76 Original Papers Antiplatelet Effects of Flavonoids Mediated by Inhibition of Arachidonic Acid Based Pathway Authors Jana Karlíčková1, Michal Říha2,Tomáš Filipský2, Kateřina Macáková1, Radomír Hrdina2,Přemysl Mladěnka2 Affiliations 1 Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic 2 Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic Key words Abstract able concentration. Contrarily, many flavonoids, l" aggregation ! particularly those possessing an isolated 7-hy- l" arachidonic acid Flavonoids, important components of human di- droxyl group and/or a 4′-hydroxyl group, acted l" cyclooxygenase et, have been claimed to possess a significant anti- as antagonists on thromboxane receptors. Inter- l" flavonoid platelet potential, in particular due to their effects estingly, the substitution of the free 7-hydroxyl l" platelet l" thromboxane on the arachidonic acid cascade. Due to variable group by glucose might not abolish the activity. and incomplete results, this study was aimed at In conclusion, the consumption of few flavonoids delivering a detailed analysis of the effects of 29 in a diet, particularly of the isoflavonoids geniste- structurally relevant, mainly natural flavonoids in and daidzein, may positively influence platelet on three consecutive steps of the arachidonic acid aggregation. cascade. Only the isoflavonoids genistein and daidzein were shown to possess a marked cyclooxygen- Abbreviations ase-1 inhibitory activity, which was higher than ! that of acetylsalicylic acid using the isolated ovine AA: arachidonic acid enzyme, and physiologically relevant, although ASA: acetylsalicylic acid lower than acetylsalicylic acid in human platelets. COX-1: cyclooxygenase-1 None of the tested flavonoids possesses an effect EDTA: ethylenediaminetetraacetic acid on thromboxane synthase in a clinically achiev- PRP: platelet rich plasma received January 5, 2015 revised July 28, 2015 Introduction in particular by collagen, but other inducers like accepted July 30, 2015 ! ADP and thrombin play a role as well [2,3]. De- Bibliography Platelets are essential components of the human spite this fact, most of the current clinically used DOI http://dx.doi.org/ blood responsible for rapid blood coagulation drugs are irreversible inhibitors of COX-1, like 10.1055/s-0035-1557902 during injuries. On the other hand, excessive ASA, and antagonists on ADP receptors (clopido- Published online September 21, platelet aggregation is associated with cardiovas- grel, ticagrelor, prasugrel) [4]. There are no other This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. 2015 cular diseases, in particular with the risk of seri- drugs acting on other levels of the AA cascade in Planta Med 2016; 82: 76–83 © Georg Thieme Verlag KG ous or fatal coronary heart disease. Because both clinical settings. Such drugs blocking thrombox- Stuttgart · New York · decreased and increased platelet aggregation are ane synthase or acting as antagonists on throm- ISSN 0032‑0943 associated with pathological states, the process boxane receptors [5] might enrich the palette of Correspondence of aggregation has to be tightly controlled. The clinically useful drugs in the future. Since they Assoc. Prof. Přemysl homeostasis is very complex and scientists have act specifically on the described steps of platelet Mladěnka, Ph.D. not been able to precisely define the process in aggregation, they may have fewer side effects in Department of Pharmacology and Toxicology all aspects up to date [1]. There are several pro- comparison to inhibitors of COX-1, which is an Faculty of Pharmacy in Hradec aggregatory factors which enhance thrombus for- enzyme with many essential physiological roles. Králové mation. The central role in this cascade appears to Flavonoids are promising candidates to be both, Charles University in Prague Heyrovského 1203 be associated with the release of AA from the cy- natural modulators of the disruption of platelet 500 05 Hradec Králové toplasmatic membrane and its transformation in- aggregation, and antiplatelet drugs. There is a vast Czech Republic to prostaglandin H2 by platelet COX-1, with fur- amount of literature on the subject showing that Phone: + 420495067295 Fax:+420495067170 ther metabolism into thromboxane A2 by platelet many flavonoids are potent inhibitors of platelet [email protected] thromboxane synthase. This process is stimulated aggregation induced by molecules such as colla- Karlíčková J et al. Antiplatelet Effects of… Planta Med 2016; 82: 76–83 Original Papers 77 Fig. 1 Chemical structure of flavonoids tested in this study. Glc: glucose, Rha: rhamnose, Glu: glucu- ronic acid. gen, AA, thromboxane receptor agonist U-46619, ADP, and epi- Results nephrine [6–10]. The data on thrombin-induced aggregation are ! controversial, some claim that flavonoids have no effect [9,11], Initially, all flavonoids were tested for their possible effects on the other report positive effects [12]. Additionally, the inhibition of inhibition of ovine COX-1 in a concentration of 100 µM and com- platelet aggregation by flavonoids is a reversible process [9], pared to ASA. ASA was moderately active at this concentration, which is another important factor when considering possible the isoflavones genistein and daidzein were more potent inhib- side effects of the current antiplatelet therapy. Although many itors (p < 0.01), while all other flavonoids were essentially inac- mechanisms responsible for antiplatelet effects have been pro- tive (l" Fig. 2A). Considering these surprising results, we retested posed [13], only a few are documented by published studies. In ASA and the active isoflavonoids for their concentration-depen- particular, their effects on the AA-based aggregation cascade are dent effects. Herein, genistein was more potent at lower concen- well documented [10,14, 15] but there are several discrepancies tration again but comparable to ASA in higher concentrations. In ensuing from the use of different protocols (e.g., animal or human the case of daidzein, a threshold effect at about 40% was found platelets, different analytical procedures, use of washed platelets, (l" Fig. 2B). To ascertain if this could be valid for humans, human This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. platelets in plasma, or in the whole blood). Nevertheless, flavo- platelet suspension in plasma was used. In these physiological noids may potentiate the effects of ASA via different mechanisms conditions, ASA showed an excellent effect in units of µM and of action [15]. completely inhibited COX-1 in higher concentrations. Both isofla- A comprehensive investigation between the flavonoid structure vonoids were significantly less active, they did not reach full inhi- and the effects on the AA cascade including COX-1, thromboxane bition even at high, pharmacologically unachievable, concentra- synthase and thromboxane receptors is still missing. In addition, tions but their activity in units of µM could have a real clinical the effects on thromboxane synthase have been analyzed only in- relevance (l" Fig. 2C). The activities of daidzein and genistein directly so far. Therefore, this study was aimed at delivering a de- were similar in this set of experiments. tailed analysis of 29 flavonoids (l" Fig. 1), representing the most The second step of the AA cascade is the transformation of pros- commonly found natural flavonoids, and some relevant synthetic taglandin H2 into thromboxane A2 via thromboxane synthase. All congeners in order to establish structure-activity relationship on flavonoids were tested for their effect on this enzyme and com- three consecutive steps of the AA-based platelet aggregation us- pared to the known inhibitor 1-benzylimidazol. At the concen- ing human platelets. tration of 100 µM, only three flavonoids showed more than 25% inhibition, but all of them were clearly less potent than 25 µM of 1-benzylimidazole (l" Fig. 3A). These partly active flavonoids, 7- Karlíčková J et al. Antiplatelet Effects of… Planta Med 2016; 82: 76–83 78 Original Papers Fig. 3 Effects of flavonoids and 1-benzylimidazol on thromboxane syn- thase. A Comparison of all compounds at the concentration of 100 µM. For comparison, 25 µM of 1-benzylimidazol (BI 25) is shown. Grey area means the error of the method. All partly active flavonoids were less potent than 1-benzyimidazol but there were no significant differences among them. B Concentration-effect curves of partly active flavonoids. Fig. 2 Effects of flavonoids and ASA on COX-1. A Comparison of the ef- fects of all tested flavonoids on ovine COX-1. Grey area shows the error of the method. Compounds were tested at a concentration of 100 µM, several bration in order to ascertain reproducibility of the results: first, flavonoids were retested at higher concentrations, too (7-hydroxyflavone, the used concentration of U-46619 in the range of 0.75–1.5 µM taxifolin, luteolin, hesperetin, luteolin and taxifolin at 200 µM and troxeru- had to produce more than 90% aggregation, and second, epicate- tin at 400 µM). B Concentration-effect curves for effective isoflavonoids and ASA on inhibition of ovine COX-1. C Concentration-effect curves for chin at the concentration of 300 µM had to decrease the aggrega- effective isoflavonoids and ASA on inhibition of human