Michał Bijak

Anticoagulant effect of polyphenols-rich extracts from black chokeberry and grape seeds

Blood coagulation consists of a series of zymogens that can be converted by limited proteolysis to active enzymes leading to the generation of thrombin. Fresh plasma and human thrombin was incubated with extracts from berries of Aronia melanocarpa or seeds of Vitis vinifera (0.5; 5; 50 μg/ml). The in vitro experiments showed that both extracts prolonged clotting time and decreased the maximal velocity of fibrin polymerization in human plasma. Moreover thrombin incubation with both extracts results in the inhibition of amidolytic activity of this enzyme. It gives hopes for development of diet supplements, which may be preventing thrombosis in pathological states.

Antithrombin effect of polyphenol-rich extracts from black chokeberry and grape seeds.

Thrombin is a serine protease that cleaves the peptide bonds in proteins located on the carboxyl side of arginine. Thrombin plays a central role in thromboembolic diseases, which are the major cause of mortality. The aim of the study was to estimate the effects of plant extracts on proteolytic properties of thrombin. Thrombin was incubated with polyphenol‐rich extracts from berries of Aronia melanocarpa or seeds of Vitis vinifera (0.5, 5, 50 µg/mL) and with polyphenols ((+)‐catechin, (−)‐epicatechin, gallic acid, chlorogenic acid, procyanidin B1, cyanidin, cyanidin 3‐glucoside, quercetin). The in vitro experiments showed that both extracts in all used concentrations inhibited proteolytic activity of thrombin observed as inhibition of thrombin‐induced fibrinogen polymerization, stabilized fibrin formation, and platelet aggregation. Moreover, thrombin amidolytic activity was inhibited by polyphenols belonging to the flavonoid class. Results presented in this study indicate that polyphenol‐rich extracts from berries of A. melanocarpa and seeds of V. vinifera may become promising dietary supplements in the prevention of thrombotic states. Copyright

The influence of conjugates isolated from Matricaria chamomilla L. on platelets activity and cytotoxicity

Cardiovascular diseases (CVD) remain the principal cause of death in both advanced and developing countries of the world. Blood platelets are involved in the pathogenesis of atherosclerosis and thrombosis. Platelet adhesion and aggregation are critical events that occur in unstable coronary syndromes. The current research is focused on the role of polysaccharide-polyphenolic conjugates isolated from chamomile (Matricaria chamomilla L.) at concentrations of 10, 25, 50 and 100 μg/mL on blood platelets (obtained from healthy donors and from patients received combined anti-platelet therapy complex with clopidogrel and acetylsalicylic acid) aggregation and experimentally induced cell toxicity. The treatment of PRP obtained from healthy donors with polyphenolic-polysaccharide conjugates from M. chamomilla (L.) (MC) resulted in a dose-dependent, decrease of platelet aggregation induced by multiple agonists (ADP, collagen and arachidonic acid). In this study we also observed that the MC reduced platelet aggregation in PRP obtained from patients with cardiovascular disorders. The result of testing the MC on human blood platelets, mouse fibroblast cultures L929 and human lung cells A549 did not show any cytotoxicity effects. Compounds obtained from M. chamomilla L. are potential composite to the development of a new anti-platelet agent, which could be an alternative to the currently used anti-platelet drugs.

Protective effects of grape seed extract against oxidative and nitrative damage of plasma proteins

Oxidative stress, vascular inflammation, endothelial dysfunction plays a crucial role in the pathogenesis of cardiovascular diseases. The aim of our in vitro study was to examine the antioxidative properties of grape seed extract, and its potential protective effect on the haemostatic function of human fibrinogen under oxidative stress conditions, induced by peroxynitrite (100 μM). The preincubation of plasma with the tested extract (0.5-50 μg/ml or 0.5-300 μg/ml) reduced the formation of 3-nitrotyrosine and diminished oxidation of thiol groups in plasma proteins. The low concentrations (0.5-50 μg/ml) of grape seed extract also decreased the level of carbonyl groups, however at higher concentrations (100-300 μg/ml) this effect was not observed. Furthermore, grape seed extract counteracted the inhibitory effect of peroxynitrite on human plasma clotting. The results obtained in this study indicate that components of the grape seed extract posses antioxidative properties and may be promising substances for the creation of new dietary supplements.

Polyphenol compounds belonging to flavonoids inhibit activity of coagulation factor X

Blood coagulation consists of series of zymogens which can be converted by limited proteolysis to active enzymes leading to the generation of thrombin and conversion of fibrinogen into fibrin by this enzyme. The activated factor X (FXa) forms prothrombinase complex on phosphatidylserine containing surface which is responsible for conversion of prothrombin to thrombin. One molecule of FXa generates more than 1000 thrombin molecules. Therefore FXa is a novel target for modern anticoagulant therapy. The aim of our present study is to examine the effects of the well-known plant polyphenolic compounds on factor Xa amidolytic activity and characterization of these interactions using bioinformatic ligand docking method. We observed that only four polyphenols belonging to flavonoids group: procyanidin B2, cyanidin, quercetin and silybin, had inhibitory effect on FXa activity. Bioinformatic analyses revealed that procyanidin B2, cyanidin, quercetin and silybin bound in the S1-S4 pockets located in vicinity of the FXa active site and blocked access of substrates to Ser195. The results presented here showed that flavonoids might be potential structural bases for design of new nature-based, safe, orally bioavailable direct FXa inhibitors.

Protective effects of (−)-epicatechin against nitrative modifications of fibrinogen

Fibrinogen appears to be particularly sensitive to toxic action of peroxynitrite; a potent oxidizing and nitrating species. An increased nitration of fibrinogen has been reported in cardiovascular diseases. The defense mechanisms against PN are crucial for complex hemostasis process. Flavonoids have antioxidative properties and could protect biomolecules against action of peroxynitrite. The aim of our studies was to establish, if (-)-epicatechin may in vitro protect fibrinogen molecule against peroxynitrite-induced nitration of tyrosines and change its thrombin-catalyzed polymerization. The exposure of purified fibrinogen (6 μM) to peroxynitrite (1-100 μM) resulted in both structural modifications and clotting ability of this glycoprotein. Peroxynitrite at the concentration of 1 μM increased maximum velocity of Fg polymerization, whereas exposure to 100 μM PN resulted in a significant decrease of Vmax. (-)-Epicatechin (1-100 μM) caused a dose-dependent inhibition of 3-nitrotyrosine formation in fibrinogen treated with peroxynitrite (100 μM) in both Western blot assays and C-ELISA assays. At the highest concentration of (-)-epicatechin (100 μM) the level of 3-NT in fibrinogen reached the control values. At lower doses (-)-epicatechin reduced tyrosine nitration by approx. 23% and 40% at the concentration of 1 μM and 10 μM, respectively. (-)-Epicatechin also abolished the pro-thrombotic effect of peroxynitrite on fibrinogen clotting. The presented in vitro results demonstrated for the first time that (-)-epicatechin might have protective effects against the impairment of structure and properties of Fg, caused by action of the strong biologic oxidant/nitration and inflammatory mediators.

Radical scavenging and antioxidant effects of Matricaria chamomilla polyphenolic-polysaccharide conjugates.

Matricaria chamomilla L. (MC), a member of the Asteraceae family, is one of the oldest medicinal plants, widely used worldwide for a variety of healing applications. Its recommendations, derived from both traditional and modern medicine, include numerous disorders such as inflammation, ulcers, wounds, gastrointestinal disorders, stomach ache, pharyngitis, rheumatic pain, as well as the other ailments. This work is focused on another aspect of the biological activity of chamomile polyphenolic-polysaccharide conjugates--their antioxidant properties in the protection of blood plasma components against in vitro oxidative stress. Measurements of DPPH and ABTS radical scavenging indicated considerable anti-free radical action of MC. Pre-incubation of blood plasma with MC considerably diminished the extent of ONOO(-)-induced oxidative modifications such as protein carbonyl groups, SH groups, 3-nitrotyrosine, as well as the formation of lipid hydroperoxides. The analysis of the FRAP assay result shows a considerable increase of ferric reducing ability of blood plasma in the presence of MC. The results obtained in this study indicate that polyphenolic-polysaccharide conjugates isolated from M. chamomilla substances possess antioxidant properties. The M. chamomilla macromolecular glycoconjugates may be useful in the creation of new natural-based medications or dietary supplements, helpful in the prevention and treatment of oxidative stress-mediated disorders.

Thrombin inhibitory activity of some polyphenolic compounds

Thrombin, also known as an active plasma coagulation factor II, belongs to the family of serine proteases and plays a crucial role in blood coagulation process. The process of thrombin generation is the central event of the hemostatic process and regulates blood coagulant activity. For this reason, thrombin inhibition is key to successful novel antithrombotic pharmacotherapy. The aim of our present study was to examine the effects of the well-known polyphenolic compounds on the activity of thrombin, by characterization of its interaction with selected polyphenols using different biochemical methods and biosensor BIAcore analyses. Only six compounds, cyanidin, quercetin, silybin, cyanin, (+)-catechin and (−)-epicatechin, of all examined in this study polyphenols caused the inhibition of thrombin amidolytic activity. But only three of the six compounds (cyanidin, quercetin and silybin) changed thrombin proteolytic activity. BIAcore analyses demonstrated that cyanidin and quercetin caused a strong response in the interaction with immobilized thrombin, while cyanin and (−)-epicatechin induced a low response. Lineweaver–Burk curves show that used polyphenol aglycones act as competitive thrombin inhibitors. Our results suggest that polyphenolic compounds might be potential structural bases and source to find and project nature-based, safe, orally bioavailable direct thrombin inhibitors.

Poststroke Depression as a Factor Adversely Affecting the Level of Oxidative Damage to Plasma Proteins during a Brain Stroke

Poststroke depression, the second most serious psychosomatic complication after brain stroke, leads to delay of the rehabilitation process and is associated with an increased disability and cognitive impairment along with increase in term mortality. Research into the biochemical changes in depression is still insufficiently described. The aim of our study was therefore to evaluate the possible association between plasma protein oxidative/nitrative damages and the development of poststroke depression. We evaluated oxidative/nitrative modifications of specific proteins by measurement of 3-nitrotyrosine and carbonyl groups levels using ELISA test. Additionally, we checked differences in proteins thiol groups by spectrophotometric assay based on reaction between DTNB and thiols. We also evaluated catalase activity in erythrocytes measured as ability to decompose H2O2. Correlation analysis was performed using Spearmans rank. We observed significant (P < 0.001) differences in all oxidative/nitrative stress parameters in brain stroke patients compared to healthy group. Our research shows that oxidative damage of proteins is correlated with the degree of poststroke depression, while nitrative changes do not show any relationship. We demonstrate a positive correlation between the concentration of carbonyl groups and the Geriatric Depression Scale and a negative correlation between the degree of depression and the concentration of -SH groups or catalase activity.

(1 → 3)-β-d-Glucan reduces the damages caused by reactive oxygen species induced in human platelets by lipopolysaccharides

LPS (lipopolysaccharide) induces platelet activation and is a well-known fundamental agent of septic shock and disseminated intravascular coagulation (DIC). Biological activity of (1→3)-β-D-glucan is related due to its anti-inflammatory, antioxidant, and antitumor properties. We focus our attention on the (1→3)-β-D-glucan (antiplatelet) properties. The main purpose of our study was to evaluate the influence of (1→3)-β-D-glucan from Saccharomyces cerevisiae on destructive activity of LPS (from Escherichia coli and Pseudomonas aeruginosa) on human blood platelets. We assess biochemically in vitro if (1→3)-β-D-glucan might combat the oxidative stress caused by LPS stroke associated with nitrative and oxidative damages of human platelet biomolecules. We also make an attempt by in silico molecular docking to determine the interactions between the molecules of (1→3)-β-D-glucan and LPS. Our conclusion is that protective mechanism of (1→3)-β-D-glucan against LPS action on blood platelets is due to as well: its antioxidant properties, as to its interaction with LPS-binding region of TLR4-MD-2 complex.