Analysis of vitamin K1 and major K2 variants in rat/human serum and lipoprotein fractions by a rapid, simple, and sensitive UHPLC-MS/MS method.

Determination of the various forms of vitamin K, which are involved in coagulation and other physiological processes in humans, is challenging and no standardized method is yet available. Therefore, a reliable and practical method was developed to quantify vitamin K levels in serum and additionally in lipoprotein fractions to clarify its distribution. The LC-MS/MS method for the determination of vitamin K1 and the three main isoforms of vitamin K2 (MK-4, MK-7, MK-9) was combined with a gradient ultracentrifugation technique to allow the separation of lipoprotein fractions. The chromatographic separation was carried out on a Kinetex™ C18 column using a mobile phase consisting mainly of methanol. The target analytes were detected by electrospray ionization mass spectrometry. The separation of all four substances was achieved after a simple sample preparation technique based on miniaturized liquid-liquid extraction. Our method of only 8.5 min revealed the levels of the major forms of vitamin K in 59 human and 12 rat sera and confirmed our hypothesis that vitamin K is primarily (about 50 %) found in the high-density lipoprotein fraction. The median concentrations of vitamin K1, MK-4, MK-7, and MK-9 were found to be 1.19, 2.98, 0.43, and < 0.71 nmol/L in human serum and 1.74, 6.75, less than 0.2, and less than 0.5 nmol/L in rat serum, respectively.

Proposed mechanisms of action of herbal drugs and their biologically active constituents in the treatment of coughs: an overview.

Various medicinal plants find their use in cough treatment, based on traditions and long-term experience. Pharmacological principles of their action, however, are much less known. Herbal drugs usually contain a mixture of potentially active compounds, which can manifest diverse effects. Expectorant or antitussive effects, which can be accompanied by others, such as anti-inflammatory or antibacterial, are probably the most important in the treatment of coughs. The aim of this review is to summarize the current state of knowledge of the effects of medicinal plants or their constituents on cough, based on reliable pharmacological studies. First, a comprehensive description of each effect is provided in order to explain the possible mechanism of action in detail. Next, the results related to individual plants and substances are summarized and critically discussed based on pharmacological in vivo and in vitro investigation.

The effects of bisphenols on the cardiovascular system ex vivo and in vivo.

The human population is regularly exposed to bisphenols. The first compound of this class, bisphenol A, is burdened by numerous reports of its potential toxicity and has been hence replaced by its analogues, so-called next generation bisphenols. Their widespread use has made them pervasive throughout the environment. These endocrine disrupting chemicals can affect the cardiovascular system, and hence the aim of this study was to test 14 bisphenols (A, AF, AP, B, BP, C, E, F, G, M, P, PH, S and Z), and compare their effects in vitro (human and rat cell lines), ex vivo (isolated rat aorta) and in vivo (Wistar Han rats, acutely or chronically exposed to low environmental and high toxic doses). The majority of the tested bisphenols relaxed rat aorta, but their potency varied markedly. The most potent compound, bisphenol AF, had an EC50 of 57 µM. The mechanism of action was likely based on the inhibition of calcium influx via L-type calcium channels. The cytotoxicity of bisphenols towards 4 human and rat cell lines (H9c2, A-10, MCF7/S0.5 and MCF7/182R-6) showed variable potencies ranging from units of micromolar to millimolar concentrations. Based on these data, an effect on arterial blood pressure and possible cardiotoxicity was expected. Contrarily, the in vivo acute effects of three doses (0.005, 0.05 and 2.5 mg/kg) of bisphenol AF and 3 other analogues (A, S and F) on the cardiovascular system were rather biologically negligible. The most potent bisphenol, AF, was also administered chronically at a dose of 2.5 mg/kg for 4 weeks to rats, but had no impact on arterial blood pressure. Our results showed that bisphenols can relax vascular smooth muscles, but the effective concentrations are too high to produce clear cardiovascular effects in relation to common biological exposure as was confirmed with the most potent bisphenol AF.

3-Hydroxyphenylacetic Acid: A Blood Pressure-Reducing Flavonoid Metabolite.

Regular intake of polyphenol-rich food has been associated with a wide variety of beneficial health effects, including the prevention of cardiovascular diseases. However, the parent flavonoids have mostly low bioavailability and, hence, their metabolites have been hypothesized to be bioactive. One of these metabolites, 3-hydroxyphenylacetic acid (3-HPAA), formed by the gut microbiota, was previously reported to exert vasorelaxant effects ex vivo. The aim of this study was to shed more light on this effect in vivo, and to elucidate the mechanism of action. 3-HPAA gave rise to a dose-dependent decrease in arterial blood pressure when administered i.v. both as a bolus and infusion to spontaneously hypertensive rats. In contrast, no significant changes in heart rate were observed. In ex vivo experiments, where porcine hearts from a slaughterhouse were used to decrease the need for laboratory animals, 3-HPAA relaxed precontracted porcine coronary artery segments via a mechanism partially dependent on endothelium integrity. This relaxation was significantly impaired after endothelial nitric oxide synthase inhibition. In contrast, the blockade of SKCa or IKCa channels, or muscarinic receptors, did not affect 3-HPAA relaxation. Similarly, no effects of 3-HPAA on cyclooxygenase nor L-type calcium channels were observed. Thus, 3-HPAA decreases blood pressure in vivo via vessel relaxation, and this mechanism might be based on the release of nitric oxide by the endothelial layer.

Synthesis of 3,3-dimethyl-6-oxopyrano[3,4-c]pyridines and their antiplatelet and vasodilatory activity.

OBJECTIVES: Both pyridine and pyrano derivatives have been previously shown to possess biologically relevant activity. In this study, we report the incorporation of these two scaffolds into one molecule. METHODS: The designed 3,3-dimethyl-6-oxopyrano[3,4-c]pyridines were synthesized by the acylation of enamine under Stork conditions followed by condensation of formed ß-diketones with 2-cyanoacetamide. The structures of these compounds were confirmed by using a wide spectrum of physico-chemical methods. Their antiplatelet, anticoagulant and vasodilatory activity together with toxicity were evaluated. KEY FINDINGS: A series of 6-oxopyrano[3,4-c]pyridines 3a-j was obtained. Four of these compounds were reported for the first time. None of the tested compounds demonstrated anticoagulant effect but 8-methyl derivative (3a) was a potent antiplatelet compound with IC50 numerically twice as low as the clinically used acetylsalicylic acid. A series of further mechanistic tests showed that 3a interferes with calcium signaling. The compound is also not toxic and in addition possesses vasodilatory activity as well. CONCLUSIONS: Compound 3a is a promising inhibitor of platelet aggregation, whose mechanism of action should be studied in detail.

Vitamin C-Sources, Physiological Role, Kinetics, Deficiency, Use, Toxicity, and Determination.

Vitamin C (L-ascorbic acid) has been known as an antioxidant for most people. However, its physiological role is much larger and encompasses very different processes ranging from facilitation of iron absorption through involvement in hormones and carnitine synthesis for important roles in epigenetic processes. Contrarily, high doses act as a pro-oxidant than an anti-oxidant. This may also be the reason why plasma levels are meticulously regulated on the level of absorption and excretion in the kidney. Interestingly, most cells contain vitamin C in millimolar concentrations, which is much higher than its plasma concentrations, and compared to other vitamins. The role of vitamin C is well demonstrated by miscellaneous symptoms of its absence-scurvy. The only clinically well-documented indication for vitamin C is scurvy. The effects of vitamin C administration on cancer, cardiovascular diseases, and infections are rather minor or even debatable in the general population. Vitamin C is relatively safe, but caution should be given to the administration of high doses, which can cause overt side effects in some susceptible patients (e.g., oxalate renal stones). Lastly, analytical methods for its determination with advantages and pitfalls are also discussed in this review.

Featuring ultimate sensitivity of high-resolution LC-MS analysis of phenolics in rat plasma.

Sensitive analysis of very low-molecular weight metabolites using liquid chromatography with quadrupole-time-of-flight mass spectrometry is challenging due to the high losses of ions in a time-of-flight analyzer. Improvement in sensitivity for these analytes via the optimization of advanced parameters, including quadrupole profile, ion guide parameters, and duty cycle, has been achieved. The optimization of the method was carried out using a large spectrum of structurally different compounds including (iso)flavonoids and their known metabolites. These compounds can be categorized into two major groups, that is, compounds with (iso)flavonoid core and low-molecular weight phenolics. The optimization of the duty cycle enabled up to a 15-fold increase in analyte responses while the contribution of tuning ion optics and quadrupole profile was negligible. The limits of quantifications of our new method were assessed using both standard solutions and rat plasma. They were decreased at least 10 times for several low-molecular weight phenolics enabling measurement of their concentrations in a range of 1-50 ng/mL in rat plasma after protein precipitation. Concurrently, the limits of quantifications for compounds with (iso)flavonoid core did not increase distinctly allowing their detection in a range of 0.5-10 ng/mL. The new method was used for the targeting of phenolics in biological samples from pharmacokinetics experiments.

Systematic review of pharmacokinetics and potential pharmacokinetic interactions of flavonolignans from silymarin.

Silymarin is an extract from the seeds (fruits) of Silybum marianum that contains flavonolignans and flavonoids. Although it is frequently used as a hepatoprotective agent, its application remains somewhat debatable, in particular, due to the low oral bioavailability of flavonolignans. Moreover, there are claims of its potential interactions with concomitantly used drugs. This review aims at a systematic summary and critical assessment of known information on the pharmacokinetics of particular silymarin flavonolignans. There are two known major reasons for poor systemic oral bioavailability of flavonolignans: (1) rapid conjugation in intestinal cells or the liver and (2) efflux of parent flavonolignans or formed conjugates back to the lumen of the gastrointestinal tract by intestinal cells and rapid excretion by the liver into the bile. The metabolism of phase I appears to play a minor role, in contrast to extensive conjugation and indeed the unconjugated flavonolignans reach low plasma levels after common doses. Only about 1%-5% of the administered dose is eliminated by the kidneys. Many in vitro studies tested the inhibitory potential of silymarin and its components toward different enzymes and transporters involved in the absorption, metabolism, and excretion of xenobiotics. In most cases, effective concentrations are too high to be relevant under real biological conditions. Most human studies showed no silymarin-drug interactions explainable by these suggested interferences. More interactions were found in animal studies, likely due to the much higher doses administered.

Biochanin A, the Most Potent of 16 Isoflavones, Induces Relaxation of the Coronary Artery Through the Calcium Channel and cGMP-dependent Pathway.

The dietary intake of flavonoids seems to be inversely related to cardiovascular mortality. The consumption of isoflavonoids is increasing in the general population, especially due to the use of food supplements and a variety of isoflavonoid-rich foods. However, detailed studies on the vascular influence of individual pure isoflavonoids are mostly missing. For this study, 16 isoflavonoids were initially screened for their vasorelaxant properties on rat aortas. The 2 most potent of them, biochanin A and glycitein, were further tested for the mechanism of action on porcine coronary arteries. They both induced an endothelium independent vascular relaxation, with EC50 below 6 and 17 µM, respectively. Biochanin A, but not glycitein, was able to block the vasoconstriction caused by KCl, CaCl2, serotonin, and U46619 in a dose-dependent manner. Another series of experiments suggested that the major mechanism of action of biochanin A was the inhibition of L-type calcium channels. Moreover, biochanin A in relatively small concentrations (2 - 4 µM) interfered with the cGMP, but not cAMP, pathway in isolated coronary arteries. These results indicate that some isoflavonoids, in particular biochanin A, are able to have vasodilatory effects in micromolar concentrations, which is of potential clinical interest for the management of cardiovascular pathologies.

A Mixture of Phenolic Metabolites of Quercetin Can Decrease Elevated Blood Pressure of Spontaneously Hypertensive Rats Even in Low Doses.

Quercetin is proven to decrease arterial blood pressure when given orally. Its bioavailability is, however, low and, therefore, its metabolites could rather be responsible for this effect. In particular, the colonic metabolites of quercetin, 3,4-dihydroxyphenylacetic acid (DHPA), 4-methylcatechol (4MC), and 3-(3-hydroxyphenyl)propionic acid (3HPPA), have been previously shown to decrease the blood pressure in spontaneously hypertensive rats (SHR). Interestingly, the mechanisms of action of these three metabolites are different. The aim of this study is hence to investigate if these metabolites can potentiate each other and thus decrease blood pressure in reduced doses. Three double-combinations of previously mentioned metabolites were administered to SHR as infusions to mimic a real biological situation. All combinations significantly decreased the blood pressure in SHR but there were important differences. The effect of DHPA and 4MC was mild and very short. A combination of DHPA with 3HPPA caused more pronounced effects, which were also rather short-lived. The last combination of 3HPPA and 4MC caused a long-lasting effect. In conclusion, certain combinations of quercetin metabolites have a more pronounced antihypertensive effect than single metabolites.

The Effect of Silymarin Flavonolignans and Their Sulfated Conjugates on Platelet Aggregation and Blood Vessels Ex Vivo.

Silymarin is a traditional drug and food supplement employed for numerous liver disorders. The available studies indicate that its activities may be broader, in particular due to claimed benefits in some cardiovascular diseases, but the contributions of individual silymarin components are unclear. Therefore, we tested silymarin flavonolignans as pure diastereomers as well as their sulfated metabolites for potential vasorelaxant and antiplatelet effects in isolated rat aorta and in human blood, respectively. Eleven compounds from a panel of 17 tested exhibited a vasorelaxant effect, with half maximal effective concentrations (EC50) ranging from 20 to 100 µM, and some substances retained certain activity even in the range of hundreds of nM. Stereomers A were generally more potent as vasorelaxants than stereomers B. Interestingly, the most active compound was a metabolite-silychristin-19-O-sulfate. Although initial experiments showed that silybin, 2,3-dehydrosilybin, and 2,3-dehydrosilychristin were able to substantially block platelet aggregation, their effects were rapidly abolished with decreasing concentration, and were negligible at concentrations ≤100 µM. In conclusion, metabolites of silymarin flavonolignans seem to have biologically relevant vasodilatory properties, but the effect of silymarin components on platelets is low or negligible.

Two flavonoid metabolites, 3,4-dihydroxyphenylacetic acid and 4-methylcatechol, relax arteries ex vivo and decrease blood pressure in vivo.

SCOPE: The flavonoid quercetin reduces arterial blood pressure in animals and humans but the mechanisms remains elusive. The aim of this study was to test the activity of flavonoid microbial metabolites, which can participate on the final vasorelaxant effect. METHODS AND RESULTS: Both ex vivo (isolated rat thoracic aorta and mesenteric artery) and in vivo (normotensive and spontaneously hypertensive rats) approaches were used in this study. 4-methylcatechol and 3,4-dihydroxyphenylacetic acid (DHPA) had greater vasorelaxant effects on mesenteric artery than 3-(3-hydroxyphenyl)propionic acid, the previously reported metabolite with vasorelaxant effect. In vivo testing confirmed their blood pressure decreasing effect given both as bolus and slow infusion. Their mechanism at molecular level was different. CONCLUSIONS: This study is the first to show that flavonoid metabolites DHPA and 4-methylcatechol decrease arterial blood pressure and hence a mixture of microbial metabolites formed in the gastrointestinal tract may be responsible for or contribute to the effect of orally ingested quercetin.

Aqueous injection of quercetin: An approach for confirmation of its direct in vivo cardiovascular effects.

Potential positive effects of flavonol quercetin on humans were suggested by many studies. However, it is not clear if these effects are mediated by quercetin or its metabolites. The in vivo confirmation of quercetin effects is largely hindered by its low water solubility and thus impossibility to test directly its impact. Therefore, a solid dispersion of quercetin with polyvinylpyrrolidone (PVP) was developed to prepare an injectable formulation of water-soluble quercetin. The optimized formulation provided a 20,000-fold increase in quercetin solubility. This formulation was tested on conventional and spontaneously hypertensive rats; it lowered their blood pressure in both short- and long-term basis. Pharmacokinetic data are also provided. This study reports for the first time an injectable water-soluble formulation of quercetin suitable for confirmation of its vascular effect in vivo.

Comprehensive review of cardiovascular toxicity of drugs and related agents.

Cardiovascular diseases are a leading cause of morbidity and mortality in most developed countries of the world. Pharmaceuticals, illicit drugs, and toxins can significantly contribute to the overall cardiovascular burden and thus deserve attention. The present article is a systematic overview of drugs that may induce distinct cardiovascular toxicity. The compounds are classified into agents that have significant effects on the heart, blood vessels, or both. The mechanism(s) of toxic action are discussed and treatment modalities are briefly mentioned in relevant cases. Due to the large number of clinically relevant compounds discussed, this article could be of interest to a broad audience including pharmacologists and toxicologists, pharmacists, physicians, and medicinal chemists. Particular emphasis is given to clinically relevant topics including the cardiovascular toxicity of illicit sympathomimetic drugs (e.g., cocaine, amphetamines, cathinones), drugs that prolong the QT interval, antidysrhythmic drugs, digoxin and other cardioactive steroids, beta-blockers, calcium channel blockers, female hormones, nonsteroidal anti-inflammatory, and anticancer compounds encompassing anthracyclines and novel targeted therapy interfering with the HER2 or the vascular endothelial growth factor pathway.

Flavonoid metabolite 3-(3-hydroxyphenyl)propionic acid formed by human microflora decreases arterial blood pressure in rats.

There are reports of positive effects of quercetin on cardiovascular pathologies, however, mainly due to its low biovailability, the mechanism remains elusive. Here, we report that one metabolite formed by human microflora (3-(3-hydroxyphenyl)propionic acid)relaxed isolated rat aorta and decreased arterial blood pressure in rats.

Novel bronchodilatory quinazolines and quinoxalines: synthesis and biological evaluation.

A series of heterocyclic derivatives analogous to (-)vasicinone, in which the vasicinone C-ring was replaced with alkyl chain terminated by tertiary amine was prepared. N3, C4-O, C4-S or C4-N were used as the sites of attachment. The 4-[3-(1-piperidyl)propylsulfanyl]derivatives displayed bronchodilatory effect at low micromolar concentrations on isolated rat trachea, and low toxicity both on Balb/c 3T3 mouse fibroblast cells and in mice.