Flavonolignans inhibit the arachidonic acid pathway in blood platelets

Bijak and Saluk-Bijak BMC Complementary and Alternative Medicine (2017) 17:396 DOI 10.1186/s12906-017-1897-7 RESEARCH ARTICLE Open Access Flavonolignans inhibit the arachidonic acid pathway in blood platelets Michal Bijak* and Joanna Saluk-Bijak Abstract Background: Arachidonic acid metabolism by cyclooxygenase (COX) is a major pathway for blood platelets’ activation, which is associated with pro-thrombotic platelet activity and the production of pro-inflammatory mediators. Inhibition of COX activity is one of the major means of anti-platelet pharmacotherapy preventing arterial thrombosis and reducing the incidence of cardiovascular events. Recent studies have presented that a silymarin (standardized extract of Milk thistle (Silybum marianum)) can inhibit the COX pathway. Accordingly, the aim of our study was to determine the effects of three major flavonolignans (silybin, silychristin and silydianin) on COX pathway activity in blood platelets. Methods: We determined the effect of flavonolignans on arachidonic acid induced blood platelet aggregation, COX pathway metabolites formation, as well as COX activity in platelets. Additionally, we analysed the potential mechanism of this interaction using the bioinformatic ligand docking method. Results: We observed that tested compounds decrease the platelet aggregation level, both thromboxane A2 and malondialdehyde formation, as well as inhibit the COX activity. The strongest effect was observed for silychristin and silybin. In our in silico study we showed that silychristin and silybin have conformations which interact with the active COX site as competitive inhibitors, blocking the possibility of substrate binding. Conclusions: The results obtained from this study clearly present the potential of flavonolignans as novel antiplatelet and anti-inflammatory agents. Keywords: Flavonolignans, Silybin, Silychristin, Silymarin, Arachidonic acid, Blood platelet, Cyclooxygenase Background Blood platelets are the smallest, un-nucleated morphotic elements of human blood that play a major role in the blood coagulation system. The biological activity of platelets, both in physiological processes as well as under pathological conditions, is dependent on the degree of their activation. A platelet’s activation process, despite the absence of a nucleus, is very complex and associated with elements of enzymatic signal transduction chains [1]. After the platelets’ activation, signal transduction leads to mobilization of intracellular calcium ions (Ca2+). High intracellular concentration of Ca2+ results in activation of phospholipases, which are responsible for the release of cell membrane phospholipids’ enzymatic hydrolyses. These include, for example, the precursor of * Correspondence: Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland essential bioactive eicosanoids – 5,8,11,14-eicosatetraenoic acid called arachidonic acid (AA), which is a 20carbon polyunsaturated fatty acid. AA released from the membranes is enzymatically oxidized, transformed by the cyclic peroxide prostaglandin synthase known as cyclooxygenase (COX) into intermediate products: proinflammatory prostaglandins and pro-thrombotic thromboxane A2 (TXA2) [2, 3]. These are accompanied by production of reactive oxygen species (ROS) [4]. TXA2 is generated from prostaglandin H2, formed by COX through thromboxane-A synthase. TXA2 is an autocrine or paracrine mediator in the nearby tissues surrounding its production site. TXA2 is a very strong blood platelet activator acting as a pro-aggregator and vasoconstrictor mediator, leading to increased platelet aggregation. This plays a pivotal role in the growth and stabilization of a coronary thrombus [5]. TXA2 is formed in platelets in © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bijak and Saluk-Bijak BMC Complementary and Alternative Medicine (2017) 17:396 response to local stimuli and exerts an activating effect within a short distance of its biosynthesis. AA metabolism by COX is a major pathway of blood platelets activation, and is associated with pro-thrombotic platelets’ activity and the production of pro-inflammatory mediators. AA addition in vitro to platelet rich plasma causes a burst of oxygen consumption, TXA2 generation and platelet aggregation [6]. One of the major method in anti-platelet pharmacotherapy of preventing arterial thrombosis is inhibition of COX activity. The results of clinical studies have shown that intake of aspirin, or different aspirin-like COXinhibitors, reduces the incidence of cardiovascular events [7]. Low-dose aspirin (40 mg per day) supplementation reduces the risk of serious cardiovascular events by 12% and non-fatal myocardial infarction by 18%. This dose is able to inhibit a large proportion of thromboxane A2 release provoked acutely by the platelets’ response. Aspirin is also able to reduce the risk of secondary thrombotic events by about 25% [8]. Experiments performed on human monocytes have shown that flavonolignans – active chemical compounds presented in a silymarin (standardized extract from of Milk thistle (Silybum marianum)) inhibit the COX pathway [9]. In accordance with this observation, the aim of our study was to determine the effects of three major flavonolignans (silybin, silychristin and silydianin) on COX pathway activity in blood platelets. Methods Reagents Dimethyl sulfoxide (DMSO), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4-(2Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), glucose, trichloroacetic acid, thiobarbituric acid, Tris, flavonolignans (silybin, silychristin and silydianin) were all obtained from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Arachidonic acid was purchased from Chrono-Log (Havertown, PA USA). All other chemicals were reagent grade or the highest-quality available. Blood samples Blood samples collected from different healthy donors were purchased from the Regional Center for Transfusion Medicine in Lodz (Poland). All samples were drawn in the morning, from fasting donors. All donors were checked by a medical doctor and found to have no cardiovascular disorders, allergy, lipid or carbohydrate metabolism disorders, nor were they being treated with any drugs [10]. Our analysis of the blood samples was performed under the guidelines of the Helsinki Declaration for (...truncated)