Characterization of the CYP3A4 Enzyme Inhibition Potential of Selected Flavonoids.

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC50 = 2.5 ± 0.6 µM, Ki = 2.4 ± 1.0 µM, kinact = 0.07 ± 0.01 min-1, kinact/Ki = 0.03 min-1 µM-1. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food-drug interactions have to be assessed clinically.

Indomethacin Increases Quercetin Affinity for Human Serum Albumin: A Combined Experimental and Computational Study and Its Broader Implications.

Human serum albumin (HSA) is the most abundant carrier protein in the human body. Competition for the same binding site between different ligands can lead to an increased active concentration or a faster elimination of one or both ligands. Indomethacin and quercetin both bind to the binding site located in the IIA subdomain. To determine the nature of the HSA-indomethacin-quercetin interactions, spectrofluorometric, docking, molecular dynamics studies, and quantum chemical calculations were performed. The results show that the indomethacin and quercetin binding sites do not overlap. Moreover, the presence of quercetin does not influence the binding constant and position of indomethacin in the pocket. However, binding of quercetin is much more favorable in the presence of indomethacin, with its position and interactions with HSA significantly changed. These results provide a new insight into drug-drug interactions, which can be important in situations when displacement from HSA or other proteins is undesirable or even desirable. This principle could also be used to deliberately prolong or shorten the xenobiotics' half-life in the body, depending on the desired outcomes.

Antithrombotic activity of flavonoids and polyphenols rich plant species.

Cardiovascular diseases represent one of the most notable health problems of the modern civilization. Stroke and heart attack often lead to lethal outcome; essential problem underneath being thrombus formation. Prophylactic approaches include acetylsalicylic acid and clopidogrel therapy on the level of primary hemostasis, i.e., primary clot formation. In the last five years, in the USA, health care expenses related to cardiovascular diseases have increased 50 %, to over 350 billion dollars. Thus, application of plant species and medicinal plants rich in polyphenols in prevention of thrombus formation are of interest. This is supported by the fact that the number of publications on antiaggregatory effect of polyphenols has doubled in the last decade. In this review we focus on antiaggregatory effect of most abundant polyphenols - flavonoids, the effect of plant extracts rich in polyphenols (propolis, species Salvia sp., Calamintha nepeta L., Lavandula angustifolia Mill., Melissa officinalis L, Mentha x piperita L., Ocimum basilicum L., Origanum vulgare L., Rosmarinus officinalis L.) on platelet aggregation, association of chemical composition and antioxidant properties with the observed biological effect, and possible clinical significance of the published results.

Screening of flavonoid aglycons' metabolism mediated by the human liver cytochromes P450.

Biological effects of flavonoids have been extensively studied in the last 80 years. As flavonoids represent a rather large group of compounds, data on metabolic biotransformations of these compounds is relatively limited to those well studied. The objective of this study was to screen the metabolism of 30 selected flavonoid aglycons mediated by the most relevant metabolic enzymes, human liver cytochromes P450. For this purpose, in vitro experiments with human liver microsomes and recombinant enzymes were conducted. To evaluate flavonoid's metabolism and structure of the products, high-performance liquid chromatography coupled with high-resolution mass spectrometry was used. Out of 30 flavonoids, 15 were susceptible to oxidative metabolism mediated by cytochromes P450. Dominant reactions were aromatic hydroxylation and O-demethylation, or a combination of these reactions. The dominant enzyme responsible for the observed metabolic reactions is CYP1A2, whereas other human liver cytochromes P450, namely, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, contribute to flavonoid metabolism to a lesser degree. These results, to some extent, contribute to the understanding of the metabolism of constituents found in antioxidant dietary supplements and their possible interactions with other xenobiotics, i.e., medicinal products.

Influence of flavonoids' lipophilicity on platelet aggregation.

Flavonoids are natural polyphenolic compounds present in a wide spectrum of plants that have a beneficial effect on human health. In the context of cardiovascular diseases related to plaque and thrombus formation, flavonoids exhibit an anti-aggregatory effect. Previously, it has been reported that all tested flavonoids exhibit an antiaggregatory effect on platelet aggregation when measured by impedance aggregometry on whole blood, in the test of aggregation induced by adenosine diphosphate (ADP). As not all flavonoids have the same targets within signaling pathways, an assumption of a common non-specific mechanism related to lipophilicity is to be considered. To test this hypothesis, reverse-phase thin layer chromatography was used to assess the lipophilicity of flavonoids; impedance aggregometry was used for testing of platelet aggregation and flow cytometry to monitor the influence of flavonoids on platelet activation. Lipophilicity analysis showed a highly negative correlation of logP and MINaAC for groups of flavones and flavanones. As determined by flow cytometry, the exposition of receptors necessary for the promotion of platelet activation and primary clot formation was diminished, i.e., lowered expression of the activated form of integrin αIIbß3 was observed in the presence of flavanone. Platelet membrane stabilization by flavonoids as a mechanism of antiaggregatory effect has been supported by impedance aggregometry experiments when specific inhibitors of platelet aggregation signaling pathways (U73122, indomethacin, verapamil) were used in the presence of a weak (ADP) and a strong (TRAP-6) agonist of aggregation. While individual flavonoids can have specific targets within aggregation signaling pathways, all flavonoids share a common non-specific mechanism of platelet aggregation inhibition related to their lipophilicity and membrane stabilization that, to some extent, contributes to their antiaggregatory effect.

The Effect of Flavonoid Aglycones on the CYP1A2, CYP2A6, CYP2C8 and CYP2D6 Enzymes Activity.

Cytochromes P450 are major metabolic enzymes involved in the biotransformation of xenobiotics. The majority of xenobiotics are metabolized in the liver, in which the highest levels of cytochromes P450 are expressed. Flavonoids are natural compounds to which humans are exposed through everyday diet. In the previous study, selected flavonoid aglycones showed inhibition of CYP3A4 enzyme. Thus, the objective of this study was to determine if these flavonoids inhibit metabolic activity of CYP1A2, CYP2A6, CYP2C8, and CYP2D6 enzymes. For this purpose, the O-deethylation reaction of phenacetin was used for monitoring CYP1A2 enzyme activity, coumarin 7-hydroxylation for CYP2A6 enzyme activity, 6-α-hydroxylation of paclitaxel for CYP2C8 enzyme activity, and dextromethorphan O-demethylation for CYP2D6 enzyme activity. The generated metabolites were monitored by high-performance liquid chromatography coupled with diode array detection. Hesperetin, pinocembrin, chrysin, isorhamnetin, and morin inhibited CYP1A2 activity; apigenin, tangeretin, galangin, and isorhamnetin inhibited CYP2A6 activity; and chrysin, chrysin-dimethylether, and galangin inhibited CYP2C8. None of the analyzed flavonoids showed inhibition of CYP2D6. The flavonoids in this study were mainly reversible inhibitors of CYP1A2 and CYP2A6, while the inhibition of CYP2C8 was of mixed type (reversible and irreversible). The most prominent reversible inhibitor of CYP1A2 was chrysin, and this was confirmed by the docking study.

The Inhibitory Effect of Flavonoid Aglycones on the Metabolic Activity of CYP3A4 Enzyme.

Flavonoids are natural compounds that have been extensively studied due to their positive effects on human health. There are over 4000 flavonoids found in higher plants and their beneficial effects have been shown in vitro as well as in vivo. However, data on their pharmacokinetics and influence on metabolic enzymes is scarce. The aim of this study was to focus on possible interactions between the 30 most commonly encountered flavonoid aglycones on the metabolic activity of CYP3A4 enzyme. 6ß-hydroxylation of testosterone was used as marker reaction of CYP3A4 activity. Generated product was determined by HPLC coupled with diode array detector. Metabolism and time dependence, as well as direct inhibition, were tested to determine if inhibition was reversible and/or irreversible. Out of the 30 flavonoids tested, 7 significantly inhibited CYP3A4, most prominent being acacetin that inhibited 95% of enzyme activity at 1 µM concentration. Apigenin showed reversible inhibition, acacetin, and chrysin showed combined irreversible and reversible inhibition while chrysin dimethylether, isorhamnetin, pinocembrin, and tangeretin showed pure irreversible inhibition. These results alert on possible flavonoid⁻drug interactions on the level of CYP3A4.

The Effect of Short-Toothed and DalmatianSage Extracts on Platelet Aggregation.

Sage (Salvia sp.) is a genus of a native Mediterranean plant used for aromatization of foods. In this study, total polyphenolic profile, antioxidant properties and antiplatelet effect of short-toothed sage (Salvia brachyodon Vandas) are analyzed and compared to most commonly used Salvia species (S. officinalis, Dalmatian sage). Content of total flavonoids was 0.08-0.23% and of phenolic acids 0.47-3.04%. Antioxidant DPPH assay showed higher antioxidant capacity of S. brachyodon (29-36 mg/mL of gallic acid equivalents) than of S. officinalis. In functional test of primary haemostasis, extracts of S. brachyodon have prevented platelet aggregation in nanomolar concentration (21 nM), thus showing potential in prevention of thrombus formation as functional food or dietary supplement. Antiplatelet activity was related to antioxidant capacity (r=0.7014, p=0.0352) indicating that prevention of aggregation is not caused by an individual component, but it is rather a result of synergistic effect of polyphenols. The obtained results are preliminary and a more detailed study of the possibility of applying the investigated plant species as supplements for the prevention of the problem of cardiovascular system and platelet aggregation is needed.

Propolis ethanolic extracts reduce adenosine diphosphate induced platelet aggregation determined on whole blood.

BACKGROUND: Propolis is a well-known bee product containing more than 2000 identified compounds. It has many beneficial effects on human health that include antibacterial, antiviral, anticancer and hepatoprotective justifying its use as a dietary supplement. Platelet aggregation plays crucial role in thrombus formation that can cause stroke or heart attacks. As cardiovascular diseases, including those caused by thrombus formation, are related to 50% of deaths of Western population, the objective of this study was to determine antiaggregatory activity of propolis on platelet aggregation on the whole blood samples. METHODS: Twenty one propolis samples from Southeast Europe were characterized by spectrophotometric methods to determine content of the total flavonoids and phenolic acids. High performance liquid chromatography coupled with diode array detection was used to identify and quantify individual polyphenols. Platelet aggregation was tested by impedance aggregometry on the whole blood samples of ten healthy volunteers. RESULTS: The mean content of total polyphenols was 136.14 mg/g and ranged from 59.23 to 277.39 mg/g. Content of total flavonoids ranged between 6.83 and 55.44 mg/g with the mean value of 19.28 mg/g. Percentage of total phenolic acids was in the range 8.79 to 45.67% (mean 26.63%). Minimal antiaggregatory concentration, representing the lowest concentration of propolis extract sample that can cause statistically significant reduction of aggregation, ranged from 5 µM to 10.4 mM. Samples of propolis with lower content of luteolin and higher content of pinocembrin-7-methyleter showed better antiplatelet activity i.e. lower values of minimal antiaggregatory concentration. CONCLUSIONS: This is the first study that shows antiaggregatory potential of propolis ethanolic extracts on the whole blood samples in the low micromolar concentrations suggesting that propolis supplementation may influence platelet aggregation and consequently thrombus formation. Further in vivo studies are needed to confirm the beneficial effects in prevention of cardiovascular diseases.

Warfarin and Flavonoids Do Not Share the Same Binding Region in Binding to the IIA Subdomain of Human Serum Albumin.

Human serum albumin (HSA) binds a variety of xenobiotics, including flavonoids and warfarin. The binding of another ligand to the IIA binding site on HSA can cause warfarin displacement and potentially the elevation of its free concentration in blood. Studies dealing with flavonoid-induced warfarin displacement from HSA provided controversial results: estimated risk of displacement ranged from none to serious. To resolve these controversies, in vitro study of simultaneous binding of warfarin and eight different flavonoid aglycons and glycosides to HSA was carried out by fluorescence spectroscopy as well as molecular docking. Results show that warfarin and flavonoids do not share the same binding region in binding to HSA. Interactions were only observed at high warfarin concentrations not attainable under recommended dosing regimes. Docking experiments show that flavonoid aglycons and glycosides do not bind at warfarin high affinity sites, but rather to different regions within the IIA HSA subdomain. Thus, the risk of clinically significant warfarin-flavonoid interaction in binding to HSA should be regarded as negligible.

Displacement of Drugs from Human Serum Albumin: From Molecular Interactions to Clinical Significance.

BACKGROUND: Human serum albumin (HSA) is the most abundant protein in human serum. It has numerous functions, one of which is transport of small hydrophobic molecules, including drugs, toxins, nutrients, hormones and metabolites. HSA has the ability to interact with a wide variety of structurally different compounds. This promiscuous, nonspecific affinity can lead to sudden changes in concentrations caused by displacement, when two or more compounds compete for binding to the same molecular site. OBJECTIVE: It is important to consider drug combinations and their binding to HSA when defining dosing regimens, as this can directly influence drug's free, active concentration in blood. CONCLUSION: In present paper we review drug interactions with potential for displacement from HSA, situations in which they are likely to occur and their clinical significance. We also offer guidelines in designing drugs with decreased binding to HSA.

Quantitative Analysis of Phenolic Acids and Antiplatelet Activity of Melissa officinalis Leaf Extracts.

The contents of total phenolic acids and rosmarinic acid were determined and the antiplatelet potential assessed of extracts of lemon balm (Melissa officinalis L., Lamiaceae) leaf samples collected during 2014 and 2015. The quantity of total phenolic acids ranged from 4.8 to 8.5%, while the rosmarinic acid content ranged from 1.78 to 4.35%. Results of statistical analysis showed that the undiluted extracts have antiplatelet activity in the ADP test (final concentration 31.25 mg/L). While there are reports on antiplatelet activity of lemon balm essential oil, to the best of our knowledge, this is the first report on antiplatelet activity of lemon balm leaf extracts.

Aromatic hydroxylation of salicylic acid and aspirin by human cytochromes P450.

Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids-salicyluric acid and gentisuric acid-and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.

Time- and NADPH-dependent inhibition of cytochrome P450 3A4 by the cyclopentapeptide cilengitide: significance of the guanidine group and accompanying spectral changes.

Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVß3 and αVß5 integrins. The candidate drug showed unexpected inhibition of cytochrome P450 (P450) 3A4 at high concentrations, that is, a 15-mM concentration caused attenuation of P450 3A4 activity (depending on the probe substrate): 15-19% direct inhibition, 10-23% time-dependent inhibition (30-minute preincubation), and 54-60% metabolism-dependent inhibition (30-minute preincubation). The inactivation efficiency determined with human liver microsomes was 0.003 ± 0.001 min(-1) mM(-1) and was 0.04 ± 0.01 min(-1) mM(-1) with baculovirus-based microsomes containing recombinant P450 3A4. Neither heme loss nor covalent binding to apoprotein could explain the observed reductions in residual activity. Slowly forming type II difference spectra were observed, with maximum spectral changes after 2 hours. Binding to both reduced and oxidized P450 3A4 was observed, with apparent Kd values of 0.66 µM and 6 µM. The significance of the guanidine group in inhibition was demonstrated using ligand binding spectral changes and inactivation assays with guanidine analogs (debrisoquine, N-acetylarginine-O-methyl ester) and the acetylated ornithine derivative of cilengitide. The observed inhibition could be explained by direct inhibition, plus by formation of stable complexes with both ferric and ferrous forms of heme iron and to some extent by the formation of reactive species capable to react to the protein or heme. Formation of the complex required time and NADPH and is attributed to the guanidino group. Thus, the NADPH-dependent inhibition is considered to be mainly due to the formation of a stable complex rather than the formation of reactive species.

A comparison of the nutritional value and food safety of organically and conventionally produced wheat flours.

Growing interest in organic agriculture has prompted this study aiming to evaluate nutritional content of wheat flours originating from organic and conventional production systems. Obtained results showed that organic samples had significantly lower protein content and lower levels of Ca, Mn and Fe compared to conventional samples. Protein digestibility and levels of K, Zn and Mo were significantly higher in organic than in conventional wheat flours. Regarding undesirable metals, significantly higher levels of As and Cd were found in conventional compared to organic wheat flours. Although the mean concentrations of zearalenone and ochratoxin A were higher in conventional than in organic flours, this difference was not significant. This study revealed that organic agriculture has the potential to yield products with some relevant improvements in terms of high quality proteins and microelements contents, while the reduction in contamination with toxic elements and mycotoxins may be accomplished.

Quantitative Determination of Flavonoids and Chlorogenic Acid in the Leaves of Arbutus unedo L. Using Thin Layer Chromatography.

The plant species Arbutus unedo shows numerous beneficial pharmacological effects (antiseptic, antidiabetic, antidiarrheal, astringent, depurative, antioxidant, antihypertensive, antithrombotic, and anti-inflammatory). For the medicinal use, standardization of extracts is a necessity, as different compounds are responsible for different biological activities. In this paper, we analyze monthly changes in the content of quercitrin, isoquercitrin, hyperoside, and chlorogenic acid. Methanolic extracts of the leaves are analyzed by HPTLC for the identification and quantification of individual polyphenol, and DPPH test is used to determine antioxidant activity. Based on the results obtained, the leaves should be collected in January to obtain the highest concentrations of hyperoside and quercitrin (0.35 mg/g and 1.94 mg/g, resp.), in June, July, and October for chlorogenic acid (1.45-1.46 mg/g), and for the fraction of quercitrin and isoquercitrin in November (1.98 mg/g and 0.33 mg/g, resp.). Optimal months for the collection of leaves with the maximum recovery of individual polyphenol suggested in this work could direct the pharmacological usage of the polyvalent herbal drugs.

Determination of Flavonoids, Phenolic Acids, and Xanthines in Mate Tea (Ilex paraguariensis St.-Hil.).

Raw material, different formulations of foods, and dietary supplements of mate demands control of the content of bioactive substances for which high performance thin layer chromatography (TLC), described here, presents simple and rapid approach for detections as well as quantification. Using TLC densitometry, the following bioactive compounds were identified and quantified: chlorogenic acid (2.1 mg/g), caffeic acid (1.5 mg/g), rutin (5.2 mg/g), quercetin (2.2 mg/g), and kaempferol (4.5 mg/g). The results obtained with TLC densitometry for caffeine (5.4 mg/g) and theobromine (2.7 mg/g) show no statistical difference to the content of total xanthines (7.6 mg/g) obtained by UV-Vis spectrophotometry. Thus, TLC remains a technique of choice for simple and rapid analysis of great number of samples as well as a primary screening technique in plant analysis.

Interference of selected flavonoid aglycons in platelet aggregation assays.

BACKGROUND: Flavonoids are widely distributed across the plant kingdom and are therefore common ingredients in an everyday diet. Some flavonoids have a potential to affect platelet aggregation; most often antiaggregatory effects of flavonoids are observed. The objective of this research was to evaluate the in vitro effect of a selected set of flavonoids on platelet aggregation in whole blood. METHODS: The effect of five selected flavonoids (pinocembrin-7-methylether, epicatechin, hesperetin, 6-hydroxyflavone and 3,6-dihydroxyflavone) on platelet aggregation was studied in the citrated whole blood samples collected from 75 healthy volunteers. A Multiplate(®) impedance analyzer and five different aggregation inducers (ADP, arachidonic acid, collagen, ristocetin and TRAP-6) were utilized for the analysis of samples. RESULTS: Minimal antiaggregatory concentrations (MINaAC) of flavonoids in individual tests were reported in the following ranges: 0.12-1.91 µM; 15.26-244.14 µM; 15.26-122.07 µM; and 0.06-15.26 µM for ADP, collagen, TRAP-6 and ristocetin aggregation-inducers, respectively. When arachidonic acid was used for induction of platelet aggregation, a proaggregatory effect was observed for pinocembrin-7-methylether, epicatechin, hesperetin and 3,6-dihydroxyflavone, while the expected antiaggregatory effect was observed only for 6-hydroxyflavone (MINaAC=7.63 µM). CONCLUSIONS: Flavonoids interfere with in vitro platelet aggregation assays exhibiting either anti- or proaggregatory effects in concentration ranges that can be achieved in circulation by dietary intake. Thus, dietary intake of flavonoids should be taken into account when interpreting the results of whole blood platelet aggregation.

Evaluation of antiaggregatory activity of flavonoid aglycone series.

BACKGROUND: Among natural compounds, present in every day diet, flavonoids have shown beneficial effect in prevention of cardiovascular diseases that can be attributed, at least partially to the described antiaggregatory activity i.e. antiplatelet effects of flavonoids. Due to the ever increasing pharmacological interest in antiplatelet agents a systematic experimental evaluation of large flavonoid series is needed. METHODS: A set of thirty flavonoid aglycones has been selected for the evaluation. All measurements of aggregation were done under standardized and firmly controlled in vitro conditions. The whole blood samples, multiple platelet functional analyzer and adenosine diphosphate (ADP) as a weak agonist of aggregation were selected for this purpose. RESULTS: The results were expressed as minimal concentration of flavonoid that can significantly lower the platelet aggregation compared to the corresponding untreated sample (minimal antiaggregatory concentration--MINaAC). All analyzed flavonoids exhibited antiaggregatory activity MINaAC ranging from 0.119 µM to 122 µM, while the most potent representatives were 3,6-dihydroxyflavone (0.119 µM) and syringetin (0.119 µM). CONCLUSIONS: Measurable antiplatelet activity established at submicromolar flavonoid concentrations suggests that even a dietary consumption of some flavonoids can make an impact on in vivo aggregation of platelets. These findings also point out a therapeutical potential of some flavonoids.