Natural products, especially alkaloids, are still a substantial resource for the drug development [1]. Based on the preliminary screening of alkaloidal extracts of various plants against Alzheimerʼs disease, Vinca minor L. (Apocynaceae) have been chosen for a detailed phytochemical and biological study. V. minor L. is an evergreen trailing subshrub common in Europe with rich content of monoterpene indole alkaloids [2]. In this study, we have isolated and identified active alkaloidal compounds and assessed their potential to inhibit hAChE, hBuChE, POP, and GSK-3β – enzymes that play a key role in the pathophysiology of Alzheimerʼs disease. Using chromatographic methods, we have isolated 23 alkaloids; 11 of them have been reported in this species for the first time. One alkaloidal structure was undescribed and was named as vincaminorudeine. The most active compound was (−)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole with IC50 = 0.65 µM for the inhibition of hBuChE, and with IC50 = 58 µM for the inhibition of POP. Other alkaloids that exhibited significant inhibition against hBuChE (IC50 < 30 µM) were vincaminoreine, minovine, 16-methoxyminovine, vincorine, and tubotaiwine. None of the isolated alkaloids was active against hAChE. The (−)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole was also further studied for its pharmacokinetic, revealing a reversible competitive type of inhibition for hBuChE with Ki = 55 nM. This compound can also penetrate the blood-brain barrier by passive diffusion, as was assessed by the PAMPA study. Additionally, the alkaloid on the panel of ten cell lines showed non-cytotoxicity. These compelling results open a possibility for further research on this indole alkaloid.

Fourteen (1-14) known Amaryllidaceae alkaloids, of various structural types, have been isolated from fresh bulbs of Hippeastrum X hybridum cv. Ferrari. The chemical structures were identified by various spectroscopic (1D and 2D NMR) and mass spectrometric (GC-MS, LC-MS) methods, and by comparison with literature data. Isolated alkaloids which, up to this date have not been studied for their inhibition potency against AChE, BuChE and POP, were screened in vitro for these inhibition activities. Unfortunatelly, all natural alkaloids were inactive in the AChE/BuChE assay; only zephyranthine (14) displayed moderate inhibition activity agains POP with an IC50 value of 142 +/- 10 mu M. Moreover, twelve new derivatives of the haemanthamine-type alkaloid vittatine (7), isolated in gram amounts from Hippeastrum cv. Ferrari, have been designed, synthesized and tested in vitro with particular emphasis on the treatment of neurodegenerative diseases. Some of the tested compounds revealed an intriguing selective hBuChE inhibitory profile with single-digit micro-molar IC50 values. The strongest hBuChE inhibition was demonstrated by 3-O-(2-methylbenzoyl)vittatine (7b), 3-O-(2-nitrobenzoyl)vittatine (7h) and 3-O-(2-chlorbenzoyl)vittatine (7m) with IC50 values 8.0 +/- 0.1 mM, 1.4 +/- 0.1 mM and 5.4 +/- 0.1 mM, respectively. The mode of hBuChE inhibition was minutely inspected using enzyme kinetic analysis in tandem with in silico experiments, the latter elucidating crucial interaction in 7b-, 7h-, and 7m-hBuChE complexes. (C) 2020 SAAB. Published by Elsevier B.V. All rights reserved.

Fifteen previously isolated Corydalis cava alkaloids have been investigated for their potential multifunctional activity on Alzheimer's disease (AD) targets. Determination of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibition was carried out using a BACE1-Immobilized Enzyme Reactor (IMER) by validating the assay with a multi-well plate format Fluorescence Resonance Energy Transfer (FRET) assay. Seven alkaloids out of fifteen were found to be active, (-)-corycavamine (IC50 FRET= 41.16 ± 7.82µM; IC50 IMER= 1690 ± 545.0µM) and (+)-corynoline (IC50 FRET = 33.59 ± 0.23µM; IC50 IMER = 89.07 ± 15.08µM) demonstrated the highest BACE1 inhibition activity, in the micromolar range, in a concentration dependent manner. BACE1-IMER was found to be a valid device for the fast screening of inhibitors and the determination of their potency. In a permeation assay (PAMPA) for the prediction of blood-brain barrier (BBB) penetration, the most active compounds (-)-corycavamine (Pe = 16.3 ± 0.9 × 10-6 cm s-1) and (+)-corynoline (Pe = 11.7 ± 0.1 × 10-6 cm s-1) were found to be able to cross the BBB. Not all compounds showed activity against glycogen synthase kinase-3β (GSK-3β) and casein kinase-1δ (CK-1δ). Furthermore, on the basis of the reported results, we found that some Corydalis cava alkaloids have multifunctional activity against AD targets (prolyl oligopeptidase, cholinesterases, and BACE1). Moreover, we tried to elucidate the treatment effectivity (rational use) of its extract in memory dysfunction in folk medicine. Based on a HPLC-UV analysis we found that due to quantitative low content of compounds with neuroprotective activity in the plant material, the use of C. cava extracts in the therapy of memory dysfunction in folk medicine is not corroborated by ascertained data [1].

Alzheimer's disease (AD) is known as one of the most common neurodegenerative diseases. The main characteristic sign of AD is progressive and irreversible loss of neurons. AD is also characterized by decreased levels of neurotransmitter acetylcholine (Ach) in the cortex, which is hydrolyzed mostly by acetylcholinesterase (AChE) in healthy brain. On the other hand, in late AD stages butyrylcholinesterase (BuChE) is the main hydrolysing enzyme as its content will increase by up to 90% in comparison to normal state. BuChE cleaves ACh in a similar way as AChE to terminate its physiological action [1].

The aim of this study was to investigate the antibacterial activity and toxicity for sperm cells of the natural substances gallic acid, methyl gallate, ethyl gallate, propyl gallate, octyl gallate, thymol, carvacrol and eugenol. The antibacterial activity of these natural substances and selected combinations of them against bacterial strains isolated from boar ejaculates was determined using the microdilution and macrodilution method in Mueller-Hinton broth. The most effective natural substances against Gram-negative and Gram-positive bacteria included in our study were thymol and carvacrol with minimum inhibitory concentration (MIC) values in the range of 300-600 µg/ml. Gallic acid exhibited the best antibacterial activity against Pseudomonas aeruginosa strains (MIC values of 300-2400 µg/ml), whereas the ranges of MIC values against Escherichia coli, Enterococcus faecalis and Staphylococcus sp. strains were higher. Octyl gallate exhibited stronger antibacterial activity against staphylococci and enterococci (MIC values of 18.8-75 µg/ml) than against Escherichia coli and Pseudomonas aeruginosa strains with MIC values in the ranges of 300-600 µg/mland 1200-2400 µg/ml, respectively. Thymol combined with carvacrol was the most effective combination against enterococci (MIC values of 75-300 : 150 thymol : carvacrol) and Pseudomonas aeruginosa (MIC values of 75-300 : 300 thymol : carvacrol), bacteria which are known to be frequently resistant to antimicrobials. Similar results were determined for the combination of carvacrol and eugenol against staphylococci and enterococci. The results of the combinations revealed more of an additive rather than a synergistic effect. Thymol and carvacrol were the most effective natural substances against the bacteria included in this study, with a low toxicity for sperm cells compared to other substances, suggesting their possible use for boar semen decontamination.

Alzheimer's disease (AD) is neurodegenerative disease with specific neuropathological changes, which are observed at molecular level. Nevertheless, ethiopathogenesis is still not clear and therapy is only symptomatic.

The use of antibiotics is currently limited within the framework of the prevention of the infectious disease in farm animals in the EU. Formerly used antibiotic growth stimulators markedly enhanced the quality of animal breeding and decreased the incidence of infectious diseases. After their ban, the new substances, which do not cause a possible resistance to pathogenic agents, are searched. One option is to use the small molecules of natural origin, particularly the primary and secondary plant metabolites. It is possible to consider the use of a new generation of cationic peptides isolated from plants or animal and bacteriocins. The use of these substances in the pig breeding is directed into the feeding stuff in a form of additive compounds, whose aim is to prevent the development of microbial infection in GIT and the area of abdomen, increasing the immunity of animals and decreasing the inflammatory processes in boar genitals. The second area is the application of these substances in the cryopreservation of boar semen and into the extenders for the short-term storage of semen prepared for AI. According to the EU recommendation, a substantially higher attention must be paid to the research of natural substances in agricultural sphere. A widespread application of antibiotics, for the prevention and therapy of infections, leads to the occurrence of the antibiotic-resistant bacterial strains in human and veterinary medicine. The farm animals (particularly bovines) may act as potential gene resources of this resistance to anti-invasive drugs and that is the cause of complications in the public health field. From the point of view of the farm animals, the situation has not been understood fully yet. At the foreground, an economic interest is more important than a later hazard which may arise from an extensive application of the antibiotics. There are views that methicillin-resistant strains of Staphylococcus aureus and multi-resistant gramnegative bacteria should be thoroughly examined, because they are broadly involved in the human field as well as in the animal production [Garcia-Alvarez et al., 2012]. An antimicrobial multi-drug resistance [Nikaido, 2009] is quite often developed, so it makes impossible to succeed in the therapy.

The tubers of Corydalis cava Schweigg. & Kort. were extracted with ethanol and the summary alkaloid extract 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 human erythrocyte acetylcholinesterase, serum butyrylcholinesterase (IC50) and for its free-radical scavenging activities (EC50).

The objective of this study was to investigate the effects of a preparation from herbal extracts (PHE) on libido and semen quality in breeding artificial insemination boars. Ten fertile boars were divided into control and experimental groups according to significant difference of libido. There were no differences in semen quality between groups. Animals were fed a commercial feeding mixture for boars. The feeding mixture for the experimental group was enriched with PHE, which was prepared from Eurycoma longifolia, Tribulus terrestris and Leuzea carthamoides. Duration of the experiment was 10 weeks. Samples of ejaculate were collected weekly. Libido was evaluated according to a scale of 0-5 points. Semen volume, sperm motility, percentage of viable spermatozoa, sperm concentration, morphologically abnormal spermatozoa, daily sperm production and sperm survival were assessed. Amounts of mineral components and free amino acids were analysed in seminal plasma. Significant differences were found in these parameters: libido (4.05 ± 0.22 vs 3.48 ± 0.78; p < 0.001), semen volume (331.75 ± 61.91 vs 263.13 ± 87.17 g; p < 0.001), sperm concentration (386.25 ± 107.95 vs 487.25 ± 165.50 × 10(3) /mm(3); p < 0.01), morphologically abnormal spermatozoa (15.94 ± 11.08 vs 20.88 ± 9.19%; p < 0.001) and Mg concentration (28.36 ± 11.59 vs 20.27 ± 13.93 mm; p < 0.05). The experimental group's libido was increased by 20% in comparison with the beginning of the experiment. Results of this study showed positive effect of PHE on libido and some parameters of boar semen quality.

Iron overload both following transfusion therapy of thalassemia and as the result of an inherited disorder, is associated with tissue damage and increased mortality. Patients suffering from iron overload can be treated by administration of iron chelators. Because of some limitations of commonly used iron chelators, the research of new iron chelators is required. In this pilot study, eight flavonoids of different basic structure and two synthetic iron chelators from aroylhydrazone group (SIH, PIH) were compared with effect of clinically used deferoxamine (DEF). Ferrous and total iron chelation activity was assessed by a simple spectrophotometric assay. Shortly: Potential iron chelators were mixed with ferrous or ferric ions in various ratios. Concentration of unbound iron was established by ferrozine at 562nm. Because ferrozine colours specifically ferrous ions, in case of ferric ions, hydroxylamine 10 mM was used for their reduction. None of tested chelators was similarly effective ferrous chelator as compared with DEF. Interestingly, apigenin was more active than synthetic chelators PIH and SIH and rutin was more effective than SIH. Other flavonoids showed only negligible activity. On the contrary, ferric ions seemed to be chelated absolutely by synthetic iron chelators PIH and SIH in ratio 10:1, chelator: iron, respectively. DEF was more effective since its activity nears 100% in ratio 1:1. Tested flavonols (rutin, kaempferol and 3-OH flavone) together with flavone apigenin brought about significant ferric chelation while flavanones (hesperetin and naringenin) with flavanols (catechin and epicatechin) were again practically inactive. This study demonstrated that some flavonoids, especially flavonols, act as iron chelators in clinically relevant concentrations. Though their effect did not approached DEF and synthetic chelators, it should be taken in consideration that additionally to their antioxidant properties they may be useful in pathological conditions associated with oxidative stress due to catalytical role of iron.

Coumarins, naturally occurring compounds derived from benzopyran, have recently been studied extensively for their antioxidant properties. A lot of coumarins have been isolated and identified from natural sources and many others have been synthesized. It is also known that pharmacological and biochemical properties and thus also therapeutic application of simple coumarins depend upon the pattern of their substitution.As a part of studies of biological effects, four naturally occurring coumarins and 18 synthesized analogs of several compounds were assayed for 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity. For this purpose the highly reliable DPPH test modified to be performed by sequential injection analysis (SIA) system was used. This in our laboratory-developed method was originally proposed for antioxidant screening of large series of plant extracts. In this assay, the DPPH test using the SIA method was used for fast and sensitive evaluation of EC50 of coumarins. The evaluation of EC50 of a single compound takes only 15 to 30 min. The structure-activity relationships of tested compounds are also established. The results verified 7,8-dihydroxy-4-methylcoumarins as excellent DPPH radical scavengers. Obtained results correspond with those of other studies and suggest the SIA procedure as a suitable method for fast and sensitive antioxidant analysis of various types of compounds.

Biologically active lignans from caulomas (stems) and leaves of Schisandra chinensis were extracted with supercritical carbon dioxide at the pressures 20–27MPa, temperatures 40, 50 and 60°C, and solvent-to-feed ratio 36–43. Six major lignans were determined by means of HPLC: schizandrin, gomisin A, deoxyschizandrin, γ-schizandrin, gomisin N and wuweizisu C. At the optimum conditions 27MPa and 50°C, the extraction yield of lignans from caulomas was 1.3% dry weight and their content in the extract was higher than 32wt.%. At the optimum conditions for CO2 extraction from S. chinensis leaves, 27MPa and 60°C, the yield of lignans was 0.22% d.w. and the yield of (E)-cinnamic acid was 0.18% d.w. Addition of 2–4% ethanol to CO2 increased the extraction rate but not the final yield of lignans. The solubility of extracted substances in supercritical CO2 was estimated from their extraction rates.

Antioxidants perform an important role in the maintenance of the integrity of the living organisms. Recently, great attention has been given to antioxidants by a reason of their medical use. It is due to the association of many human diseases with oxidative stress. The present study briefly surveys the types of antioxidants, reactive oxygen and nitrogen species and describes the basic mechanisms of their activity. The application of antioxidants in the therapy of the injury caused by sulfur mustard, a vesicant type chemical warfare agent introduced in World War I, is also given.

Free radical scavenging activity of crude ethanol extracts from the fruiting bodies of 32 taxons of Polyporales mushrooms was evaluated by means of DPPH (2,2'-diphenyl-1-picrylhydrazyl radical) test using SIA (PC-controlled Sequential Injection Analysis system) method developed in our laboratory [1]. The most active samples were tested by means of hydrogen peroxide-induced luminol chemiluminescence assay using SIA [2]. The DPPH radical scavenging activity of the samples was expressed as 50% effective concentration (EC50): Fomitopsis pinicola 0.086mg/ml, Osmoporus odoratus 0.276mg/ml, Phaeolus schweinityii 0.297mg/ml, Ganoderma lipsiense 0.379mg/ml, Daedaleopsis confragnosa 0.42mg/ml, Albatrellus ovinus 0.435mg/ml and Oligoporus ptychogaster 0.823mg/ml, and this values were compared with known antioxidants: acidum ascorbicum 0,006mg/ml, and trolox 0,014mg/ml. The results of the second method was also expressed as EC50: F. pinicola 0,124mg/ml, G. lipsiense 0,273mg/ml, O. odoratus 0,393mg/ml and P. schweinitzii 0,573mg/ml and this values were compared with known antioxidants: trolox 0,025mg/ml, rutin 0,190mg/ml and caffeic acid 0,008mg/ml. The results at this point are promising for further research.

Acetylcholinesterase (AChE) reactivators are employed for the prophylaxis and treatment of intoxications with organophosphorus AChE inhibitors, including nerve agents and pesticides. For the recovery of inhibited enzyme, derivatives from the group of pyridinium or bispyridinium aldoximes (called oximes) are used. Adverse effects of these substances are not well elucidated, because of their narrow and one-shot usage. Owing to this fact, the study evaluated the influence of some currently applied oximes on human platelet aggregation in vitro. The antiplatelet activity of pralidoxime, obidoxime, HI-6, methoxime and HLö 7 was assayed in human platelet rich plasma (2.5 x 10(8) platelets.ml(-1)) at a concentration of 1.35 mM. Arachidonic acid (AA), adenosine diphosphate (ADP), collagen (COL) and thrombin (TR) were used as agonists of platelet aggregation. All tested substances, except pralidoxime and methoxime, caused a significant inhibition of the aggregation process induced by AA, ADP and COL. Of the oximes assayed, none was found to influence TR triggered aggregation. Since reduced platelet aggregation can play an important role as an adverse effect in reactivator administration, further evaluation is needed for the estimation of the real impact of active oximes to the aggregation process in humans.

β-Sitosterol (22,23-dihydrostigmasterol) and scopoletin (7-hydroxy-6-methoxycoumarin) were extracted from roots of stinging nettle (Urtica dioica L.) with supercritical and liquid CO2. Extraction rates were measured for both oleoresin and the individual substances in dependence on extraction conditions. The solvent modified with ethanol (0–9.4wt.%) was applied at the pressures of 100, 200, 250 and 280bar and the temperatures of 25, 40 and 60°C. Extracts were analysed by means of HPLC method. The maximum yields of β-sitosterol and scopoletin were 0.63 and 0.058mg/g dry mass, respectively. In comparison with the extract obtained with diethyl ether, the maximum yields obtained with CO2 were at least two times higher for both investigated substances. A model describing the extraction of a solute that is present in two forms, free and interacting with matrix, was applied to interpret the experimental extraction curves.

The drug Wuweizi (dried fruits of Schisandra chinensis or S. sphenantherd) is one of important medicinal means used in the Oriental medicine. The lignans of dibenzo[a,c]cyklooctadiene type are major constituents, a volatile oil with mono- and ses-quiterpens, an oil, organic acids and small amounts of additional compounds are also present. The content of major lignans (schizandrin, deoxyschizandrin, gomisin A, gomisin N, γ-schizandrin, wuweizisu C) in commercially available drugs ranges usually between 3 and 5%. The present paper biefly comments the isolation and biological activity of the lignans and is especially concerned with analytical methods (TLC and HPLC) for the determination of the drug fingerprint and methods for the determination of constituents in drugs, mixtures and biological materials. HPLC methods using RP-silica bonded phases and diluted methanol, acetonitrile (or a mixture of both), are most important for these purposes. Electromigration methods are less suitable and the importance of hyphenation procedures is practically negligible.

At the beginning of a new millennium, several important questions have come to the fore connected with the utilization of natural resources for the investigation of new medicaments (necessity of cooperation of both highly developed and developing countries in the protection of natural resources, necessity to justly evaluate and reward the contributions of the individual cultures in the case that a knowledge of traditional medicine was employed in the development of a new medicament, necessity to achieve the maximal chemical variety of real and virtual libraries of substances from which potential drugs are selected for screening tests). The research of chalcones and its analogues which are relatively easily available not only by isolation from natural material, but also by the methods of classical and combinatorial synthesis should not be substantially affected by the above-described changes. The present communication links up with previous papers (Opletalová, V., Sedivý, D.: Ces. slov. Farm. 48, 252 (1999); Opletalová, V.: Ces. slov. Farm. 49, 278 (2000); Opletalová, V. et al.: Folia Pharm. Univ. Carol. 25, 21 (2000)) and is devoted to the stabilizing action of chalcones on the vascular wall, vasodilating and anti-aggregating effects, and a favourable effect of their antioxidative activity on the functions of the cardiovascular system. The introduction also briefly discusses the nomenclature of this group of compounds.

The thiophene polyine (E)-2-[5-(hept-5-en-1,3-diynyl)-thien-2-yl]-ethan-1,2-diol was isolated from an ethanolic extract of underground parts of Leuzea carthamoides. The thiophene polyine demonstrated significant antifungal activity against all tested species.