Trifolium pallidum and Trifolium scabrum extracts in the protection of human plasma components Joanna Kolodziejczyk-Czepas, Beata Olas, Joanna Malinowska, Barbara Wachowicz, Barbara MoniuszkoSzajwaj, Iwona Kowalska, et al. Journal of Thrombosis and Thrombolysis A Journal for Translation, Application and Therapeutics in Thrombosis and Vascular Science ISSN 0929-5305 Volume 35 Number 2 J Thromb Thrombolysis (2013) 35:193-199 DOI 10.1007/s11239-012-0792-9 1 23 Your article is protected by copyright and all rights are held exclusively by Springer Science+Business Media, LLC. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy J Thromb Thrombolysis (2013) 35:193–199 DOI 10.1007/s11239-012-0792-9 Trifolium pallidum and Trifolium scabrum extracts in the protection of human plasma components Joanna Kolodziejczyk-Czepas • Beata Olas • Joanna Malinowska Barbara Wachowicz • Barbara Moniuszko-Szajwaj • Iwona Kowalska • Wieslaw Oleszek • Anna Stochmal • Published online: 4 August 2012  Springer Science+Business Media, LLC 2012 Abstract Clovers (genus: Trifolium) have been used in traditional medicine by many cultures, but the biological activity of the most of these plants still remains unknown. The aim of our in vitro study was to assess the antioxidative action of phenolic extracts from aerial parts of Trifolium scabrum and Trifolium pallidum in human blood plasma, exposed to oxidative stress. In the present study we also demonstrate, for the first time the effects of the tested extracts on coagulative properties and fibrinolytic activity of blood plasma. The protective properties of the examined extracts (0.5–50 lg/ml) against peroxynitrite-induced oxidative stress were estimated by the measurements of 3-nitrotyrosine, thiol groups and the thiobarbituric acid-reactive substances levels. The extracts considerably prevented the oxidative and nitrative damage to plasma proteins. Even the lowest doses of the Trifolium extracts (0.5 lg/ml) were able to markedly reduce 3-nitrotyrosine formation (by about 50 %) and to increase the level of –SH groups (by about 30 %), in comparison to the plasma exposed to ONOO- in the absence of the extracts. The protective action of all the used concentrations of the Trifolium extracts in the prevention of lipid peroxidation was also found. The tested extracts influenced neither the coagulative properties nor fibrinolytic J. Kolodziejczyk-Czepas (&)  B. Olas  J. Malinowska  B. Wachowicz Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland e-mail: joannak@biol.uni.lodz.pl B. Moniuszko-Szajwaj  I. Kowalska  W. Oleszek  A. Stochmal Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, Czartoryskich 8, 24-100 Pulawy, Poland activity of plasma. Moreover, the extracts were able to significantly reduce the inhibitory effect of ONOO- on fibrinolytic activity of plasma (assessed with the use of a chromogenic substrate for plasmin). Keywords Clover  Plasma  Fibrinolysis  Oxidative stress  Trifolium Introduction Oxidative stress, a consequence of the imbalance between the enhanced production of reactive oxygen and nitrogen species (ROS/RNS) and their elimination, plays an important role in the pathogenesis of atherosclerosis, ischemic heart disease, hypertension, cardiomyopathies, cardiac hypertrophy and congestive heart failure [1]. Oxidants generated in the cardiovascular system may significantly affect all components of the haemostatic system and lead to dysfunction of vascular endothelium, alterations in the coagulation process as well as impaired fibrinolytic activity of blood plasma [2–4]. The use of exogenous substances displaying antioxidative properties is one of prophylactic and therapeutic strategies for the protection of the cardiovascular system. On the other hand, clinical administration of synthetic exogenous antioxidants is controversial, since it has been shown that two synthetic antioxidants, such as butylated hydroxyanisole and butylated hydroxytoluene may evoke toxic effects [5]. Plant-derived substances, enhancing the physiological antioxidative defence are believed to be non-toxic and able to effectively counteract the biological consequences of oxidative stress. Investigation of therapeutic properties of extracts derived from clovers (genus: Trifolium) is a promising trend in phytopharmacological research for a variety of reasons, including the diversity of 123 Author's personal copy 194 chemical components, widespread occurrence of these plants as well as low costs of cultivation. Trifolium species synthesize numerous biologically active substances: isoflavones (phytoestrogens), clovamides (caffeic acid esters), phenolic acids and other compounds [6, 7]. Therapeutic administration of the Trifolium-derived extracts and supplements is mainly based on the traditional medicine recommendations; however some data from the scientific research are also available. The vast majority of these reports concerns Trifolium pratense (red clover) and its phytoestrogenic action, being a result of isoflavone content [8]. In the present in vitro study, we assessed the antioxidative properties of plant extracts, obtained from two clovers: Trifolium scabrum (rich in isoflavones) and Trifolium pallidum (rich in phenolic acids and clovamides) and their possible role in the protection of blood plasma components, particularly fibrinolytic proteins. Despite the fact that herbal mixtures and medicines are thought to be safe, it should be noted that they may influence haemostasis, and even cause thrombosis [9]. In the available literature, both the information on the anti[10] and procoagulative [11] effects of various plant-derived extracts are present. Therefore, to exclude possibility of the interaction of the examined extracts with haemostatic proteins, we assessed their effect on coagulometric parameters and the fibrinolytic activity of human plasma. Materials and methods Plant material and reagents Seeds of T. pallidum Waldst. et Kit. (TRIF 253/95) and T. scabrum L. (TRIF 120/79) were obtained from GeneBank, Leibniz Institute of Plant Genetics and Crop Plant Research (Gatersleben, Germany). Seeds were planted at experimental plots at Institute of Soil Science and Plant Cultivation, State Research Institute in Pulawy, Poland. The voucher samples have been deposited in the Department of Biochemistry and Crop Quality of Institute. Peroxynitrite was synthesized according to the method of Pryor and Squadrito [12]. Anti-3-nitrotyrosine polyclonal antibody were purchased from Abcam (Cambridge, UK). StreptABComplex/HRP polyclonal swine anti-goat, mouse, rabbit immunoglobulins, multi-link were from DAKO (Glostrup, Denmark). Chromogenic substrate for plasmin (S-2238TM) was purchased from Chromogenix (Italy). Other reagents were obtained from Sigma (St. Louis, MO, USA). Separation of phenolic fractions of T. pallidum and T. scabrum The isolation of phenolic fractions was performed according to previously developed procedures for Medicago 123 J. Kolodziejczyk-Czepas et al. sativa L. [13]. Dried phenolic fractions of T. pallidum and T. scabrum contained: phenolic acids—14.20 mg/g; 0.66 mg/g (equivalent of chlorogenic acid), flavonoids— 0.61 mg/g; 7.67 mg/g (equivalent of quercetin glucoside), isoflavones—7.31 mg/g; 72.76 mg/g (equivalent of daidzein) and clovamides—12.94 mg/g (equivalent of chlorogenic acid); lack, respectively [7]. Stock solutions of tested extracts were prepared in 50 % dimethylsulfoxide, and its effects were excluded. Blood plasma isolation and samples preparation Blood was obtained from young (20–25 years) healthy, non-smoking volunteers and collected on ACD solution (citric acid/citrate/dextrose; 5:1; v/v; blood/ACD). Plasma samples were preincubated for 5 min at 37 C with the examined extracts, added to the final concentration range of 0.5–50 lg/ml, and then exposed to 100 lM peroxynitrite (ONOO-). To assess the antioxidative effect Trifolium extracts, the samples of plasma treated with peroxynitrite in the absence of T. scabrum or T. pallidum extracts were performed. For the measurements of the pro-thrombin time (PT) and thrombin time (TT), three the most representative concentrations (0.5, 5 and 50 lg/ml) from the concentration range of the tested extracts, were chosen. The control samples (plasma untreated with the extracts and/or peroxynitrite), were also prepared. The experiments with blood plasma and the extracts only (without adding ONOO-) were carried out; no prooxidative effect was found. Determination of 3-nitrotyrosine in human plasma proteins by the competitive ELISA test The detection of 3-nitrotyrosine in blood plasma was performed according to the method described by Khan et al. [14] and modified by Olas et al. [15]. Concentrations of nitrated proteins were estimated from the standard curve, as the nitro-fibrinogen equivalents. Determination of thiols The concentration of thiol groups in blood plasma was determined by using 5,50 -dithio-bis(2-nitro-benzoic acid) (DTNB) [16]. Thiobarbituric acid-reactive substances (TBARS) generation in blood plasma (analysis of lipid peroxidation) The procedure was performed according to the protocol described previously [17]. The obtained results were expressed as nanomoles of TBARS per milliliter of plasma. Author's personal copy Trifolium pallidum and Trifolium scabrum extracts Measurements of pro-thrombin time and thrombin time PT and TT were determined coagulometrically (Optic Coagulation Analyser model K-3002; Kselmed, Grudziadz, Poland). Human plasma (50 ll) was incubated with 50 ll of thromboplastin (Sis Biomed; commercial preparation was dissolved in 2 ml of deionized water) for 1 min at 30 C on block heater. Then, cuvettes were transferred to the measuring holes and 50 ll of 25 mM CaCl2 was added. In the measurements of thrombin time, after the 1 min of preincubation at 30 C on block heater, cuvettes (containing 50 ll of plasma) were transferred to the measuring holes, and then 100 ll of thrombin was added (final concentration—1 U/ml). 195 these assays, a kinetic protocol in a microplate reader at 415 nm, was used. Data analysis To eliminate uncertain data, the Q-Dixon test was used. The statistical analysis was performed with one-way ANOVA test and POST Hoc test (Bonferoni). Statistical differences were confirmed using the Student t test; p \ 0.05 was considered as statistically significant. Results The activity of fibrinolytic system was induced by streptokinase, and assessed by the amidolytic method with the use of chromogenic substrate for plasmin, S-2251TM. Plasma was diluted ten times in 0.05 M Tris/HCl buffer (pH 7.4) and pre-incubated with streptokinase (at the final concentration of 100 U/ml) for 10 min, at 37 C. Then, substrate was added to the final concentration of 0.6 mM. Measurements of the amidolytic activity were performed in untreated (control) plasma, plasma samples treated with ONOO- in the presence or absence of the tested extracts, and plasma samples treated with the extracts only. For The exposure of blood plasma to peroxynitrite, used at the concentration of 100 lM, induced oxidative and nitrative alterations of plasma components (Figs. 1, 2, 3). A noticeable decrease of plasma amidolytic activity (by about 60 %) was also observed (p \ 0.001). A strong increase of 3-nitrotyrosine level (a marker of peroxynitrite action) in plasma proteins was detected (p \ 0.001), while the level of thiol groups was reduced by about 50 %, in comparison to the control samples (untreated plasma) (Figs. 1, 2). Measurements of the TBARS generation revealed approximately twofold increase of lipid peroxidation in blood plasma (Fig. 2; p \ 0.001). In plasma samples incubated with ONOO- in the presence of the extract T. scabrum or T. pallidum (0.5–50 lg/ml), the extent of oxidative/nitrative damage of blood plasma proteins was significantly reduced. Fig. 1 Effects of phenolic extracts from T. scabrum and T. pallidum on protein tyrosine nitration in blood plasma exposed to peroxynitrite. Concentration of 3-nitrotyrosine in plasma proteins was measured by a competitive enzyme-linked immunosorbent assay (c-ELISA) test. Values obtained for plasma samples exposed to 100 lM ONOO- in the absence of the tested extracts, were assumed as 100 % of nitration. Results are representative of five independent experiments and are expressed as mean ± SD. The influence of all used concentrations of both extracts was statistically significant: *p \ 0.02, **p \ 0.001; ONOO- treated plasma versus control plasma: p \ 0.001 Analysis of amidolytic activity of plasmin in plasma samples 123 Author's personal copy 196 J. Kolodziejczyk-Czepas et al. Fig. 2 Effects of phenolic extracts from T. scabrum and T. pallidum on the thiol groups level in blood plasma exposed to peroxynitrite. Results are representative of five independent experiments, and are expressed as mean ± SD. The statistical significance of the used extracts was found: *p \ 0.02, **p \ 0.01, ***p \ 0.001; ONOO- treated plasma versus control plasma: p \ 0.001 Fig. 3 Effects of phenolic extracts from T. scabrum and T. pallidum on lipid peroxidation in blood plasma exposed to peroxynitrite action, estimated by the level of TBARS. Results are expressed as means ± SD of five independent experiments. The antioxidative effect of all used concentrations of both extracts was statistically significant: *p \ 0.02, **p \ 0.01, ***p \ 0.001; ONOO- treated plasma versus control plasma: p \ 0.001 Extracts from T. scabrum and T. pallidum effectively diminished the nitration of tyrosine residues, as well as the oxidation of thiol groups in plasma proteins, caused by peroxynitrite (Figs. 1, 2). The peroxynitrite-induced peroxidation of plasma lipids was distinctly inhibited by all the used concentrations of T. scabrum and T. pallidum extracts. In plasma samples exposed to ONOO- action in the presence of the highest concentrations of the extracts, the levels of lipid peroxidation were comparable to the control plasma (Fig. 3). The results included in Table 1 shows that the tested plant extracts at concentrations of 0.5, 5 and 50 lg/ml did not change the plasma clotting ability. Furthermore, the examined extracts did not alter the activity of the fibrinolytic system in plasma samples (Table 1, p [ 0.05), but significantly diminish the peroxynitrite-induced inhibition of plasma amidolytic activity (Fig. 4). 123 Discussion Physiological effects of Trifolium species are only partly recognized and the most of biological activities of various clovers, including T. scabrum and T. pallidum, have been not known yet. Previously, we have demonstrated that the clovamide fraction from T. pallidum possesses the antioxidative properties, and partly protects blood platelets and plasma against the oxidative stress-induced damage [17]. In the present study, we demonstrate for the first time the antioxidative effect of phenolic extracts (0.5–50 lg/ml) from T. scabrum (rich in isoflavones) and T. pallidum (rich in both phenolic acids and clovamides). To induce oxidative stress we used peroxynitrite (ONOO-), a strong oxidative and nitrative agent. The formation of peroxynitrite is a result of a reaction between superoxide anion (O•2 ) and Author's personal copy Trifolium pallidum and Trifolium scabrum extracts 197 Table 1 Effects of phenolic extracts from T. pallidum and T. scabrum (0.5–50 lg/ml), on coagulative properties and amidolytic activity of blood plasma The concentration of phenolic fraction (lg/ml) Prothrombin time (s) Thrombin time (s) Plasma amidolytic activity (Vmax) Control (untreated) plasma 14.8 ± 1.1* 59.5 ± 3.8* 52.8 ± 3.9* T. pallidum 0.5 14.6 ± 1.4* 60.6 ± 5.9* 52.4 ± 1.9* 5 14.2 ± 1.2* 59.7 ± 5.7* 50.4 ± 2.4* 50 14.4 ± 1.4* 58.5 ± 4.7* 53.7 ± 1.3* T. scabrum 0.5 15.0 ± 0.8* 60.2 ± 5.4* 54.3 ± 1.2* 5 14.7 ± 0.9* 60.9 ± 3.2* 54.5 ± 2.2* 50 14.1 ± 1.0* 59.9 ± 3.3* 55.0 ± 2.4* Results are representative of 16 independent experiments, and are expressed as mean ± SD. The amidolytic activity of plasmin in plasma samples was measured by the kinetic method, and is expressed as Vmax values. Effects of T. pallidum and T. scabrum were not statistically significant according to one-way ANOVA test; * p [ 0.05 nitric oxide (NO) [18]. In the cardiovascular system, ONOO- may be formed under pathological conditions, such as inflammation, ischaemia and reperfusion, as well as endogenously in blood platelets, during their activation [19, 20]. The concentration of ONOO-, used in our study (100 lM), may correspond to its concentrations in vivo. Beckmann et al. [21] have established that 250 lM bolus of ONOO- is an equivalent of 7-min action of 1 lM peroxynitrite—this concentration has been reported to occur in vivo [21, 22]. The lower concentrations of the examined extracts also correspond to the range of physiological level of plant-derived phenolic compounds, detected after a dietary intake or isoflavone supplementation. For example, an oral daily dose of two T. pratense-derived isoflavone tablets (one tablet contained 24.5 mg of biochanin, 1.5 mg of genistein, 16 mg of formononetin, and 1.5 mg of daidzein) results in 0.25 lM concentration of daidzein and 0.42 lM concentration of genistein in blood plasma [23]. Antioxidative properties of the examined clover extracts are a result of a high content of phenolic acids and numerous polyphenolic compounds. Isoflavones and their glycosides display free radical scavenging activity, attributed to the presence of hydroxyl groups in the structure of these compounds. Phenolic acids present in Trifoliumderived extracts are also able to scavenge superoxide anion [24] and peroxynitrite [25]. Clovamides display strong antioxidant activity due to the presence of two catechol moieties, strongly promoting free radical scavenging action [26, 27]. Our results indicate that the extracts from T. scabrum and T. pallidum may protect blood plasma proteins and lipids against peroxynitrite-induced damage. Since in our work a significant reduction of both oxidative and nitrative ONOO--induced damage was observed, it seems that phenolic compounds naturally occurring in T. scabrum and T. pallidum are able to scavenge peroxynitrite or the secondary radicals formed from peroxynitrite. Additionally, the present study provides more information about the effects of Trifolium extracts on haemostatic properties of human plasma. Our studies demonstrates that phenolic extracts from T. scabrum and T. pallidum do not induce changes in coagulative properties of human plasma, their influence on the activity of the fibrinolytic system (measured by the hydrolysis of a chromogenic substrate for plasmin) in plasma was also excluded. A new aspect of the biological activity the examined clovers was their possible protective action against oxidative stress-induced decrease of plasmin activity. The fibrinolytic system, as the control mechanism is crucial for maintaining the haemostatic Fig. 4 Effects of phenolic extracts from T. scabrum and T. pallidum on amidolytic activity of plasmin, induced in plasma treated with peroxynitrite. Human plasma samples were preincubated with the extracts (0.5–50 lM), and then ONOO- was added (100 lM). The streptokinaseinduced hydrolysis of chromogenic substrate for plasmin (amidolytic activity) was measured by using a kinetic protocol (A415). The amidolytic activity of plasmin in plasma samples was expressed as Vmax values 123 Author's personal copy 198 balance, but under pathological conditions (such as chronic and acute inflammation) its activity may be significantly diminished. Oxidative stress influences haemostasis and shifts the haemostatic mechanisms in favour of thrombosis [28]. The dysfunction of endothelium results in a decrease of its antithrombotic properties and lead to the hypercoagulability [29] and, in consequence, the impaired activity of fibrinolytic enzymes becomes an additional pro-thrombotic factor. According to Lind et al. [30] plasmin, the key enzyme of the fibrinolytic system may undergo the oxidative inactivation. The susceptibility of plasminogen (plasmin proenzyme) to ONOO- action was demonstrated in several studies. The most likely cause of the inhibition of plasminogen and plasmin functions is tyrosine nitration [31–33]. Therefore, the antioxidative protection of the fibrinolytic system seems to be an important strategy for the maintaining of haemostatic balance of blood plasma. We have observed a significant decrease of ONOO--induced inhibition of plasmin amidolytic activity in the presence of either T. scabrum or T. pallidum. These results may suggest a possible protective role of these extract in the prevention of the oxidative damage to fibrinolytic proteins. In conclusion, the present study demonstrates for the first time the protective effect of the phenolic extracts from aerial parts of T. scabrum (rich in isoflavones) and T. pallidum (rich in phenolic acids and clovamides) on plasma components, including fibrinolytic proteins. The protective action of Trifolium-derived extracts seems to be attributed to their antioxidative properties; however, the clarification of molecular mechanisms of the observed effect requires further studies. Acknowledgments This work was supported by Grant 506/810 and 545/217 from University of Lodz, Poland and statutory activities of Institute of Soil Science and Plant Cultivation, State Research Institute, Pulawy, Poland. Special thanks to Dr Pawel Nowak for supplying of peroxynitrite and helpful suggestions. References 1. Dhalla NS, Temsah RM, Netticadan TJ (2009) Role of oxidative stress in cardiovascular diseases. J Hypertens 18:655–673 2. Olas B, Wachowicz B (2007) Role of reactive nitrogen species in blood platelet functions. Platelets 18:555–565 3. Ferroni P, Basili S, Paoletti V, Davı̀ G (2006) Endothelial dysfunction and oxidative stress in arterial hypertension. Nutr Metab Cardiovasc Dis 16:222–233 4. Nielsen VG, Crow JP, Mogal A, Zhou F, Parks DA (2004) Peroxynitrite decreases hemostasis in human plasma in vitro. Anesth Analg 99:21–26 5. Madhavi DL, Salunkhe DK (1995) Toxicological aspects of food antioxidants. In: Madavi DL, Deshpande SS, Salunkhe DK (eds) food antioxidants. Dekker, New York, p 267 6. Oleszek W, Stochmal A (2002) Triterpene saponins and flavonoids in the seeds of Trifolium species. Phytochemistry 61: 165–170 123 J. Kolodziejczyk-Czepas et al. 7. Oleszek W, Stochmal A, Janda B (2007) Concentration of isoflavones and other phenolics in the aerial parts of Trifolium species. J Agric Food Chem 55:8095–8100 8. Kolodziejczyk-Czepas J (2012) Trifolium species-derived substances and extracts—biological activity and prospects for medicinal applications. J Ethnopharmacol (in press) 9. Thorat JD, Ng I (2007) Acute dural sinus thrombosis following ingestion of an herbal tonic: case report. J Stroke Cerebrovasc Dis 16:232–235 10. Pawlaczyk I, Czerchawski L, Kuliczkowski W, Karolko B, Pilecki W, Witkiewicz W, Gancarz R (2011) Anticoagulant and anti-platelet activity of polyphenolic-polysaccharide preparation isolated from the medicinal plant Erigeron canadensis L. Thromb Res 127:328–340 11. Huang L, Lin C, Li A, Wei B, Teng J, Li L (2010) Pro-coagulant activity of phenolic acids isolated from Blumea riparia. Nat Prod Commun 5:1263–1266 12. Pryor WA, Squadrito GL (1995) The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am J Physiol 268:699–722 13. Stochmal A, Piacente S, Pizza C, De Riccardis F, Leitz R, Oleszek W (2001) Alfalfa (Medicago sativa L.) flavonoids. Apigenin and luteolin glycosides from aerial parts. J Agric Food Chem 49:753–758 14. Khan J, Brennan DM, Bradley N, Gao B, Bruckdorfer R, Jacobs M (1998) 3-nitrotyrosine in the proteins of human plasma determined by an ELISA method. Biochem J 330:795–801 15. Olas B, Nowak P, Kolodziejczyk J, Ponczek M, Wachowicz B (2006) Protective effects of resveratrol against oxidative/nitrative modifications of plasma proteins and lipids exposed to peroxynitrite. J Nutr Biochem 17:96–102 16. Rice-Evans CA, Diplock AT, Symons MCR (1991) Techniques in free radicals research. In: Burdon RH, van Knippenberg PH (eds) Laboratory techniques in biochemistry and molecular biology. Elsevier, Amsterdam, pp 147–148 17. Kolodziejczyk J, Olas B, Wachowicz B, Szajwaj B, Stochmal A, Oleszek W (2011) Clovamide-rich extract from Trifolium pallidum reduces oxidative stress-induced damage to blood platelets and plasma. J Physiol Biochem 67:391–399 18. Pacher P, Beckman JS, Liaudet L (2007) Nitric oxide and peroxynitrite in health and disease. Physiol Rev 87:315–424 19. Szabó C, Ischiropoulos H, Radi R (2007) Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov 6:662–680 20. Mazzanti L, Raffaelli F, Vignini A, Nanetti L, Vitali P, Boscarato V, Giannubilo SR, Tranquilli AL (2012) Nitric oxide and peroxynitrite platelet levels in gestational hypertension and preeclampsia. Platelets 23:26–35 21. Beckmann JS, Chen J, Ischiropoulos H, Crow JP (1994) Oxidative chemistry of peroxynitrite. Methods Enzymol 233:229–240 22. Bartosz G (1996) Peroxynitrite: mediator of the toxic action of nitric oxide. Acta Biochim Pol 43:645–659 23. Howes J, Waring M, Huang L, Howes LG (2002) Long-term pharmacokinetics of an extract of isoflavones from red clover (Trifolium pratense). J Altern Complement Med 8:135–142 24. Gulcin I (2006) Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology 217:213–220 25. Pannala A, Razaq R, Halliwell B, Singh S, Rice-Evans C (1998) Inhibition of peroxynitrite dependent tyrosine nitration by hydroxycinnamates: nitration or electron donation? Free Radic Biol Med 24:594–606 26. Arlorio M, Locatelli M, Travagilia F, Coisson JD, del Grosso E, Minassi A, Appendino G, Martelli A (2008) Roasting impact on the contents of clovamide (N-caffeoyl- L-DOPA) and the antioxidant activity of cocoa beans (Theobroma cacao L.). Food Chem 106:967–975 Author's personal copy Trifolium pallidum and Trifolium scabrum extracts 27. Ley JP, Bertram H-J (2003) Synthesis of lipophilic clovamide derivatives and their antioxidative potential against lipid peroxidation. J Agric Food Chem 51:4596–4602 28. Esmon CT (2004) Crosstalk between inflammation and thrombosis. Maturitas 47:305–314 29. Fenster BE, Tsao PS, Rockson SG (2003) Endothelial dysfunction: clinical strategies for treating oxidant stress. Am Heart J 146:218–226 30. Lind SE, McDonagh JR, Smith CJ (1993) Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate. Blood 82:1522–1523 199 31. Gugliucci A (2003) Human plasminogen is highly susceptible to peroxynitrite inactivation. Clin Chem Lab Med 41:1064–1068 32. Hathuc C, Hermo R, Schulze J, Gugliucci A (2006) Nitration of human plasminogen by RAW 264.7 macrophages reduces streptokinase-induced plasmin activity. Clin Chem Lab Med 44:213–219 33. Nowak P, Kolodziejczyk J, Wachowicz B (2004) Peroxynitrite and fibrinolytic system: the effect of peroxynitrite on plasmin activity. Mol Cell Biochem 400:141–146 123 View publication stats