxidants and antimicrobial additives by natural ones has fostered research on vegetable sources and on the screening of raw materials, for identifying new antioxidants and antimicrobial natural agents. The aim of the present study was to assess total phenolic contents and determine polyphenolic composition, related antioxidative and antimicrobial properties of plum leaves extracts from six cultivars. It was observed that the content of total phenolic compounds was cultivar dependent. High antioxidant capacity has been observed and related to the relative amounts of polyphenolic compounds with good antioxidant properties. The antimicrobial activity was confirmed against Listeria innocua and Escherichia coli, and it has found to be related with the high phenolic contents. Our results suggest that the use of plum leaf extracts is a feasible alternative as antibacterial and antioxidant agents to prevent the deterioration of stored foods by bacteria and oxidation.     

Antioxidant and antiacetylcholinesterase activities of chard (Beta vulgaris L. var. cicla)

Plants have been used for many years as a source of traditional medicine to treat various diseases and conditions. Many of these medicinal plants are also excellent sources for phytochemicals, many of which contain potent antioxidant and antiacetylcholinesterase activities. Chard (Beta vulgaris L. var. cicla) is widely spread in Turkey and used as an antidiabetic in traditional medicine. In the present study, the antioxidant activity and acetylcholinesterase inhibitor capacity of chard were examined. In addition, proline level of chard was determined. The antioxidant activity of water extract of chard was evaluated using different antioxidant tests. The results were compared with natural and synthetic antioxidants. The results suggest that chard may provide a natural source of antioxidant and antiacetylcholinesterase activities and proline content.     

Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex-GS, 0.22 microm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax and Potter-Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 microM) or HI 6 (30 microM) of sarin-inhibited human muscle acetylcholinesterase (0.142+/-0.004 min(-1) and 0.166+/-0.008 min(-1), respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker.     

Comparative kinetics of organophosphates and oximes with erythrocyte, muscle and brain acetylcholinesterase

There is an ongoing debate whether oximes can effectively counteract the effects of organophosphorus compounds (OP) on brain acetylcholinesterase (AChE) activity and whether there are differences in the kinetic properties of brain and erythrocyte AChE. In order to investigate the kinetics of AChE from different tissues and species the well established dynamically working in vitro model with real-time determination of membrane-bound AChE activity was adapted for use with brain AChE. The enzyme reactor, that was loaded with brain, erythrocyte or muscle AChE, was continuously perfused with substrate and chromogen while AChE activity was on-line analyzed in a flow-through detector. It was possible to determine the Michaelis-Menten constants of human erythrocyte, muscle and brain AChE which were almost identical. In addition, the inhibition kinetics of sarin and paraoxon as well as the reactivation kinetics of obidoxime and HI 6 were determined with human, swine and guinea pig brain and erythrocyte AChE. It was found that the inhibition and reactivation kinetics of brain and erythrocyte AChE were highly comparable in all tested species. These data support the view that AChE from different tissue has similar kinetic properties and that brain AChE is comparably susceptible toward reactivation by oximes.     

Antioxidant and anti-neoplastic activities of Picrorhiza kurroa extracts

Picrorhizakurroa Royle ex Benth., a well-known traditional herb from the Scrophulariaceae family has a remarkable reputation among the indigenous medical practitioners. The antioxidant and anti-neoplastic activities of methanolic and aqueous extracts of P. kurroa rhizome were investigated in the present study. The total phenolic content was determined by a spectrophotometric method. The antioxidant efficacies of the extracts were studied employing radical scavenging assays (DPPH and OH), ferric reducing antioxidant property (FRAP) and thiobarbituric acid (TBA) assay for testing inhibition of lipid peroxidation. Furthermore, the cytotoxicity of the extracts was tested by XTT assay in MDA-MB-435S (human breast carcinoma), Hep3B (human hepatocellular carcinoma) and PC-3 (human prostate cancer) cell lines. The ability of the extracts to induce apoptosis was also investigated. Both extracts exhibited promising antioxidant potentials. The extracts were also observed to be cytotoxic at the tested dosage and were able to target cells towards apoptosis. The study concludes that P. kurroa possess diverse therapeutic potentials which might be useful in development of drugs or their precursors.     

Effects of organophosphorus compounds, O,O-dimethyl O-(2,2-dichlorovinyl)phosphate (DDVP) and O,O-dimethyl O-(3-methyl 4-nitrophenyl)phosphorothioate (fenitrothion), on brain acetylcholine content and acetylcholinesterase activity in Japanese quail

Effects of 2 organophosphorus compounds, O,O-dimethyl O-(2,2-dichlorovinyl)phosphate (DDVP) and O,O-dimethyl O-(3-methyl 4-nitrophenyl)phosphorothioate (fenitrothion), on the brain cholinergic system were investigated in Japanese quail. Cholinergic signs, such as salivation and convulsions in legs and wings, were seen 7–15 min after administration with DDVP (3–4 mg/kg) or 6–120 min after administration with fenitrothion (250–350 mg/kg). In the DDVP-treated quail (10 min after dosage of 3 mg/kg), free acetylcholine (ACh), labile-bound ACh, increased significantly and acetylcholinesterase (AChE) decreased to 28% of the value determined in untreated quail. In the fenitrothion-treated group (60 min after dosage of 300 mg/kg), only free ACh increased and AChE activity decreased to 20% of the control value. In vitro, DDVP and fenitrothion inhibited AChE activity in brain homogenate with an I₅₀ of 10^?8 M and 10^?5 M, respectively. It appeared that both organophosphorus compounds might have essentially the same effect on the brain cholinergic system. There were only small differences in the effect on various fractions of ACh between the 2 compounds, although there was a hundred-fold range in dose.     

Antioxidant,cytoprotective and antibacterial effects of Sea buckthorn (Hippophae rhamnoides L.) leaves

The present study was carried out to investigate the antioxidant, cytoprotective and antibacterial effects of aqueous and hydroalcoholic extracts of Hippophae rhamnoides L. (Sea buckthorn) (SBT) leaves by using various invitro systems and analysis of marker compounds by reverse phase-high performance liquid chromatography (RP-HPLC). The chemical composition of the leaf extracts was quantified by colorimetric reaction in terms of total phenol and flavonoids contents. Further, some of its bioactive phenolic constituents, such as quercetin-3-O-galactoside, quercetin-3-O-glucoside, kaempferol and isorhamnetin were also quantified in both SBT leaf extracts by RP-HPLC. The SBT leaf extracts exhibited potent antioxidant activity determined by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Further, both extracts were observed to have cytoprotective activity against hydrogen peroxide and hypoxanthine-xanthine oxidase induced damage to BHK-21 cell line. The SBT leaf extracts showed growth inhibiting effect against Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis. These observations suggest that aqueous and hydroalcoholic extracts of Sea buckthorn leaves have marked antioxidant, cytoprotective and antibacterial activities.     

Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes

The wide-spread use of organophosphorus compounds (OP) as pesticides and the availability of highly toxic OP-type chemical warfare agents (nerve agents) underlines the necessity for an effective medical treatment. Acute OP toxicity is primarily caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment, however, the commercially available compounds, obidoxime and pralidoxime, are considered to be rather ineffective against various nerve agents. The antidotal efficacy of new oximes is primarily tested in animals for ethical reasons. However, the various interactions between AChE, OP and oximes can be investigated with human AChE which enables the direct assessment of oxime potency, thus excluding species differences. The kinetics of inhibition, reactivation and aging were investigated with human erythrocyte AChE, various structurally different OP (organophosphates, -phosphonates and phosphoramidates) and oximes (obidoxime, pralidoxime, HI 6, HL? 7). The inhibitory potency of OPs, reactivating potency of oximes and spontaneous reactivation and aging were strongly affected by the structural characteristics of the OPs and of the phosphyl-AChE-complex. The kinetic data emphasize the superior inhibitory potency of organophosphonates. AChE inhibited by various phosphoramidates was mostly resistant towards reactivation by oximes while phosphonylated AChE was easily reactivated. HL? 7 was most potent with phosphonylated AChE and obidoxime with AChE inhibited by organophosphates and phosphoramidates. With the exception of soman, OP-inhibited AChE aged rather slowly (t(1/2) 3-231 h) and reactivated spontaneously with some compounds. These results indicate that there is obviously no direct structure-activity relationship for the various interactions of human AChE, OPs and oximes.     

Antioxidant and pro-oxidant properties of acylated pelargonidin derivatives extracted from red radish (Raphanus sativus var. niger,Brassicaceae)

The antioxidant and pro-oxidant potential of an extract from red radish, in which the major compounds were acylated pelargonidin derivatives, were assessed with a variety of assays in vitro. The extract appeared to form a complex with Fe³⁺ or Cu²⁺. It displayed a concentration-dependant reducing power (1.16 OD_{700 nm} at a concentration of 4 mM) and scavenging effect against 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals (with IC₅₀ = 1.74 ± 0.03 mM). It could promote the cleavage of plasmid DNA with Cu(II)/H₂O₂ or Cu(II) alone. This DNA damage could be inhibited by horseradish peroxidase, catalase, and EDTA, respectively. The extract also showed growth inhibition of Bel-7402 cells at lower concentration. The results suggested that the formation of reactive oxygen species might be involved in the mechanism of DNA damage. The acylated pelargonidin derivatives extracted from red radish could act as antioxidant and pro-oxidant and their antioxidant and pro-oxidant properties were relative to the reaction conditions. It might provide novel antioxidant and anticarcinogenic agents.     

Chemical composition,antimicrobial and antioxidant activities of the essential oils of Sideritis erythrantha Boiss. and Heldr. (var. erythrantha and var. cedretorum P.H. Davis) endemic in Turkey

In the present study, chemical compositions, antimicrobial and antioxidant activities of the essential oils of Sideritis erythrantha var. erythrantha (SE) and Sideritis erythrantha var. cedretorum (SC), which are endemic taxa in Turkey, were investigated. The essential oils obtained by hydrodistillation were analyzed by gas chromatography–mass spectrometry (GC–MS). α-Pinene was the major component of the essential oils of SC and SE. SC essential oil was as effective as antibiotic against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus faecalis (VRE), ampicillin resistant Haemophilusinfluenzae and vancomycin sensitive E. faecalis. Similarly, SE essential oil was also as effective as antibiotic against VRE and ampicillin resistant H. influenzae. Antioxidant activities of the essential oils of SC and SE were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH), β-carotene/linoleic acid and reducing power. Both essential oils exhibited weak antioxidant activity. This is the first report on antimicrobial and antioxidant activities of the essential oils of SC and SE.     

Improving the ex vivo stability of drug ester compounds in rat and dog serum: Inhibition of the specific esterases and implications on their identity

In drug development, it has been noticed that some drug compounds, especially esters, are unstable in serum samples ex vivo. This can lead to a substantial underestimation of the actual drug concentration.     

Medicinal properties of mangosteen (Garcinia mangostana)

Many tropical plants have interesting biological activities with potential therapeutic applications. Garcinia mangostana Linn. (GML) belongs to the family of Guttiferae and is named “the queen of fruits”. It is cultivated in the tropical rainforest of some Southeast Asian nations like Indonesia, Malaysia, Sri Lanka, Philippines, and Thailand. People in these countries have used the pericarp (peel, rind, hull or ripe) of GML as a traditional medicine for the treatment of abdominal pain, diarrhea, dysentery, infected wound, suppuration, and chronic ulcer.     

Inhibition of acetylcholinesterase by two genistein derivatives: kinetic analysis,molecular docking and molecular dynamics simulation

In this study two genistein derivatives (G1 and G2) are reported as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and differences in the inhibition of AChE are described. Although they differ in structure by a single methyl group, the inhibitory effect of G1 (IC₅₀=264 nmol/L) on AChE was 80 times stronger than that of G2 (IC₅₀=21,210 nmol/L). Enzyme-kinetic analysis, molecular docking and molecular dynamics (MD) simulations were conducted to better understand the molecular basis for this difference. The results obtained by kinetic analysis demonstrated that G1 can interact with both the catalytic active site and peripheral anionic site of AChE. The predicted binding free energies of two complexes calculated by the molecular mechanics/generalized born surface area (MM/GBSA) method were consistent with the experimental data. The analysis of the individual energy terms suggested that a difference between the net electrostatic contributions (ΔE_ele+ΔG_GB) was responsible for the binding affinities of these two inhibitors. Additionally, analysis of the molecular mechanics and MM/GBSA free energy decomposition revealed that the difference between G1 and G2 originated from interactions with Tyr124, Glu292, Val294 and Phe338 of AChE. In conclusion, the results reveal significant differences at the molecular level in the mechanism of inhibition of AChE by these structurally related compounds.KEY WORDS: Genistein derivatives, Acetylcholinesterase (AChE), Kinetics analysis, Molecular docking, Molecular dynamics simulation, MM/GBSAAbbreviations: ACh, acetylcholine; AChEIs, acetylcholinesterase inhibitors; AChE, acetylcholinesterase; AD, Alzheimer׳s disease; BuChE, butyrylcholinesterase; BuSCh, S-butyrylthiocholine chloride; CAS, catalytic active site; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); GAFF, generalized AMBER force field; G1, 3-(4-methoxyphenyl)-7-(2-(piperidin-1-yl)ethoxy)-4H-chromen-4-one; G2, (S)-3-(4-methoxyphenyl)-7-(2-(2-methylpiperidin-1-yl)ethoxy)-4H-chromen-4-one; iso-OMPA, tetraisopropyl pyrophosphoramide; MD, molecular dynamics; MM/GBSA, molecular mechanics/generalized born surface area; PAS, peripheral anionic site; PDB, protein data bank; PME, particle mesh Ewald; RMSD, root-mean-square deviation; S-ACh, acetylthiocholine iodide; ΔE_ele, electrostatic energy contribution; ΔE_MM, gas-phase interaction energy between receptor and ligand; ΔE_vdw, van der Waals energy contribution; SASA, solvent accessible surface area; ΔG_exp, experimental binding free energy; ΔG_GB, polar desolvation energy term; ΔG_pred, total binding free energy; ΔG_SA, nonpolar desolvation energy term; ΔS, conformational entropy contribution     

Oxathiolene oxides: a novel family of compounds that induce ferritin,glutathione S-transferase,and other proteins of the phase II response

Compounds that induce the synthesis of cytoprotective phase II enzymes have shown promise as cancer chemopreventive agents. Although chemically diverse, phase II enzyme inducers are capable of participating in Michael reaction chemistry. We have synthesized a novel class of organosulfur compounds, termed oxathiolene oxides (OTEOs). Based on their chemical properties, we hypothesized that these compounds could function as phase II enzyme inducers. Northern blot analysis showed that oxathiolene oxides induce the phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), and ferritin H and L mRNA in a concentration-dependent fashion in a normal embryonic mouse liver cell line, BNLCL.2. OTEO-562 (3-cyclohexenyl-4-methyl-1,2-oxathiol-3-ene-2-oxide) was the strongest inducer. Western blot analysis demonstrated that GST-alpha and ferritin H protein levels were also induced in cells treated with OTEO-562, as was total GST and NQO1 enzyme activity. Further, induction of NQO1 activity by OTEO-562 was equivalent in aromatic hydrocarbon (Ah) receptor wild-type and Ah receptor mutant cell lines, suggesting that oxathiolene oxides activate phase II enzymes by an Ah receptor-independent mechanism. Consistent with this observation, OTEO-562 failed to induce cytochrome P450 1A1 mRNA. These results suggest that oxathiolene oxides may merit further investigation as candidate chemopreventive agents.