Experimental determination and computational design of antiaggregatory effect of polyphenolics

Među prirodnim spojevima prisutnim u svakodnevnoj prehrani, flavonoidi su pokazali povoljan učinak u prevenciji kardiovaskularnih bolesti koji se, barem djelomično, može pripisati antiagregacijskom učinku. S obzirom na farmakološki interes, u potrazi za antitrombocitnim lijekovima, potrebna je sustavna eksperimentalna procjena antiagregacijskog učinka flavonoida. Takvi podaci mogli bi služiti za QSAR modeliranje antiagregacijskog učinka, istraživanje signalnih putova i procjenu utjecaja na invitro testove agregacije trombocita. Skup od trideset flavonoida, odabran je za procjenu antiagregacijskog učinka, na uzorcima pune krvi pomoću Multiplate® funkcionalnog analizatora (Dynabyte, Njemačka) i ADP-a (ADPtest) kao slabog agonistaagregacije. Pet učinkovitih flavonoida iz ADPtesta je analizirano s četiri dodatna agonistaagregacije (arahidonska kiselina, kolagen, ristocetin i TRAP-6). Za računalno simuliranje antiagregacijskog učinka, generirano je 155 molekulskih deskriptora koji opisuju fizikalno-kemijska, odnosno globalna svojstva molekula i supstituenata. Od računalnih metoda za procjenu mehanizama djelovanja primijenjeno je hijerarhijsko formiranje klasteravišedimenzijskim ponovnim uzorkovanjem. Slučajna šuma, metoda statističkog učenja, korištena je za QSAR modeliranje. Laboratorijski rezultati su iskazani kao minimalna koncentracija flavonoida koja dovodi do statistički značajnog smanjenja agregacije trombocita u odnosu na netretirani uzorak (minimalna antiagregacijska koncentracija ‒ MINaAC). MINaACflavonoida, u pojedinim testovima agregacijekoja je potaknuta ADP-om, kolagenom, TRAP-6 i ristocetinom, bila je u sljedećim rasponima: 0,12‒122,07 μM; 15,26‒244,14 μM; 15,26‒122,07 μM i 0,06‒15,26 μM. U testu agregacije trombocita koja je potaknuta arahidonskom kiselinom, proagregacijski učinak je zapažen kod pinocembrin-7-metiletera, epikatehina, hesperetina i 3,6- dihidroksiflavona. Literaturni su podaci bili nedostatni za interpretaciju rezultata dobivenih tvorbom hijerarhijskih klastera uslijed raznolikih mehanizama djelovanja pojedinih flavonoida. Validacija predviđanja učinka koja se temelji na metodi slučajne šume, rezultirala je niskom točnošćupredviđanja od 40,67%. Mjerljivantiagregacijski učinak, na submikromolarnoj razini koncentracija flavonoida, sugerira da i svakodnevna konzumacija flavonoida prehranom može utjecati na in vivoagregaciju trombocita, što ukazuje i na njihovu moguću terapijsku primjenu. Temeljem antiagregacijskih testova s različitim induktorima agregacije, može se zaključiti da flavonoidi interferiraju s invitroagregacijom trombocita, ili antiagregacijski ili proagregacijski, što može utjecati na interpretaciju testova agregacije trombocita na punoj krvi. Razvoj pouzdanog QSAR modela onemogućuju raznovrsni mehanizmi djelovanja flavonoida kojima se ostvaruje antiagregacijski učinak. Stoga se daljnja istraživanja trebaju usmjeriti na pojedinačne mete i na povećanje broja analiziranih supstancija.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 their antiaggregatory activity. Due to the ever increasing pharmacological interest in antiplatelet agents, a systematic experimental evaluation of large flavonoid series is needed. This will serve as possible data set for QSAR modeling of antiaggregatory activity, assessment of signaling pathways and evaluation of the in vitro effects of flavonoids on platelet aggregation in whole blood. A set of thirty flavonoid aglycones was selected for the evaluation. Aggregation measurements were performed on the whole blood samples with multiple platelet functional analyzer (Dynabyte, Germany) and adenosine diphosphate (ADPtest) as a weak agonist of aggregation. Five potent flavonoids from the ADPtest were further analyzed using the four additional aggregation inducers (arachidonic acid, collagen, ristocetin and TRAP-6). Computational design of antiaggregatory effect was based on 155 molecular descriptors of physical and chemical properties; global properties of molecule and substituents. Method for the assessment of the possible mechanisms of action used was hierarchical clustering with multiscale bootstrap resampling. Random forest, a statistical learning method, was used for QSAR modeling. Laboratory 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). MINaAC of flavonoids in individual tests were reported in the following ranges: 0.12‒122.07 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. Literature data was inconclusive for proper interpretation of hierarchical clustering due to different mechanism by which flavonoids achieve antiaggregatory effect. Validation of random forest prediction model resulted in 40.67% accuracy. 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 therapeutic potential of some flavonoids. Based on the test with different agonists of aggregation it can be concluded that flavonoids interfere with in vitro platelet aggregation assays exhibiting either anti- or pro-aggregatory influencing the interpretation of the results of platelet aggregation. Development of reliable QSAR model under described settings is not possible due to different mechanisms responsible for antiaggregatory effect. Further studies should focus on specific targets; number of the analyzed substances should be increased

Experimental determination and computational design of antiaggregatory effect of polyphenolics

Među prirodnim spojevima prisutnim u svakodnevnoj prehrani, flavonoidi su pokazali povoljan učinak u prevenciji kardiovaskularnih bolesti koji se, barem djelomično, može pripisati antiagregacijskom učinku. S obzirom na farmakološki interes, u potrazi za antitrombocitnim lijekovima, potrebna je sustavna eksperimentalna procjena antiagregacijskog učinka flavonoida. Takvi podaci mogli bi služiti za QSAR modeliranje antiagregacijskog učinka, istraživanje signalnih putova i procjenu utjecaja na invitro testove agregacije trombocita. Skup od trideset flavonoida, odabran je za procjenu antiagregacijskog učinka, na uzorcima pune krvi pomoću Multiplate® funkcionalnog analizatora (Dynabyte, Njemačka) i ADP-a (ADPtest) kao slabog agonistaagregacije. Pet učinkovitih flavonoida iz ADPtesta je analizirano s četiri dodatna agonistaagregacije (arahidonska kiselina, kolagen, ristocetin i TRAP-6). Za računalno simuliranje antiagregacijskog učinka, generirano je 155 molekulskih deskriptora koji opisuju fizikalno-kemijska, odnosno globalna svojstva molekula i supstituenata. Od računalnih metoda za procjenu mehanizama djelovanja primijenjeno je hijerarhijsko formiranje klasteravišedimenzijskim ponovnim uzorkovanjem. Slučajna šuma, metoda statističkog učenja, korištena je za QSAR modeliranje. Laboratorijski rezultati su iskazani kao minimalna koncentracija flavonoida koja dovodi do statistički značajnog smanjenja agregacije trombocita u odnosu na netretirani uzorak (minimalna antiagregacijska koncentracija ‒ MINaAC). MINaACflavonoida, u pojedinim testovima agregacijekoja je potaknuta ADP-om, kolagenom, TRAP-6 i ristocetinom, bila je u sljedećim rasponima: 0,12‒122,07 μM; 15,26‒244,14 μM; 15,26‒122,07 μM i 0,06‒15,26 μM. U testu agregacije trombocita koja je potaknuta arahidonskom kiselinom, proagregacijski učinak je zapažen kod pinocembrin-7-metiletera, epikatehina, hesperetina i 3,6- dihidroksiflavona. Literaturni su podaci bili nedostatni za interpretaciju rezultata dobivenih tvorbom hijerarhijskih klastera uslijed raznolikih mehanizama djelovanja pojedinih flavonoida. Validacija predviđanja učinka koja se temelji na metodi slučajne šume, rezultirala je niskom točnošćupredviđanja od 40,67%. Mjerljivantiagregacijski učinak, na submikromolarnoj razini koncentracija flavonoida, sugerira da i svakodnevna konzumacija flavonoida prehranom može utjecati na in vivoagregaciju trombocita, što ukazuje i na njihovu moguću terapijsku primjenu. Temeljem antiagregacijskih testova s različitim induktorima agregacije, može se zaključiti da flavonoidi interferiraju s invitroagregacijom trombocita, ili antiagregacijski ili proagregacijski, što može utjecati na interpretaciju testova agregacije trombocita na punoj krvi. Razvoj pouzdanog QSAR modela onemogućuju raznovrsni mehanizmi djelovanja flavonoida kojima se ostvaruje antiagregacijski učinak. Stoga se daljnja istraživanja trebaju usmjeriti na pojedinačne mete i na povećanje broja analiziranih supstancija.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 their antiaggregatory activity. Due to the ever increasing pharmacological interest in antiplatelet agents, a systematic experimental evaluation of large flavonoid series is needed. This will serve as possible data set for QSAR modeling of antiaggregatory activity, assessment of signaling pathways and evaluation of the in vitro effects of flavonoids on platelet aggregation in whole blood. A set of thirty flavonoid aglycones was selected for the evaluation. Aggregation measurements were performed on the whole blood samples with multiple platelet functional analyzer (Dynabyte, Germany) and adenosine diphosphate (ADPtest) as a weak agonist of aggregation. Five potent flavonoids from the ADPtest were further analyzed using the four additional aggregation inducers (arachidonic acid, collagen, ristocetin and TRAP-6). Computational design of antiaggregatory effect was based on 155 molecular descriptors of physical and chemical properties; global properties of molecule and substituents. Method for the assessment of the possible mechanisms of action used was hierarchical clustering with multiscale bootstrap resampling. Random forest, a statistical learning method, was used for QSAR modeling. Laboratory 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). MINaAC of flavonoids in individual tests were reported in the following ranges: 0.12‒122.07 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. Literature data was inconclusive for proper interpretation of hierarchical clustering due to different mechanism by which flavonoids achieve antiaggregatory effect. Validation of random forest prediction model resulted in 40.67% accuracy. 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 therapeutic potential of some flavonoids. Based on the test with different agonists of aggregation it can be concluded that flavonoids interfere with in vitro platelet aggregation assays exhibiting either anti- or pro-aggregatory influencing the interpretation of the results of platelet aggregation. Development of reliable QSAR model under described settings is not possible due to different mechanisms responsible for antiaggregatory effect. Further studies should focus on specific targets; number of the analyzed substances should be increased

Stoljeće Coca-Cole - od biljnih droga do svjetskog brenda

In 1886 pharmacist John Pemberton introduced new medicine called Coca- Cola. Even though today it is not available at the pharmacy shelves, during past 130 years it has become one of the best selling soft carbonated drinks all over the world. The core of its success lays in its natural flavour, obtained mainly from the coca leaves and kola nuts. In this paper a brief history of Coca-Cola is given, accompanied with an overview of herbal drugs used for its preparation Coca (Erythroxylum spp., Erythroxylaceae) and Kola (Cola spp., Sterculiaceae)

Pripravci ljekovitih biljaka u liječenju prehlade i gripe

Influenza viruses represent a permanent global health threat with approximately one billion people infected each year. Although influenza is usually self-limited infection, there are specific populations in which complications are common and sometimes fatal. Therefore, antiviral therapy with neuraminidase inhibitors and less often with adamantanes is becoming preferable course of action. Nevertheless, herbal plants, reviewed in this article, and plant preparations are still a widespread option for influenza and other viral diseases both in developed and developing countries

Vino i ljudsko zdravlje

History of wine consumption begins in prehistoric periods; a vast majority of early nations was giving divine status to one of the most popular alcoholic drinks, simply made by fermentation of Vitis vinifera grapes. Nowadays, a unique consensus on the harmfulness of alcohol consumption exists among whole scientific society, but wine stays an exception. Numerous epidemiological studies are showing its tremendous benefit on consumer’s cardiovascular health status, and numerous in vitro studies show antimicrobial, anticancer, antidiabetic, hormonal and other beneficial biological effects. Researchers are attributing healing properties of wine to its high content of herbal secondary metabolites, like tannins and polyphenols with the emphasis on phytoalexin resveratrol. However, it is still hard to determine the exact physiological mechanisms of those presumably beneficial compounds and therefore, more effort in new sophisticated research is required

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 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 HSA-indomethacin- quercetin interactions, spectrofluorometric, docking, molecular dynamics studies, and quantum chemical calculations were performed. Results show that 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 xenobiotics’ half-life in the body, depending on the desired outcomes

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

Determination of the content of total polyphenols, non-tannin polyphenols and tannins in five species of the genus Teucrium L.

Background and Purpose: Many species of the genus Teucrium L. are used in folk medicine. These species contain various chemical compounds that contribute to its biological activities. No literature could be found in respect of the determination of total polyphenols, non-tannin polyphenols and tannins in the species T. halacsyanum Heldr., T. hyrcanicum L. and T. scorodonia L. Quantitative analysis of polyphenols and tannins was therefore performed. Materials and Methods: Plant material was collected over two consecutive seasons in the Pharmaceutical Botanical Garden “Fran Kušan” of the Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia. The content of total polyphenols, non-tannin polyphenols and tannins was determined by combination of spectrophotometric and hide powder method. Results: The quantity of total polyphenols of investigated species ranged from 0.87 to 4.1%. The content of polyphenols unadsorbed on hide powder (non-tannin polyphenols) was between 0.83 and 3.4%, while the quantity of tannins ranged from 0.04 to 1.4%. The sample of T. scorodonia that grows along the hedge contained the highest content of total polyphenols (4.1%) and tannins (1.4%), while the highest quantity of non-tannin polyphenols was found in the sample of T. chamaedrys (3.4%) collected in 2013. The sample of T. botrys collected in 2012 contained the lowest amounts of all investigated compounds. Conclusion: For the first time the content of total polyphenols, nontannin polyphenols and tannins in the species T. halacsyanum, T. hyrcanicum and T. scorodonia is reported. The content of all investigated compounds depended on the plant species investigated and year of collection

Antioksidacijski učinak i HPLC analiza listova planike - Arbutus unedo L.

Some of the major constituents of strawberry tree are flavonoids and phenolic acids that are responsible for some of the pharmacological activities i.e. adstringent, antihypertensive, antidiarrheal and antibacterial. Objective of this work was to determine flavonoid aglycones and phenolic acids in strawberry tree leaves collected during 12 months of 2013 using RP-HPLC-DAD and assess antioxidant activity using ABTS stable free radical. Antioxidant activity of strawberry tree leaves varied between 11% and 28%. The content of individual polyphenols was: chlorogenic acid 0.8 to 6.5 mg/g, caffeic acid 0.6–1.0 mg/g, p-coumaric acid 0.2–6.6 mg/g and quercetin 0.5–10.7 mg/g. The highest content of polyphenols was during winter months. Compared to the thin-layer chromatography analysis we have determined presence of additional phenolic acids (caffeic and p-coumaric) and quercetin – aglycone of quercitrin, isoquercitrin and hyperoside

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, and 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 mini-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