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Serious health risks associated with the consumption of food products contaminated with aflatoxins (AFs) are worldwide recognized and depend predominantly on consumed AF concentration by diet. A low concentration of aflatoxins in cereals and related food commodities is unavoidable, especially in subtropic and tropic regions. Accordingly, risk assessment guidelines established by regulatory bodies in different countries help in the prevention of aflatoxin intoxication and the protection of public health. By assessing the maximal levels of aflatoxins in food products which are a potential risk to human health, it's possible to establish appropriate risk management strategies. Regarding, a few factors are crucial for making a rational risk management decision, such as toxicological profile, adequate information concerning the exposure duration, availability of routine and some novel analytical techniques, socioeconomic factors, food intake patterns, and maximal allowed levels of each aflatoxin in different food products which may be varied between countries.

Metal nanoparticles (MNPs) are popular in different research fields due to their unique physical and chemical properties and superior antimicrobial, anticancer, antioxidant, larvicidal, and catalytic potentials. Although conventional wet methods like colloidal synthesis, calcination, and spark ablation for synthesizing MNPs are effective, their synthesis uses an array of reducing and stabilizing agents and gases, making the process tedious. Additionally, metal nanoparticles induce oxidative stress through reactive oxygen species (ROS), showing high toxicity. Research and development in green chemistry have gained momentum and massive attention because of being efficient, clean, economical, environment-friendly, and free of hazardous byproducts. Recently, seed extracts in-lieu of chemical stabilizers and reducing agents have become popular because of the single-step green synthesis of MNPs. Seeds provide the researchers with a cost-effective alternate to other biological methods due to low maintenance costs, culture/growth independence for biomass, and diversity of phytochemicals as reducing and capping agents. Thus, effective green synthesis approaches are considered sustainable for MNP synthesis. This review depicts the literature on the challenges associated with metal and metal oxide nanoparticles and discusses their synthesis using seed extracts. The application section of the review discusses the antimicrobial, anticancer, and larvicidal activities of seed extracted-synthesized metallic nanoparticles. Furthermore, insights into the different biological potentials of the synthesized green MNPs have also been discussed.

Fresh fruit and vegetables are highly utilized commodities by health-conscious consumers and represent a prominent segment in the functional and nutritional food sector. However, food processing is causing significant loss of nutritional components, and the generation of waste is creating serious economic and environmental problems. Fruit and vegetables encompass husk, peels, pods, pomace, seeds, and stems, which are usually discarded, despite being known to contain potentially beneficial compounds, such as carotenoids, dietary fibers, enzymes, and polyphenols. The emerging interest in the food industry in the nutritional and biofunctional constituents of polyphenols has prompted the utilization of fruit and vegetable waste for developing enriched and functional foods, with applications in the pharmaceutical industry. Moreover, the utilization of waste for developing diverse and crucial bioactive commodities is a fundamental step in sustainable development. Furthermore, it provides evidence regarding the applicability of fruit and vegetable waste in different food formulations especially bakery, jam, and meat based products.

Organophosphorus poisoning caused by some pesticides and nerve agents is a life-threating condition that must be swiftly addressed to avoid casualties. Despite the availability of medical countermeasures, the clinically available compounds lack a broad spectrum, are not effective towards all organophosphorus toxins, and have poor pharmacokinetics properties to allow them crossing the blood-brain barrier, hampering cholinesterase reactivation at the central nervous system. In this work, we designed and synthesised novel isatin derivatives, linked to a pyridinium 4-oxime moiety by an alkyl chain with improved calculated properties, and tested their reactivation potency against paraoxon- and NEMP-inhibited acetylcholinesterase in comparison to the standard antidote pralidoxime. Our results showed that these compounds displayed comparable in vitro reactivation also pointed by the in silico studies, suggesting that they are promising compounds to tackle organophosphorus poisoning.

Detection of pathogenic microbes as well as antibiotic residues in food animals, especially in chicken, has become a matter of food security worldwide. The association of various pathogenic bacteria in different diseases and selective pressure induced by accumulated antibiotic residue to develop antibiotic resistance is also emerging as the threat to human health. These challenges have made the containment of pathogenic bacteria and early detection of antibiotic residue highly crucial for robust and precise detection. However, the traditional culture-based approaches are well-comprehended for identifying microbes. Nevertheless, because they are inadequate, time-consuming and laborious, these conventional methods are not predominantly used. Therefore, it has become essential to explore alternatives for the easy and robust detection of pathogenic microbes and antibiotic residue in the food source. Presently, different monitoring, as well as detection techniques like PCR-based, assay (nucleic acid)-based, enzyme-linked immunosorbent assays (ELISA)-based, aptamer-based, biosensor-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry-based and electronic nose-based methods, have been developed for detecting the presence of bacterial contaminants and antibiotic residues. The current review intends to summarize the different techniques and underline the potential of every method used for the detection of bacterial pathogens and antibiotic residue in chicken meat.

The inability of standard therapy to provide adequate protection against poisoning by organophosphorus compounds (pesticides and nerve agents) motivated us to search for new, more effective oximes. We investigated the pharmacotoxicological properties of six experimental K-oximes (K027, K033, K048, K074, K075, and K203) in vivo. The therapeutic efficacy of K-oximes (at doses of 5 or 25 % of their LD50) combined with atropine was assessed in paraoxon-poisoned mice and compared with conventionally used oximes HI-6 and TMB-4. The bisoxime K074 was the most toxic (LD50=21.4 mg kg-1) to mice, while monoxime K027 was the least toxic (LD50=672.8 mg kg-1). With the exception of K033, all of the tested K-oximes showed better therapeutic efficiency than HI-6 and TMB-4. K027 and K048 stood out by demonstrating low acute toxicities and ensuring protective indices ranging from 60.0 to 100.0 LD50 of paraoxon. Taking into account that these two oximes showed a similar therapeutic efficacy regardless of the applied doses, our results suggest that K027 and K048 could be antidotes for paraoxon intoxication.

The toxicity of organophosphorus compounds (OPs) caused by the phosphylation of AChE active center serine results in the accumulation of the neurotransmitter acetylcholine (ACh), consequently causing seizures and in severe cases even death. Immediate therapeutic treatment usually consists of a combined administration of anticholinergic drugs and an oxime as the reactivator of AChE. The nerve agent soman is an extremely toxic OP and acts almost instantaneously. AChE inhibited by soman is less accessible to reactivation due to the rapid dealkylation phenomenon of the soman- AChE conjugate called ˝ageing˝. Earlier studies have shown that amino acid substitutions in the AChE active center could slow down ageing and enable oximes to reactivate the soman-AChE conjugate prior to its ageing. The human AChE mutant hY337A/F338A combines the increase in the active center accessibility of the Y337A mutant to oximes with the ageing resistance of the F338A mutation. Therefore, its administration to potentially exposed individuals in the form of a pre-treatment is suggested so that it acts as a pseudo catalytic bioscavenger. Nevertheless, apart from ageing, the oxime reactivation of the soman- AChE conjugate is effected by the steric hindrance of the soman’s alkoxy side chain which limits the effectiveness of pyridinium oximes. In the 1970s, it was found that potential reactivators have an oxime group in the ortho position on the pyridinium ring and/or a CH2-O-CH2 linking chain between two pyridinium rings. Oxime HI-6, containing both characteristics, especially stood out. The search for novel oxime reactivators is still ongoing. We tested a number of novel and some previously known oximes in order to challenge the 1970s findings. Nevertheless, among all the tested oximes, HI-6 and its structural analogs, when taking kobs as the main criterion, have proven to be the most potent reactivators of the soman-Y337A/F338A conjugate. Acknowledgments: This work was supported by the NIH (Grant No. U01 NS058046) and by the Croatian Ministry of Science, Education and Sports (Grant No.022-0222148-2889).

Neuritic plaques, which are situated in the brain of Alzheimer's disease (AD) patients, are composed mainly of peptides containing 40 or 42 amino acid residues known as ß-amyloid plaques (Aß). The Aß peptide is the result of the enzymatic cleavage of the amyloid precursor protein (APP). In the so-called amyloidogenic pathway, the ß-secretase enzyme releases a protein fragment (C99), which is subsequently metabolized by the enzyme γ-secretase. Monomer forms of Aß are turned into oligomer forms, which are the main cause of cellular neuronal death in AD patients. The following study is focused on γ-secretase inhibitors that can slow down the production or accumulation of pathologic Aß deposits. γ secretase inhibitors that reached different phases of clinical trials are particularly reported as well as other promising groups of these analogues.

In the event of poisoning by organophosphorus compounds (OP), immediate therapeutic treatment usually consists of combined administration of an anticholinergic drug, such as atropine, and an oxime-reactivator of acetylcholinesterase (AChE, E.C. 3.1.1.7). However, the treatment is very limiting in case of nerve agent soman poisoning due to extremely rapid aging of phosphorylated enzyme. For now, HI-6 is the best known reactivator of soman inhibited AChE. Today, because of limited reactivation of phosphorylated AChE and fast ageing, researches are pointed towards AChE bioscavangers, human AChE mutants among other enzymes. The mutant of our interest is Y337A/F338A where increased accessibility of the Y337A mutation to oximes is combined with aging resisting the F338A mutation. We screened 35 oximes (1mM) for the reactivation activity of soman inhibited human AChE mutant, Y337A/F338A. Only 15 oximes were able to restore more than 30% of soman inhibited Y337A/F338A activity. None of the tested oximes restored Y337A/F338A activity fully nor any of those oximes were better than HI- 6 despite the fact that some of these oximes have CH2-O-CH2 linking chain and/or oxime group in position 2, which is known to be characteristic for the most potent reactivators of soman inhibited AChE. In fact, some of the most potent reactivators among tested oximes have benzene ring in the linking chain so our findings could point the quest for reactivators of soman inhibited AChE and AChE mutants in new direction.

The widespread use of organophosphorous compounds (OP) as pesticides and the misuse of OP nerve agents in military conflicts or terrorist attacks emphasize the need for effective antidotal preparedness. The toxic symptomatology of OP is caused by irreversible inhibition of the serine-protease acetylcholinesterase (AChE), a vitally important enzyme that hydrolyses the neurotransmitter acetylcholin. Generally, anticholinergics such as atropine and AChE reactivators - oximes are used as first aid antidotes for OP intoxications. However, reactivation by oximes is not possible in all cases of OP poisoning, especially in the case of nerve agent tabun. Recent in vitro and in vivo studies on mice draw attention to oxime [(E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide] (K203) as a very potent reactivator of tabun-inhibited AChE. In this study we used rats as experimental model to broaden our knowledge on antidotal potency of K203 as well as to examine impact of possible oxidative stress on OP toxicity and efficacy of applied therapy. Animals were injected subcutaneously with sublethal dose of tabun, and treated intraperitoneally 1min later with oxime K203 (5 or 25% of its LD50) alone or in combination with atropine. All animals were sacrificed after 0.5, 1, 6 and 24 h. Plasma samples were analyzed for cholinesterase (ChE) activity and levels of oxidative stress markers (the concentration of thiobarbituric reactive substances, TBARS, as a measure of lipid peroxidation and activity of an antioxidant enzyme superoxide dismutase, SOD). Either alone or in combination with atropine, K203 significantly increased ChE activity at 0.5 and 1 h, in respect to untreated rats poisoned with tabun. In addition, intraperitoneal administration of oxime alone as a control didn't disturb enzyme activity in plasma. Determined TBARS concentrations suggest that this acute treatments resulted with free radical-mediated lipid peroxidation. Considering SOD activity, a notable correlation exists between ChE depression and SOD elevation. Together with published data on K203's efficacy our results indicate the high potency of this oxime in counteracting tabun poisoning. Based on simultaneous measurements of TBARS and SOD we can conclude that the existence of oxidative stress have a role in OP toxicity.

Some compounds, although not primarily designed as supportive drugs in chemotherapy, are promising candidates for clinical use. The ability of HI-6 oxime to relieve the side effects of irinotecan was recently determined in vitro. In this animal study, we investigated the efficacy of HI-6 in vivo, when given as a pre-treatment and concomitantly with irinotecan. We evaluated the cholinesterase (ChE)/acetylcholinesterase (AChE) activity, the levels of oxidative stress markers, DNA damage and the radical scavenging capacity of HI-6. Both HI-6 and irinotecan inhibited ChE/AChE activity but showed different levels of ChE inhibition in plasma and AChE inhibition in the liver and brain tissue. We also observed a weak antioxidant capacity of HI-6, undiscovered until now, and found an acceptable genotoxicity profile in three types of somatic cells in rats. The in vivo erythrocyte micronucleus assay showed that HI-6 did not significantly change either the frequency of micronuclei or the ratio of polychromatic and normorchromatic erythrocytes. Taken together, our results provide a good argument in favour of HI-6 as a promising molecule for further studies and eventual use in humans.

We studied bispyridinium oxime K203 [(E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyimino methylpyridinium)- but-2-ene dibromide] with tabun-inhibited human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro, and its antidotal effect on tabun-poisoned mice and rats in vivo. We compared it with oximes K048 and TMB-4, which have proven the most effi cient oxime antidotes in tabun poisoning by now. Tabun-inhibited AChE was completely reactivated by K203, with the overall reactivation rate constant of 1806 L mol-1 min-1. This means that K203 is a very potent reactivator of tabun-inhibited AChE. In addition, K203 reversibly inhibited AChE (Ki = 0.090 mmol L-1) and BChE (Ki = 0.91 mmol L-1), and exhibited its protective effect against phosphorylation of AChE by tabun in vitro. In vivo, a quarter of the LD50 K203 dose insured survival of all mice after the application of as many as 8 LD50 doses of tabun, which is the highest dosage obtained compared to K048 and TMB-4. Moreover, K203 showed high therapeutic potency in tabun-poisoned rats, preserving cholinesterase activity in rat plasma up to 60 min after poisoning. This therapeutic improvement obtained by K203 in tabun-poisoning places this oxime in the spotlight for further development., Proučavali smo bispiridinijski oksim K203 [(E)-1-(4-karbamilpiridinij)-4-(4-hidroksiiminometilpiridinij)- but-2-ene dibromid] u uvjetima in vitro - studirajući njegove interakcije s ljudskom acetilkolinesterazom (AChE) i butirilkolinesterazom (BChe) inhibiranim tabunom te u uvjetima in vivo - određivanjem njegova antidotskog učinka na miševe i štakore otrovane tabunom. Radi usporedbe uključili smo rezultate dobivene s oksimima K048 i TMB-4 kao najučinkovitijim oksimima kod otrovanja tabunom. K203 je potpuno reaktivirao AChE inhibiranu tabunom sa sveukupnom brzinom reaktivacije od 1806 L mol-1 min-1 što ga svrstava u najučinkovitije reaktivatore AChE inhibirane tabunom. K203 je reverzibilno inhibirao AChE (Ki = 0,090 mmol L-1) i BChE (Ki = 0,91 mmol L-1) pokazujući svoja in vitro zaštitna svojstva od inhibicije tabunom. Terapija dozom K203 od ¼ njegove LD50 omogućila je preživljavanje svih miševa nakon otrovanja dozom tabuna od 8,0 LD50. Time je K203 pokazao bolju učinkovitost u usporedbi s K048 ili TMB-4. K tome, K203 je značajno zaštitio štakore od otrovanja tabunom kompenzirajući toksični učinak tabuna na aktivnost kolinesteraze i do 60 min nakon trovanja. Pokazano poboljšanje terapeutske učinkovitosti K203 ističe ovaj oksim pretečom za daljnji razvoj antidota u otrovanju tabunom.

A new approach to reduce the in vivo toxicity of chemical warfare nerve agents includes the use of bioscavengers – enzymes that could react with nerve agent before it inhibits acetylcholinesterase (AChE ; EC 3.1.1.7) at the physiologically important target sites. Butyrylcholinesterase (BChE ; EC 3.1.1.8), naturally present in plasma, liver, small intestine, smooth muscle, hearth, adipose tissue and in the brain, is considered an endogenous stochiometric bioscavenger of anticholinesterase compounds. Due to a limited concentration of BChE in organism, a stochiometric reduction of nerve agents is not always sufficient. This can be improved by creating a pseudo-catalytic scavenger adding oxime as a reactivator of inhibited BChE. In order to discuss BChE endogenous bioscavenging function in tabun and paraoxon poisoning, we tested in vitro reactivation of phosphorylated human plasma BChE by several bispyridinium oximes. In the case of tabun-inhibited BChE, the most potent reactivators were K117 [1, 1'-(2, 2'-oxybis(ethane-2, 1-diyl))bis(4-hydroxyiminomethyl pyridinium) bromide] and K127 [4-carbamoyl-1-(2-(2-(4-(hydroxyiminomethyl) pyridinium-1-yl)ethoxy)ethyl)pyridinium bromide]. The reactivation by these oximes (1 mM) reached about 50 % after only 20 min, but reactivation stopped at 70 %. Observed first order reactivation rate constants (kobs) were 0.05 min-1 for K117 and 0.03 min-1 for K127. On the other hand, reactivation of paraoxon-inhibited BChE, was much slower by all of the selected oximes. The maximum of 70-95 % was obtained in 8 h with kobs ranging from 0.004-0.006 min-1. Nevertheless, reactivation results obtained for K117 and K127, pointed out these oximes as promising leads in the search and development of new BChE reactivators.

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still very important task. Organophosphates-nerve agents are characterized as compounds influencing cholinergic nerve transmission via inhibition of acetylcholinesterase (AChE), an extremely active enzyme that hydrolyses acetylcholin. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsions and seizures. Signs are dose-dependent, leading to severe incapacitation and rapid death. Pyridinium oximes are nowadays used as successful treatment of poisoning with many organophosphates. Reason for that lies in the mechanism of oxime activity, which is based on protection of unphosphorylated AChE and/or reactivation of phosphorylated AChE. The aim of this investigation was to define the biochemical parameters of three bispyridinium oximes K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K033 [1, 4-bis (2-hydroxyiminomethylpyridinium) butane dibromide] and K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide]. For each of the compounds it was determined the toxic effect on human erythrocytes AChE in vitro (IC50) and their reactivating (%R) and protective potency (P50) with respect to soman- and tabun- inhibited AChE. Earlier we described in vivo antidotal activity of tested oximes against soman and tabun in male mice. The new tested oximes were toxic to human erythrocytes AChE ; in decreasing order from K033, K048 to K027. IC50 ranged from 0.02 to 1.0 mM. The best reactivating potency was obtained with K048 when AChE was inhibited by tabun. The protective potency of all oximes on human erythrocytes AChE inhibited by soman and tabun could not be determined. Our results indicate a good affinity of the tested compounds for AChE. The oximes are reversible ihibitors of AChE in vitro and show pharmacological properties which are related to reactivation of AChE.

Organophosphorus compounds are widely used as pesticides and unfortunately as nerve agents in chemical warfare. They are known inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7) an enzyme that hydrolizes the neurotransmitter acetylcholine in the nervous system. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsion and seizures. Signs are dose-dependent, leading to severe incapacitation and rapid death. Together with atropine, pyridinium oximes are known to be successfully used to treat poisoning with many organophosphorus compounds. In this paper three new bis-pyridinium compounds: K033 [1, 4-bis(2-hydroxyiminomethylpyridinim)butane dibromide], K027 [1, 4-hydroksyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] were tested as potential antidotes in tabun poisoned mice. Their antidotal effect was compared with TMB-4 [1, 3-bis(4-hydrxyiminomethylpyridinium) propane dibromide], which is the best-known antidote in tabun poisoning. In all experiments, oxime K033 in doses of 1/4 or 5% of its LD50 was used for the pre-treatment 15 minutes before tabun-intoxication. Also, one or 5 minutes after tabun application experimental animals received oxime K027, K033 or K048 (5% or 1/4 of its LD50) plus atropine sulphate as therapy. The antidotal efficacy of tested compounds was expressed as therapeutic factor (TF) and therapeutic dose (TD). Under same experimental conditions, our experiment selected compound K048 as the most reactivator of tabun inhibited AChE. Namely, this study has shown that the therapeutic regimen consisting of K033 in dose 5% of its LD50 as preatretment and &frac14 ; of LD50 of K048 plus atropine as treatment had the highest TF and TD. The TF was 13.3 LD50 of tabun, TD was 10 LD50 of tabun and insurance survival of all tested animals. In conclusion, treatment with these new bis-pyridinium oximes seems to be a very good alternative for current treatment in tabun poisoning. For this reason, these and other similar compounds require further investigation.

The mechanism of nerve agent tabun poisoning involves phosphorylation of a serine hydroxyl group in the active site of human acetylcholinesterase (AChE ; EC 3.1.1.7), leading to inactivation of this essential enzyme. The therapeutic approach to organophosphate poisoning is to reactivate AChE with a site-directed nucleophile such as an oxime. The ability of three bispyridinium oximes K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] and K033 [1, 4-bis(2-hydroxyiminomethylpyridinium) butane dibromide] to reactivate tabun-inhibited human erythrocyte AChE was evaluated. Reversible inhibition of native AChE by these oximes and their protective index from inactivation by tabun were determined as well. Tabun-inhibited AChE was completely reactivated by micromolar concentrations of K027 and K048 within two hours with the overall reactivation rate constants 303 min-1M-1 and 640 min-1M-1, respectively. The reactivation by K033 reached 60 % after 26 hours. The enzyme-oxime dissociation constants for K027, K048 and K033 binding to native AChE were 130 μ M, 130 μ M and 15 μ M, respectively. Although K033 did not show significant reactivation ability, this oxime might be of interest as a pretreatment drug due to its high affinity for the native AChE. The theoretically calculated protective index of oxime against AChE phosphorylation by tabun was as high as 7.6 for 0.1 mM K033, and only 1.8 for 0.1 mM K027 and K048. Our experiments pointed out K048 and K027 as promising reactivators in tabun poisoning and K033 as a promising protective agent.

Multi-target drug discovery is one of the most followed approaches in the active central nervous system (CNS) therapeutic area, especially in the search for new drugs against Alzheimer's disease (AD). This is because innovative multi-target-directed ligands (MTDLs) could more adequately address the complexity of this pathological condition. In a continuation of our efforts aimed at a new series of anti-AD MTDLs, we combined the structural features of the cholinesterase inhibitor drug tacrine with that of resveratrol, which is known for its purported antioxidant and anti-neuroinflammatory activities. The most interesting hybrid compounds (5, 8, 9 and 12) inhibited human acetylcholinesterase at micromolar concentrations and effectively modulated Aβ self-aggregation in vitro. In addition, 12 showed intriguing anti-inflammatory and immuno-modulatory properties in neuronal and glial AD cell models. Importantly, the MTDL profile is accompanied by high-predicted blood-brain barrier permeability, and low cytotoxicity on primary neurons.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Eliška Potůčková Supervisor: Doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of Doctoral Thesis: Study of antiproliferative effects of novel iron chelators Recent studies demonstrate that iron chelators possess marked potential as anticancer agents and also as substances protecting sensitive tissues against oxidative stress. This thesis is dealing mainly with design and synthesis of new antiproliferative iron chelators based on the structure of salicylaldehyde isonicotinoyl hydrazone (SIH) and with a characterization of their pharmacological and toxicological properties. Antiproliferative effects of these substances were studied on human breast adenocarcinoma cell line and human leukemic cell line and the non-specific toxicity on neonatal rat cardiac tissue-derived cells. During these studies some novel structure- activity relationships have been found. Furthermore, the suitability of simultaneous administration of iron chelators with antineoplastic agents used in breast cancer treatment was studied. In this project the anthracycline antibiotic, doxorubicin, and, in particular, the estrogen receptor antagonist, tamoxifen, were identified as the most suitable agents for potential treatment...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Mgr. Jakub Chlebek Supervisor: Doc. RNDr. Lubomír Opletal, CSc. Title of Doctoral Thesis: Study of biological activity of isolated alkaloids from Corydalis cava (Fumariaceae). Key words: Corydalis cava, isoquinoline alkaloids, DPPH, AChE, BuChE, BACE1, IMER. Tubers of Corydalis cava were selected on the basis of bio-guided spectrophotometric Ellman's method as a source of isoquinoline alkaloids for study of their selected biological activities. The tubers of Corydalis cava were extracted with ethanol and the mixture of summary tertiary alkaloids (fraction A) was fractionated in silica gel chromatography column using step gradient elution with hexane, chloroform and ethanol. Repeated column chromatography, preparative TLC and crystallization led to the isolation of fifteen isoquinoline alkaloids. The chemical structures of isolated compounds were determined on the basis of spectroscopic techniques and by comparison with literature data. Isolated alkaloids were tested on ability to inhibit AChE, serum BuChE (IC50), BACE1 and for its free-radical scavenging activity (EC50). The cholinesterase inhibitory activity was determined in vitro by modified spectrophotometric Ellman's...

Mood disorders are serious diseases. Nevertheless, their pathophysiology is not sufficiently clarified. Biological markers that would facilitate the diagnosis or successful prediction of pharmacotherapy are still being sought. The aim of the study was to find out whether mitochondrial functions are affected by antidepressants, mood stabilizers and depression. Our research is based on recent hypotheses of mood disorders, the advanced monoamine hypothesis, the neurotrophic hypothesis, and the mitochondrial dysfunction hypothesis. We assume that impaired function of mitochondria leads to neuronal damage and can be related to the origin of mood disorders. Effects of antidepressants and mood stabilizers on mitochondrial functions can be related to their therapeutic or side effects. In vitro effects of pharmacologically different antidepressants and mood stabilizers on the activities of mitochondrial enzymes were measured in mitochondria isolated from pig brains (in vitro model). Activity of monoamine oxidase (MAO) isoforms was determined radiochemically, activities of other mitochondrial enzymes were measured spectrophotometrically. Overall activity of the system of oxidative phosphorylation was measured electrochemically using high- resolution respirometry. Methods were modified to measure the same...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Chemistry and Drug Control Candidate Mgr. Veronika Nováková Supervisor doc. PharmDr. Petr Zimčík, Ph.D. Title of Doctoral Thesis Synthesis and study of properties of aza-analogues of phthalocyanines Phthalocyanines (Pc) and their azaanalogues azaphthalocyanines (Azapc) are planar macrocyclic compounds with significant absorption band at 620-720 nm finding application in the areas such as photodynamic therapy, chemical catalysis and solar cells. Photophysical (fluorescence) and photochemical (singlet oxygen production) parameters are important in above mentioned applications. Peripheral substituents of Pc/AzaPc play key role in this case. Alkylsulfanyl and alkyloxy derivatives exerted high fluorescence quantum yields (ΦF) as well as quantum yields of singlet oxygen (ΦΔ). On the other hand, alkylamino substitution led to almost zero values of both ΦF and Φ∆. These derivatives have recently found new application as dark quenchers in DNA hybridization probes. The first part of this thesis demonstrated that intramolecular charge transfer (ICT) is responsible for zero values of quantum yields of alkylamino AzaPc and that the lone electron pair of peripheral nitrogen is a donor for ICT in AzaPc....

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of pharmaceutical chemistry Candidate: Mgr. Pochopová Lenka Consultant: Ing. Kubíček Vladimír, CSc. Tittle of Thesis: ,,ELECTROSYNTHESIS OF DELTA-VALERO LACTONES" In this project electrolysis has been used as a method for a synthesis of new compounds. The main aim is cathodic reduction of the carbon-chlorine bond in a series of aldol trichloroacetates. Formed carbanion would form a 6 member ring by attaching the carbonyl group. Two structures can be formed: α, α-dichloro-β-hydroxy-δ-lactones and α, β-unsaturated-α-chloro -δ-lactones from the starting esters using electrolysis. The whole process was performed at controlled potential in a separated cell, on mercury pool as a cathode and platinum as an anode in an argon atmosphere with dichloromethane/tetraethylamonium system. During the process two electrons have been passed. The synthesized lactones are completely new compounds. In this report also electrochemical and spectroscopy properties of those compounds have been characterized to provide for their other use.

Charles University in Prague, Faculty of Pharmacy in Hradec Kralove Department of Pharmaceutical Chemistry and Drug Control SYNTHESIS OF PRECURSORS FOR BIOLOGICALLY ACTIVE LACTONES IV. Rigorous Thesis Hana Piskacova A high width of biological activity of natural or synthetic unsaturated five membered lactones from the family of furan-2(5H)-ones, is the main reason why to synthesize new substances of this structure. The introductory part of the thesis deals with antineoplastic natural compounds of this type. Except of antitumour activity some unsaturated five membered lactones exhibit antifungal, antiviral, antibacterial effects, or inhibit the synthesis of cholesterol, for example. The experimental project is an extension of my diploma thesis concerned with the synthesis of precursors for lactones. These precursors - methyl (E)- and (Z)-2-bromo-5- (subst.)arylpent-2-en-4-ynoates - were prepared by Sonogashira couplings. The parent substances for couplings were arylethynes (1-ethynyl-4-(methoxymethoxy)benzen, 1-ethynyl-2-nitrobenzene, 3-ethynylaniline, 2-ethynylpyridine) and methylesters of dihalogenated prop-2-enoic acid. The dominant products of the couplings were β-monoalkynylated esters, apart from them side products of homocouplings were obtained. Reactions of E-methylesters of dihalogenated...

Markéta Komlóová: Synthesis of bisquarternary asymmetrical reactivators of acetylcholinesterase Nine potential bisquaternary reactivators of AChE have been prepared and their reactivation activity against paraoxon or tabun-inhibited enzyme has been tested in vitro. Three reactivators of AChE proved to be suitable for further testing against paraoxon. One of these compounds also demonstrated satisfactory efficiency when reacting against tabun. The reactivation abilities of prepared compounds depend on structural factors such as the presence of oxime group, its location, the presence of quaternary nitrogen and other functional groups. Also four monoquaternary inhibitors have been prepared and their inhibitive activity tested in vitro.

The aim of present work was a comparison of effectiveness of oximes chosen in the experimental therapy of nerve agents intoxication (soman and russian VX). All experiments were performed in male Wistar rats. All oximes tested were administered intramuscularly either in a separate injection or in combination with atropine. The preparation of the brain parts for determination of acetylcholinesterase was performed in several time intervals after drugs tested administration. Antidotal potency of oxime HI-6 was superior in comparison with the rest of oximes tested.

QUESTIONS OF THE IN VITRO INHIBITION OF CHOLINESTERASES BY SELECTED ORGANOPHOSPHORUS PESTICIDES Helena Lázenská Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05, Hradec Kralove, Czech Republic. The aim of this study was to describe organophosphorus inhibitors paraoxone, DDVP and DFP from the aspect of the kinetics of their reaction with human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BuChE), to find IC50, and to test the in vitro potency of five selected oximes (pralidoxime, methoxime, trimedoxime, obidoxime and HI-6) to reactivate AChE and BuChE inhibited by three mentioned organophosphorus inhibitors. The inhibition of AChE and BuChE was performed by incubation with organophosphorus inhibitors at a convenient concentration and such time, that would result in about 90% activity of an enzyme. A solution of a reactivator at a final concentration 1 μM or 10 μM was added to an enzyme, after 10 min of reactivation, a solution of a substrate - acetylthiocholine-iodide or butyrylthiocholine-iodide was added, and the activity of an enzyme was measured by the spectrophotometric Ellman's method. The experiment was performed at 25 žC, pH 7,4 and in a 0,1 M phosphate buffer. According...

SOUHRN Spolu s ostatními diplomantkami (Šárkou Brožovou, Janou Nagyovou a Evou Vítkovou) jsem provedla extrakci 41,8 kg suché nati s kořeny. Primární extrakt jsme vyčistily filtrací a následným oddestilováním rozpouštědla na vakuové odparce. Dále jsme připravily sekvenčním postupem výtřepky s jednotlivými typy alkaloidů: šlo o dva diethyletherové výtřepky (získané po předchozí alkalizaci primárního extraktu uhličitanem sodným a hydroxidem draselným), které byly následně okyseleny kyselinou chlorovodíkovou, vzniklé kvartérní jodidy po přidání jodidu draselného vytřepány chloroformem a po následné alkalizaci extraktu amoniakem získány další kvartérní jodidy vytřepáním do směsi chloroform + ethanol 8,5:1,5. Já jsem se zabývala výtřepkem B, který obsahoval silně basické alkaloidy. Provedla jsem separaci výtřepku B pomocí sloupcové chromatografie. Zpracovávala jsem frakci 27- 46, jejíž odparek jsem po vysušení rozdělila na dvě části. První část (0,02 g) byla podrobena barevným reakcím s činidly, které prokázaly, že izolovaný alkaloid CH-M/B-1 je berberin.Dále jsem změřila teplotu tání Z druhé části odparku jsem získala 0,346 g žlutého prášku, který byl v závěru práce podroben testu vlivu látky na aktivitu AChE. Z naměřené hodnoty IC50= 0,221 (0,146- 0,228) μM je patrné, že látka CH-M/B-1 je vysoce aktivní v...