Antithrombotic Effects of Pyridinium Compounds ... - ACS Publications

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Mar 13, 2014 - Antithrombotic Effects of Pyridinium Compounds Formed from. Trigonelline upon Coffee Roasting. Bartlomiej Kalaska,*. ,†. Lukasz Piotrowski,.
Article pubs.acs.org/JAFC

Antithrombotic Effects of Pyridinium Compounds Formed from Trigonelline upon Coffee Roasting Bartlomiej Kalaska,*,† Lukasz Piotrowski,‡ Agnieszka Leszczynska,† Bartosz Michalowski,‡ Karol Kramkowski,† Tomasz Kaminski,† Jan Adamus,‡ Andrzej Marcinek,‡ Jerzy Gebicki,‡ Andrzej Mogielnicki,† and Wlodzimierz Buczko†,§ †

Department of Pharmacodynamics, Medical University of Bialystok, Mickiewicza Str. 2C, 15-222 Bialystok, Poland Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland § Higher Vocational School, Noniewicza 10, 16-400 Suwalki, Poland ‡

S Supporting Information *

ABSTRACT: Coffee may exert a preventive effect on arterial thrombosis. Trigonelline is one of the most abundant compounds in coffee that undergoes pyrolysis upon roasting of coffee beans. The aim of the present study was to identify pyridinium compounds formed upon trigonelline pyrolysis and coffee roasting and to investigate the effect of three of them, i.e., 1methylpyridine and 1,3- and 1,4-dimethylpyridine, on experimentally induced arterial thrombosis in rats. 1,3- and 1,4dimethylpyridine but not 1-methylpyridine inhibited arterial thrombus formation. 1,3-Dimethylpyridine inhibited platelet aggregation and reduced fibrin formation in platelet-rich plasma, whereas 1,4-dimethylpyridine increased the plasma level of 6keto-PGF1α. 1,4-Dimethylpyridine slightly increased rat tissue plasminogen activator plasma activity. In summary, we demonstrated that pyridinium compounds display mild antithrombotic properties due to stimulation by prostacyclin release (1,4dimethylpyridine) and inhibition of platelet aggregation (1,3-dimethylpyridine). Those pyridinium compounds may, to some extent, be responsible for the beneficial effects of coffee drinking. KEYWORDS: coffee, pyridinium salts, thrombosis, prostacyclin, platelet aggregation



INTRODUCTION The effect that coffee, one of the most commonly consumed beverages, exerts on human health has long been discussed and widely studied. Even small effects of coffee drinking on human body could have a large impact on public health. It has long been suspected that coffee consumption may have adverse effects on the cardiovascular system. One of the best known compounds in coffee is caffeine. An intake of caffeine is associated with a rise in blood pressure,1,2 increased systemic vascular resistance,3 arterial stiffness,4,5 plasma activity of renin, epinephrine, and norepinephrine,6 and unfavorable effects on endothelial function in healthy subjects.7 However, the results of several studies demonstrated that coffee consumption is associated with a lower risk of cardiovascular disease.8 The subanalysis of the Framingham study suggests that regular intake of caffeinated coffee may reduce the risk of myocardial infarction.9 Coffee consumption has been associated with better glucose tolerance in nondiabetic subjects.10 Interestingly, a significant acute favorable dose-dependent effect of decaffeinated espresso coffee on endothelial function has also been reported.11 Coffee drinking (irrespective of caffeine) inhibited collagen and arachidonic acid induced platelet aggregation ex vivo.12 Furthermore, a recent study suggests that it is coffee rather than caffeine intake that may have a preventive effect on arterial occlusive thrombus formation.13 Apart from caffeine, coffee also contains a number of compounds that may affect the cardiovascular system.14 Noordzij et al.2 already showed that the blood pressure elevations appeared to be larger in the subjects who ingested © 2014 American Chemical Society

caffeine but not in the subjects who ingested coffee, suggesting that other compounds present in coffee could potentially counterbalance the pressor effect of caffeine. Several phenolic compounds with antioxidant properties,15 affecting the process of atherosclerosis favorably by preventing oxidation of LDLcholesterol16 and inhibiting platelet aggregation and thrombogenesis,12,17 have already been found. Trigonelline is one of the most abundant compounds in coffee beans. We have shown in our previous studies that trigonelline did not induce thrombolytic activity itself.18 However, about 50−80% of the trigonelline is decomposed during roasting, forming nicotinic acid and some aromatic nitrogen compounds, including pyridines, pyrroles, and bicyclic compounds. Main nonvolatile products of trigonelline pyrolysis are 1-methylpyridine and dialkylpyridinium compounds.19 In several studies, it has been demonstrated that N-methyl derivatives of pyridine have antioxidant,20 hepatoprotective,21 and vasoprotective22 effects. These compounds show such effects probably due to their structural similarity to the metabolite of nicotinamide-1-methylnicotinamide (Figure 1). We have previously shown that 1-methylnicotinamide exerts antithrombotic, antiplatelet, and fibrinolytic activity by releasing endothelial prostacyclin.18,23 Received: Revised: Accepted: Published: 2853

October 24, 2013 March 11, 2014 March 13, 2014 March 13, 2014 dx.doi.org/10.1021/jf5008538 | J. Agric. Food Chem. 2014, 62, 2853−2860

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Minolta, Tokyo, Japan) spectrophotometer in the CIE Lab scale (L*, a*, and b*).25 The spectrophotometer had a 4-mm diameter viewing area and was equilibrated and calibrated with a standard tile before experiments. The samples were illuminated with standard D65 light, and the colorimetric normal observer angle was 10°. Coffee samples were made into pellets (13 × 1 mm) by a hydraulic press at a pressure of 6 tons. Color attributes were measured at six different points of each pellet on white and black backing. Each value reported is the mean of 12 measurements ± SEM. Three different roasting degrees were obtained: light (roasting time 14.5 min, L* = 51.62 ± 0.23, a* = 12.44 ± 0.08, and b* = 27.09 ± 0.15), medium-dark (roasting time 19 min, L* = 32.01 ± 0.29, a* = 9.00 ± 0.09, and b* = 11.12 ± 0.16), and dark roasted coffee (roasting time 23 min, L* = 25.98 ± 0.44, a* = 3.67 ± 0.08, and b* = 4.24 ± 0.10). The coffee samples were ground using a MKM 6003 coffee grinder (Bosch, Stuttgart, Germany). Extract Preparation. Roasted and ground coffee were prepared using different extraction methods. Ground coffee (1.875 g) was suspended in a final volume of 50 mL of (A) medium temperature water at 60 °C or (B) hot water at 90 °C. Extracts were then cooled down to room temperature, filtered using a paper filter, and centrifuged for 15 min at 5500 rpm and a temperature of 15 °C. The supernatant was brought up to pH 8 with 0.5 M NaOH prior to further purification with the use of solid-phase extraction. Trigonelline Pyrolysate Preparation. Crystalline trigonelline was ground in a porcelain mortar and placed as a thin layer (2−3 mm) on a Petri dish; the sample was then placed in a laboratory oven LE/4/ 11/R6 (Nabertherm, Bremen, Germany) and heated in a dry nitrogen atmosphere at 100 °C for 1 h. In the next step, the temperature was raised to 200 °C, and the sample was heated for additional 25 min. Trigonelline pyrolysate was cooled down to room temperature and extracted two times by chloroform. Extracted samples were dried at reduced pressure (0.1 mbar, RT). Samples of 2 mg and 0.2 mg of pyrolysate per 1 mL of Millipore-grade water were used for further analysis. Solid-Phase Extraction (SPE) of Pyridinium Compounds from Coffee Extracts. Because of the cationic nature of pyridinium compounds, ion exchange chromatography with the use of propylcarboxylic acid Bakerbond CBA columns (J.T. Baker, Phillipsburg, NJ, USA) was chosen as a method of extraction. A portion of supernatant (5 mL) obtained after coffee extract preparation was loaded into a CBA column (500 mg), preconditioned with a methanol and water (each 5 mL). After penetration of column packing by a coffee solution, the column was washed with water followed by methanol (5 mL). The analyte was eluted with 0.2 M formic acid in water (5 mL). This acid fraction was transferred into a glass vial prior to UPLC/MS analyses. Ultraperformance Liquid Chromatography−Electrospray Ionization Mass Spectrometry of Pyridinium Compounds in Trigonelline Pyrolysate and Coffee Extracts. All analyses of pyridinium compounds in coffee extracts and trigonelline pyrolysate were performed with an Aquity ultraperformance liquid chromatography system UPLC (Waters, Milford, MA, USA) coupled online to a photodiode array (PDA) spectrometer for UV/vis measurements and an LCT Premier XE oa-TOF mass spectrometer (Waters MicroMass, Manchester, UK). Separations were accomplished on 100 mm × 2.1 mm, i.d., 1.7 μm Acquity UPLC BEH C18 column with a 5 mm × 2.1 mm, i.d. guard column of the same material (Waters, Milford, MA, USA) maintained at 40 °C. Samples were eluted using 90% of 2 mM nonafluoropentanoic acid aqueous solution and 10% of methanol solution at a flow rate of 0.3 mL/min. The injection volumes and temperature for both sample and standard solutions were 0.2−2 μL and 4 °C, respectively. The mass spectrometer was operated in “W mode” with lock mass correction. Lock mass solution (leucineenkephalin, reference mass: [M]+ 556.2771 and [M+H]+ 557.2801) was prepared fresh at a concentration of 0.5 ng/μL in water/ acetonitrile (50:50; v/v) and stored at 4 °C until further use. Standards were prepared in Millipore-grade water. Ion chromatograms of pyridine derivative standards obtained at different m/z from mass spectrometer are presented in Figure 2. External calibration curves were established in the concentration range from 0.05 to 5 μM for

Figure 1. Chemical structures of trigonelline, nicotinamide, 1methylnicotinamide, methyl derivatives of pyridine, and trigonelline methyl ester.

Therefore, the aim of the present study was to identify pyridinium compounds formed upon coffee roasting and to investigate their influence on experimentally induced arterial thrombosis in rats.



MATERIALS AND METHODS

Animal Studies. Animals were purchased from and housed in the Centre of Experimental Medicine of Medical University in Bialystok according to Good Laboratory Practice rules. Male Wistar rats (180− 200 g) were used in all experiments. Animals were housed in a room with a 12 h light/dark cycle, grouped in cages as appropriate, and allowed to have access to tap water and a standard rat chow. All of the procedures involving animals and their care were approved by a local bioethics committee and conducted in accordance with the institutional guidelines that are in compliance with national and international laws and Guidelines for the Use of Animals in Biomedical Research.24 Chemicals and Materials. Trigonelline hydrochloride was obtained from Fluka (Steinheim, Germany). Methanol and formic acid (LC-LC-MS grade) were from Sigma-Aldrich (Schnelldorf, Germany). All other reagents were from Sigma-Aldrich, unless noted otherwise, and were of the highest purity available. 1-Methylpyridinium chloride (1-methylpyridine, 4), 1,2-dimethylpyridinium chloride (1,2-dimethylpyridine, 5), 1,3-dimethylpyridinium chloride (1,3-dimethylpyridine, 6), 1,4-dimethylpyridinium chloride (1,4dimethylpyridine, 7), 1-ethylpyridinium chloride (1-ethylpyridine, 8), and trigonelline methyl ester (9) (see Figure 1), as standards for chemical analysis and drugs for pharmacological experiments, were synthesized using the methods in the literature. The 6-Keto-PGF1α ELISA kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA) and rat plasminogen activator inhibitor −1 (PAI-1) ag/activity Zymutest Kits from HYPHEN BioMed (Neuville sur Oise, France), whereas the rat tissue plasminogen activator (t-PA) activity kit was obtained from Innovative Research Inc. (Novi, MI, USA). Reagents to prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen (Fg) level measurements were purchased from Bio-Ksel (Grudziadz, Poland). Moreover, collagen (Chrono-log Corporation, Havertown, PA, USA), pentobarbital (Biovet, Pulawy, Poland), and trisodium citrate (Avantor Performance Materials, Gliwice, Poland) were used in the study. Solid-phase extraction columns Bakerbond CBA, 500 mg and 3 mL (J.T. Baker, Phillipsburg, NJ, USA), were obtained from Witko (Lodz, Poland). The column used was 100 mm × 2.1 mm, i.d., 1.7 μm Acquity UPLC BEH C18 with a 5 mm × 2.1 mm, i.d. guard column of the same material (Waters, Milford, MA, USA). Coffee Samples. Coffee (C. arabica) samples (150 g) were roasted using coffee roaster KN-8828D (Hottop, Cranston, RI, USA) at 220 °C. The roasting process was manually controlled by following audible cues of the beginning of a “first crack” (14.5 min), the beginning of a “second crack” (19 min), and the end of a “second crack” (23 min). Objective color measurements were obtained by a CM-3600d (Konica 2854

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Arterial thrombosis was induced by electrical stimulation of the common carotid artery, according to methods previously described.23,26−28 Briefly, the anode, a stainless steel L-shaped wire, was inserted under the artery and connected to a circuit with a constant current generator. The cathode was a subcutaneous metal needle attached to the hindlimb. The stimulation (1 mA) took 10 min. Forty-five minutes after stimulation, the segment of the common carotid artery with the formed thrombus was clipped at both sides, dissected, opened lengthwise, and the thrombus completely removed, air-dried in 37 °C, and weighed 24 h after the end of the experiment. After the removal of the formed thrombus, blood samples were taken from the heart. Platelet aggregation was assessed in whole blood directly after the experiment. The rest of the blood samples were drawn into 3.13% trisodium citrate in a volume ratio of 10:1, then centrifuged at 3500g in 4 °C for 20 min, and plasma was deep-frozen (−70 °C) in aliquots of 1 mL until further assays could be performed. Measurement of Platelet Aggregation. Platelet aggregation was assessed in whole blood directly after the experiment by measuring electrical impedance in a Chronolog aggregometer (Chrono-log Corporation, Havertown, PA, USA) according to the method described by Cardinal et al.29 Blood samples were drawn into 3.13% trisodium citrate in a volume ratio of 10:1. Collagen (5 μg/mL) was added after 15 min of incubation at 37 °C with 0.9% NaCl in a volume ratio of 1:2. Then changes in impedance were registered for 6 min. The results were expressed as percentages using four parameters: maximal extension, slope of platelet aggregation, lag phase, and area under the curve. In addition, in an in vitro study blood samples were mixed with 1,3-dimethylpyridine, 1,4-dimethylpyridine, or vehicle solution before incubation. Plasma Level of 6-Keto-PGF1α, a Stable Metabolite of Prostacycline (PGI2), Fibrynolysis, and Coagulation Parameters, and Blood Cell Count. Plasma level of 6-keto-PGF1α was measured by ELISA technique in a microtiter plate at 25 °C using a microplate reader (Dynex Technologies, Chantilly, VA, USA) to monitor the changes in absorbance at 405 nm according to the manufacturer’s directions. Concentrations of active rat PAI-1 and active rat t-PA were analyzed using ELISA techniques at 25 °C using a microplate reader according to the kit manufacturer’s instructions. Both rat PAI-1 and rat t-PA kits were used in a bioimmunoassay for measuring rat PAI-1 and rat t-PA activities in rat plasma collected on trisodium citrate anticoagulant. The stop solutions changed the color from blue to yellow, and the intensity of the color was measured at 450 nm. PT and aPTT were automatically determined with a Coag-Chrom 3003 coagulometer (Bioksel, Grudziadz, Poland) adding routine laboratory reagents to the collected rat plasma. Fg was evaluated according to Clauss method.30 Blood cell count was assessed by an animal blood counter (ABC Vet, Horiba ABX, Monpellier, France). Fibrin Generation. Fibrin generation was estimated according to our modification32,33 of the method of He et al.31 Briefly, blood samples were drawn from the left ventricle of thrombotic rats, and platelet-rich plasma (PRP) was gained by centrifugation of citrated blood (1:10, 3.13%) at 300g at 4 °C for 20 min. A fibrin generation curve was created by adding thrombin (1 IU/mL) and CaCl2 (36 mM) to the Tris buffer (66 mM Tris and 130 mM NaCl, pH 7.4) and

Figure 2. Ion-chromatograms of 500 nM standards of pyridine derivatives obtained at different m/z. Retention times: trigonelline, (1, tR = 1.44 min); nicotinic acid, (2, tR = 1.85 min); 1-methylpyridine, (4, tR = 4.27 min); 1-ethylpyridine, (8, t R = 5.10 min); 1,2dimethylpyridine, (5, tR = 5.62 min); 1,3-dimethylpyridine, (6, tR = 6.27 min); 1,4-dimethylpyridine, (7, tR = 6.38 min), and trigonelline methyl ester, (9, tR = 7.00 min). mass spectrometry and 5−1000 μM for UV/vis measurements. Analytes not falling within the given range were adequately diluted with water and reinjected. The amount of the compounds in the samples was calculated from the respective linear regression equation, r2 > 0.999. Data acquisition and analyses were performed using MassLynx 4.1 data system software (Waters, Milford, MA, USA) and OriginPro 9.0c (OriginLab, Northampton, MA, USA). Drugs Administration. 1-Methylpyridine (100 mg/kg/d), 1,3dimethylpyridine (30, 100, 300 mg/kg/d), 1,4-dimethylpyridine (30, 100, 300 mg/kg/d), or vehicle was administered per os once a day for 10 days preoperatively. The animals received the examined drugs dissolved in drinking water or drinking water (vehicle) daily for 10 days via an intragastric probe. Induction of Arterial Thrombosis. Male Wistar rats were anesthetized by intraperitoneal injection of pentobarbital (40 mg/ kg) and placed in a supine position on a heated operation table.

Table 1. Quantitative Analysis of Alkylpyridinium Derivatives Present in the Roasted Coffee Extractsa concentration of roasting time (min) 14.5 19.0 23.0

extraction method water water water water water water

60 90 60 90 60 90

°C °C °C °C °C °C

1-ethylpyridine (μM) ND ND 0.07 0.23 0.50 0.69

± ± ± ±

0.02 0.03 0.03 0.02

1-methylpyridine (μM)

1,2-dimethylpyridine (μM)

1,3-dimethylpyridine (μM)

1,4-dimethylpyridine (μM)

4.8 ± 0.8 4.0 ± 0.3 206 ± 3 218 ± 3 302 ± 2 320 ± 10

ND ND NQ NQ 0.05 ± 0.03 0.11 ± 0.03

ND ND 0.03 ± 0.01 0.14 ± 0.02 0.94 ± 0.06 1.8 ± 0.2

ND ND ND NQ NQ NQ

All values represent the mean ± SEM for at least n = 3. The calibration curve was r2 > 0.999 within the given concentration range. ND, concentration below detection level; NQ, concentration below quantitation level.

a

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Figure 3. Influence of coffee roasting time and extraction procedure on the concentration of the main trigonelline thermal degradation product, 1methylpyridine, in the coffee extracts samples. (A) Chromatograms obtained at 264 ± 1.2 nm with the use of a photodiode array detector (PDA) for 50 μM 1-methylpyridine standard and coffee samples of different roasting degrees. (B) Concentration of 1-methylpyridine in the coffee extract. mixing with the platelet-rich plasma samples directly in the microplate wells. Optical density was measured via a microplate reader (Dynex Technologies, Chantilly, VA, USA) at 405 nm in 1 min intervals for 6 min and expressed as area under the curve (AUC). Statistical Analysis. The data are shown as the mean ± SEM and analyzed using the nonparametric Mann−Whitney test. P values less than 0.05 were considered significant, less than 0.01 highly significant, and less than 0.001 extremely significant.



RESULTS AND DISCUSSION The discrepancies between the results of the studies on coffee consumption in relation to the risk of cardiovascular disease and at the same time clear evidence for the beneficial role of coffee drinking from both epidemiologic34 and experimental studies11,13 encouraged us to search for new compounds in roasted coffee that could possibly improve endothelial function.

Figure 5. Difference between the profile of trigonelline thermal degradation products in coffee extracts and trigonelline pyrolysate. Ion chromatograms obtained at m/z 108 ± 0.05 for 500 nM standards of 1-ethylpyridine, 1,2-, 1,3-, and 1,4-dimethylpyridine, a dark roasted coffee sample, and trigonelline pyrolysate.

Table 2. Results of the Quantitative Analysis of Thermal Degradation Products of Trigonellinea

Figure 4. Influence of coffee roasting time on the profile of trigonelline thermal degradation products. Ion chromatograms obtained at m/z 108 ± 0.05 for 500 nM standards of 1-ethylpyridine, 1,2-, 1,3-, and 1,4dimethylpyridine, and coffee extract samples (roasting time and extraction temperature are as indicated).

a

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compd

quantity (mg)

trigonelline nicotinic acid 1-methylpyridine 1,2-dimethylpyridine 1,3-dimethylpyridine 1,4-dimethylpyridine 1-ethylpyridine trigonelline methyl ester

0.91 ± 0.02 0.012 ± 0.001 0.015 ± 0.001 (2.6 ± 0.4) × 10−6 (1.8 ± 0.2) × 10−6 (4.4 ± 0.5) × 10−5 (6.8 ± 0.8) × 10−6 (6.3 ± 1) × 10−5 0.94 ± 0.02

The Major Products Identified in 1 mg of Trigonelline Pyrolysate.

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As various factors could be responsible for the conflicting results of the studies on coffee drinking such as daily consumption and the type of coffee35 or the role of coffee preparation,36,37 we have focused our attention only on a selected group of compounds, pyridine derivatives (see Figure 1), present in the roasted coffee extracts and formed upon trigonelline pyrolysis. As was shown previously by Stadler et al.,19 during the roasting process nonvolatile alkylpyridinium salts are formed. Under a specific physiochemical condition, trigonelline, one of the most abundant compounds in green coffee, undergoes thermal degradation with subsequent formation of 1-methylpyridine and nicotinic acid, as major products, and dialkylpyridinium salts as minor products. There is a lack of information on the adverse effects of alkylpyridinium, especially dialkylpyridinium compounds. Some studies indicate that 1-methyl substituted analogues are less toxic than pyridine itself 38 but may activate cytochrome P450 CYP1A enzyme.39 However, Szafarz et al.40 did not observe any changes in basic biological parameters after intravenous administration of 1,4-dimethylpyridine at a dose of 100 mg/kg. We have monitored the formation of 1-methylpyridine in coffee extracts using a photodiode array detector (PDA) coupled with a liquid chromatograph, by an increase in absorbance at 264 ± 1.2 nm. The concentration of 1methylpyridine found in coffee extracts, expressed in μmol/L, is presented in Table 1 and Figure 3. Our findings are consistent with the previous results presented by Stadler et al.19 Decomposition of trigonelline and subsequent formation of 1methylpyridine increases with roasting time and higher temperature of extraction. The formation of 1-methylpyridine, as expected, is also accompanied by the formation of dialkylpyridinium compounds as shown in Figure 4. Using a new chromatographic method with the mobile phase modifier, ion-pairing reagent, we were able to differentiate four different isomers with ions of identical m/z 108.08 ± 0.05 (Figure 4). As the formation of three dimethylpyridinium salts is known in the literature, the new entity was characterized and assigned to 1-ethylpyridine, mainly by comparison with its authentic standard. The formation of compounds specified by m/z equals 108.08 was also correlated with the formation of 1-methylpyridine and the degree of coffee roasting. The concentration of dialkylpyridinium compounds

Figure 6. Columns represent the dry thrombus weight in Wistar rats treated (per os for 10 days) with vehicle, 1,3-dimethylpyridine (30, 100, and 300 mg/kg/d), 1,4-dimethylpyridine (30, 100, and 300 mg/ kg/d), and 1-methylpyridine (100 mg/kg/d). Digits inside columns indicate n number. * p < 0.05 vs vehicle, Mann−Whitney test. Results are shown as the mean ± SEM.

Figure 7. Columns represent the 6-keto-PGF1α concentration in Wistar rats treated (per os for 10 days) with vehicle, 1,3dimethylpyridine (30, 100, and 300 mg/kg/d), 1,4-dimethylpyridine (30, 100, and 300 mg/kg/d), and 1-methylpyridine (100 mg/kg/d). Digits inside columns indicate n number. ** p < 0.01 vs vehicle, Mann−Whitney test. Results are shown as the mean ± SEM.

Table 3. Platelet Aggregation, Platelet Count, and Fibrinolysis Parameters Measured in Rats Treated (per os for 10 days) with Vehicle, 1,3-Dimethylpyridine (30, 100, and 300 mg/kg/d), 1,4-Dimethylpyridine (30, 100, and 300 mg/kg/d), and 1Methylpyridine (100 mg/kg/d)a 1,3-dimethylpyridine n MaxA Slp Lag AUC PLT (103/mm3) PAI-1 activity t-PA activity

1,4-dimethylpyridine

1-methylpyridine

vehicle

30

100

300

30

100

300

100

10 100 ± 4.9 100 ± 4.1 100 ± 4.2 100 ± 7.1 555.3 ± 13.7

8 85.0 ± 6.1 93.7 ± 7.9 130.6 ± 6.1** 78.8 ± 8.4 556.1 ± 16.8

8 85.3 ± 4.7 88.0 ± 4.0 130.8 ± 6.8** 75.1 ± 7.2 546.6 ± 16.5

8 77.2 ± 5.1** 80.9 ± 5.6* 143.1 ± 8.4*** 68.2 ± 7.5** 537.2 ± 18.7

7 108.7 ± 3.7 105.3 ± 2.8 92.1 ± 3.2 106.5 ± 7.6 533.6 ± 20.6

5 111.0 ± 7.9 111.7 ± 6.0 98.4 ± 3.2 109.0 ± 5.9 580.4 ± 16.2

7 106.3 ± 3.9 109.3 ± 4.4 92.1 ± 6.0 105.2 ± 14.3 562.4 ± 16.3

6 109.4 ± 7.7 112.9 ± 5.1 99.0 ± 8.6 105.2 ± 14.3 560.8 ± 21.3

100 ± 5.6 100 ± 9.7

89.1 ± 33.1 98.7 ± 10.7

119.1 ± 14.9 103.7 ± 19.4

101.1 ± 22.8 105.7 ± 8.2

101.4 ± 15.6 131.6 ± 6.4*

107.5 ± 11.7 108.5 ± 20.5

104.5 ± 9.7 91.1 ± 7.2

88.1 ± 14.7 133.9 ± 29.9

a n, number of animals; MaxA, maximal extension; Slp, slope of platelet aggregation; Lag, lag phase; and AUC, area under the curve. PAI-1 activity and t-PA activity expressed as % of vehicle; PLT, platelet count; * p < 0.05, ** p < 0.01, *** p < 0.001 vs vehicle, Mann−Whitney test. Results are shown as the mean ± SEM.

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did 1,3- and 1,4-dimethylpyridine appear to inhibit the progress of electrically induced carotid artery thrombosis but also was a more significant effect observed when we administered these pyridines in higher doses. These results indicate a new class of compounds with mild antithrombotic properties which can be found in coffee extracts. We have further examined possible mechanisms of pharmacological activity of pyridine derivatives formed from trigonelline thermal decomposition. Since we have previously proven an important role of prostacyclin in the effect of many substances influencing endothelial function including structurally related pyridine derivatives such as 1-methylnicotinamide, we tried to elicit the contribution of prostacyclin to the antithrombotic effect of dimethylpyridinium compounds. Prostacyclin is produced in endothelial cells from prostaglandin H2 by the action of the enzyme prostacyclin synthase and is a potent vasodilator and inhibitor of platelet aggregation.42 We have found a dose-dependent increase in plasma level of 6-ketoPGF1α (a stable metabolite of prostacyclin) in rats receiving 1,4-dimethylpyridine. 1,4-Dimethylpyridine increased the plasma level of 6-keto-PGF1α to 130.3 ± 14.2% (p < 0.01) and 160.2 ± 18.9% (p < 0.01), at doses of 100 and 300 mg/kg/d, respectively. 1-Methylpyridine and 1,3-dimethylpyridine showed only a trend to increase the plasma level of 6-ketoPGF1α (Figure 7). Prostacyclin (PGI2) is thought to be one of the most important prostanoids in regulating the homeostasis of the cardiovascular system.43,44 It is clear that pharmacological stimulation of PGI2 in vivo might be beneficial in vascular diseases. So far, prostacyclin or its stable analogues have been widely used in the treatment of pulmonary hypertension45 and have been proven effective in cases of peripheral arterial disease46 or liver injury.47 We simultaneously measured collagen induced platelet aggregation after thrombus formation in rat arteries. We found that 1,3-dimethylpyridine exerted an antiaggregatory effect, decreasing collagen induced platelet aggregation (Table 3) in thrombotic animals. Both 1,3-dimethylpyridine and 1,4dimethylpyridine (10−5, 10−4, and 10−3 M) did not significantly change platelet aggregation in in vitro experiments (data not shown). Since platelet activation is crucial in arterial thrombosis development, the antiplatelet activity of 1,3-dimethylpyridine could be responsible for its antithrombotic effect. The lack of influence of 1,4-dimethylpyridine on platelets, while stimulating prostacyclin release, was rather surprising and suggests a different mechanism of action. Thus, we performed in vitro experiments in which we incubated whole blood with various concentrations of examined compounds, but they both failed to directly influence platelet aggregation. It seems that the site of 1,3-dimethylpyridine action is not direct or that it acts through active metabolites. Furthermore, we measured typical coagulation parameters, like prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen (Fg) level using routine laboratory methods. 1-Methylpyridine and 1,3- and 1,4dimethylpyridine failed to influence the activity of rat plasminogen activator inhibitor-1 (PAI-1) (Table 3), PT, aPTT, Fg, and blood cell count (data not shown). 1,3Dimethylpyridine decreased fibrin formation in platelet-reach plasma, but not in platelet-poor plasma (data not shown), while 1-methylpyridine and 1,4-dimethylpyridine produced no effect (data not shown). 1,4-Dimethylpyridine increased slightly tissue plasminogen activator (t-PA) activity only at lowest

was 2 to 3 orders of magnitude lower than that of 1methylpyridine. The main dialkylpyridinium compound, which is formed during coffee roasting is 1,3-dimethylpyridine. The concentrations of alkylpyridiniums salts present in the coffee extracts are listed in Table 1. We were not able to quantitate the amount of 1,4-dimethylpyridine due to the high concentration of 1,3-dimethylpyridine present in the extract. As trigonelline, in contrast to caffeine, undergoes significant thermal degradation upon coffee roasting, including decarboxylation and demethylation (both processes take place in the temperature range typical for coffee roasting), it may be expected that similar products could be formed upon coffee roasting and trigonelline pyrolysis. Indeed, the analysis of trigonelline pyrolysate indicated the formation of similar products. The main dialkylpyridinium product formed from pyrolysis of trigonelline was not, however, as expected 1,3dimethylpyridine but 1,4-dimethylpyridine, which is shown in Figure 5. The formation of 1-ethylpyridine and trigonelline methyl ester was also observed. The data of quantitative analysis of products formed during pyrolysis of trigonelline are presented in Table 2. For some reason, the formation of 1,3-dimethylpyridine is favored during coffee roasting compared to pyrolysis of trigonelline itself, probably due to a great variety of other compounds being present in coffee beans. We assume that the formation of 1,3-dimethylpyridine rather than 1,4-dimethylpyridine in roasted coffee might be due to the direct reduction of trigonelline. In the case of thermal decomposition of pure trigonelline, bimolecular reactions might be preferred. The influence of 1-methylpyridine, and 1,3- and 1,4dimethylpyridine on the hemostasis and thrombotic processes is not known. To investigate that, we chose the model of experimental arterial thrombosis induced by electrical stimulation of carotid artery of rat. This experimental model enables simultaneous measurement of platelet aggregation and many other coagulation and fibrinolysis parameters. Although thrombus formation was initiated by electrical stimulation producing arterial injury that is unrelated to clinical thrombosis, platelet-rich thrombus morphology suggests that the growth of intravascular thrombotic material in response to electric injury is physiologically relevant.27 Importantly, in this model platelets make a major contribution to the development of thrombosis as the inhibition of platelet activation by ASA and carbon monoxide or stimulation of endothelial release of PGI2 by angiotensin-converting enzyme inhibitors or 1-methylnicotinamide afforded pronounced antithrombotic effects.18,23,25,33,41 The collagen-induced platelet aggregation, and the markers of coagulation and fibrinolysis were also examined ex vivo. We administered these compounds at a dose of 100 mg/kg/d per os for 10 days to rats before the development of arterial thrombosis. 1-Methylpyridine had no effect on thrombus formation. Since we observed rather a trend to enhance the antithrombotic effect of the 100 mg/kg/d dose of 1-methylpyridine (106.3 ± 5.6% of control, for doses of 100 mg/kg/d; ns, Figure 6), other doses of this compound have not been elucidated. Moreover, we did not further evaluate this compound as a potential antithrombotic agent. By contrast, 1,3- and 1,4-dimethylpyridine inhibited arterial thrombus formation. 1,4-Dimethylpyridine at doses of 100 and 300 mg/kg/d decreased thrombus weight to 77.0 ± 5.2% (p < 0.05) and 71.3 ± 10.3% (p < 0.05), respectively. 1,3-Dimethylpyridine was effective at a dose of 300 mg/kg/d decreasing thrombus weight to 78.4 ± 6.9%. Not only 2858

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(5) Karatzis, E.; Papaioannou, T. G.; Aznaouridis, K.; Karatzi, K.; Stamatelopoulos, K.; Zampelas, A.; Papamichael, C.; Lekakis, J.; Mavrikakis, M. Acute effects of caffeine on blood pressure and wave reflections in healthy subjects: should we consider monitoring central blood pressure? Int. J. Cardiol. 2005, 98, 425−430. (6) Robertson, D.; Frölich, J. C.; Carr, R. K.; Watson, J. T.; Hollifield, J. W.; Shand, D. G.; Oates, J. A. Effects of caffeine on plasma renin activity, catecholamines and blood pressure. N. Engl. J. Med. 1978, 298, 181−186. (7) Papamichael, C. M.; Aznaouridis, K. A.; Karatzis, E. N.; Karatzi, K. N.; Stamatelopoulos, K. S.; Vamvakou, G.; Lekakis, J. P.; Mavrikakis, M. E. Effect of coffee on endothelial function in healthy subjects. The role of caffeine. Clin. Sci. 2005, 109, 55−60. (8) Wu, J. N.; Ho, S. C.; Zhou, C.; Ling, W. H.; Chen, W. Q.; Wang, C. L.; Chen, Y. M. Coffee consumption and risk of coronary heart diseases: a meta-analysis of 21 prospective cohort studies. Int. J. Cardiol. 2009, 137, 216−225. (9) Greenberg, J. A.; Chow, G.; Ziegelstein, R. C. Caffeinated coffee consumption, cardiovascular disease, and heart valve disease in the elderly (from the Framingham Study). Am. J. Cardiol. 2008, 102, 1502−1508. (10) Bidel, S.; Hu, G.; Sundvall, J.; Kaprio, J.; Tuomilehto, J. Effects of coffee consumption on glucose tolerance, serum glucose and insulin levels-a cross-sectional analysis. Horm. Metab. Res. 2006, 38, 38−43. (11) Buscemi, S.; Verga, S.; Batsis, J. A.; Tranchina, M. R.; Belmonte, S.; Mattina, A.; Re, A.; Rizzo, R.; Cerasola, G. Dose-dependent effects of decaffeinated coffee on endothelial function in healthy subjects. Eur. J. Clin. Nutr. 2009, 63, 1200−1205. (12) Natella, F.; Nardini, M.; Belelli, F.; Pignatelli, P.; Di Santo, S.; Ghiselli, A.; Violi, F.; Scaccini, C. Effect of coffee drinking on platelets: inhibition of aggregation and phenols incorporation. Br. J. Nutr. 2008, 100, 1276−1282. (13) Toda, E.; Ishida, H.; Aoki, T.; Urano, T.; Takahari, Y.; Tamura, N.; Goto, S. Possible mechanism of preventive effects of coffee intake on the formation of arterial occlusive thrombosis. Tokai J. Exp. Clin. Med. 2010, 35, 133−136. (14) Spiller, M. A. The Chemical Components of Coffee. In Caffeine; Spiller, G. A., Ed.; CRC Press Inc.: Boca Raton, FL, 1998; pp 97−161. (15) Bonita, J. S.; Mandarano, M.; Shuta, D.; Vinson, J. Coffee andcardiovascular disease: in vitro, cellular, animal, and human studies. Pharmacol. Res. 2007, 55, 187−198. (16) Yukawa, G. S.; Mune, M.; Otani, H.; Tone, Y.; Liang, X. M.; Iwahashi, H.; Sakamoto, W. Effects of coffee consumption on oxidative susceptibility of low-density lipoproteins and serum lipid levels in humans. Biochemistry 2004, 69, 70−74. (17) Bydlowski, S. P.; Yunker, R. L.; Rymaszewski, Z.; Subbiah, M. T. Coffee extracts inhibit platelet aggregation in vivo and in vitro. Int. J. Vitam. Nutr. Res. 1987, 57, 217−223. (18) Chlopicki, S.; Swies, J.; Mogielnicki, A.; Buczko, W.; Bartus, M.; Lomnicka, M.; Adamus, J.; Gebicki, J. 1-Methylnicotinamide (MNA), a primary metabolite of nicotinamide, exerts anti-thrombotic activity mediated by a cyclooxygenase-2/prostacyclin pathway. Br. J. Pharmacol. 2007, 152, 230−239. (19) Stadler, R. H.; Varga, N.; Hau, J.; Vera, F. A.; Welti, D. H. Alkylpyridiniums. 1. Formation in model systems via thermal degradation of trigonelline. J. Agric. Food Chem. 2002, 50, 1192−1199. (20) Somoza, V.; Lindenmeier, M.; Wenzel, E.; Frank, O.; Erbersdobler, H. F.; Hofmann, T. Activity-guided identification of a chemopreventive compound in coffee beverage using in vitro and in vivo techniques. J. Agric. Food Chem. 2003, 51, 6861−6869. (21) Gebicki, J.; Marcinek, A.; Chlopicki, S.; Adamus, J. PCT International Application. WO 2008104920 A1, 2008. (22) Lang, R.; Wahl, A.; Skurk, T.; Yagar, E. F.; Schmiech, L.; Eggers, R.; Hauner, H.; Hofmann, T. Development of a hydrophilic liquid interaction chromatography-high-performance liquid chromatographytandem mass spectrometry based stable isotope dilution analysis and pharmacokinetic studies on bioactive pyridines in human plasma and urine after coffee consumption. Anal. Chem. 2010, 82, 1486−1497.

dose. 1-Methylpyridine and 1,3- and 1,4-dimethylpyridine at higher doses failed to influence t-PA activity (Table 3). Inhibition of fibrin generation in platelet-reach but not in platelet-poor plasma by 1,3-dimethylpyridine suggests platelet involvement in the antithrombotic effect of this compound. This effect is in line with the inhibitory effect of 1,3dimethylpyridine on platelet aggregation in whole blood. According to the fibrinolytic parameters analysis, we may also assume that the fibrinolytic system is not involved in the anithrombotic effect of dimethylpiridinium salts. In conclusion, we demonstrated here that pyridinium compounds present in roasted coffee possess the antithrombotic prostacyclin releasing (1,4-dimethylpyridine) and platelet inhibiting (1,3-dimethylpyridine) properties. Although they have a cationic chemical nature and their permeability through cell membranes might be difficult, they can reach relatively high dose-dependent concentration in blood.22 This property would be important for all orally administered formulations. We may hypothesize that 1,3-dimethylpyridine could be a promising agent preventing atherothrombosis development, where the platelets play a crucial role, whereas 1,4-dimethylpyridine could be beneficial in states with impaired prostacyclin endothelial production. Our findings on the novel in vivo biological activity of dimethylpiridinium compounds may have potentially important therapeutic implications and warrant further studies, although low concentration of dialkylpyridinium salts in the roasted coffee probably limits their contribution to the overall antithrombotic functions of coffee beverages.



ASSOCIATED CONTENT

S Supporting Information *

Description of the synthesis of pyridinium derivatives (1methylpyridine, 1,2-, 1,3-, and 1,4-dimethylpyridine, 1-ethylpyridine, and trigonelline methyl ester). This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Tel: +48 857485601. Fax: +48 857485601. E-mail: kalaskab@ gmail.com. Funding

This work was supported by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, No. POIG.01.01.02-00-069/09). Notes

The authors declare no competing financial interest.



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