C-phycocyanin, a very potent and novel platelet aggregation inhibitor from Spirulina platensis

The aim of this study was to systematically examine the inhibitory mechanisms of C-phycocyanin (C-PC), one of the major phycobiliproteins of Spirulina platensis (a blue-green alga), in platelet activation. In this study, C-PC concentration-dependently (0.5-10 nM) inhibited platelet aggregation stimulated by agonists. C-PC (4 and 8 nM) inhibited intracellular Ca2+ mobilization and thromboxane A2 formation but not phosphoinositide breakdown stimulated by collagen (1 microg/mL) in human platelets. In addition, C-PC (4 and 8 nM) markedly increased levels of cyclic GMP and cyclic GMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser(157) phosphorylation. Rapid phosphorylation of a platelet protein of Mw 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (150 nM). This phosphorylation was markedly inhibited by C-PC (4 and 8 nM). In addition, C-PC (4 and 8 nM) markedly reduced the electron spin resonance (ESR) signal intensity of hydroxyl radicals in collagen (1 microg/mL)-activated platelets. The present study reports on a novel and very potent (in nanomolar concentrations) antiplatelet agent, C-PC, which is involved in the following inhibitory pathways: (1) C-phycocyanin increases cyclic GMP/VASP Ser157 phosphorylation and subsequently inhibits protein kinase C activity, resulting in inhibition of both P47 phosphorylation and intracellular Ca2+ mobilization, and (2) C-PC may inhibit free radicals (such as hydroxyl radicals) released from activated platelets, which ultimately inhibits platelet aggregation. These results strongly indicate that C-PC appears to represent a novel and potential antiplatelet agent for treatment of arterial thromboembolism.     

Mechanisms involved in the antiplatelet activity of rutin, a glycoside of the flavonol quercetin, in human platelets

The aim of this study was to systematically examine the inhibitory mechanisms of rutin, a well-known flavonoid in platelet aggregation. In this study, rutin concentration-dependently (250 and 290 microM) inhibited platelet aggregation in human platelets stimulated by agonists (i.e., collagen). Rutin (250 and 290 microM) did not significantly interfere with the binding of FITC-triflavin to the glycoprotein IIb/IIIa complex in human platelets. Rutin (250 and 290 microM) markedly inhibited intracellular Ca(2+) mobilization and thromboxane A(2) formation in human platelets stimulated by collagen. Rapid phosphorylation of a platelet protein of M(r) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/mL). This phosphorylation was markedly inhibited by rutin (250 and 290 microM). On the other hand, rutin (250 and 290 microM) did not significantly increase the formations of cyclic AMP and nitric oxide/cyclic GMP in platelets. In conclusion, these results indicate that the antiplatelet activity of rutin may involve the following pathways: rutin inhibited the activation of phospholipase C, followed by inhibition of protein kinase C activity and thromboxane A(2) formation, thereby leading to inhibition of the phosphorylation of P47 and intracellular Ca(2+) mobilization, finally resulting in inhibition of platelet aggregation.     

Inhibitory effect of α-lipoic acid on platelet aggregation is mediated by PPARs

Peroxisome proliferator-activated receptors (PPARs) isoforms (α, β/δ, and γ are present in human platelets, and activation of PPARs inhibits platelet aggregation. α-Lipoic acid (ALA), occurring naturally in human food, has been reported to exhibit an antiplatelet activity. However, the mechanisms underlying ALA-mediated inhibition of platelet aggregation remain unknown. The aim of this study was to investigate whether the antiplatelet activity of ALA is mediated by PPARs. ALA itself significantly induced PPARα/γ activation in platelets and increased intracellular amounts of PPARα/γ by blocking PPARα/γ secretion from arachidonic acid (AA)-activated platelets. Moreover, ALA significantly inhibited AA-induced platelet aggregation, Ca(2+) mobilization, and cyclooxygenase-1 (COX-1) activity, but increased cyclic AMP production in rabbit washed platelets. Importantly, ALA also enhanced interaction of PPARα/γ with protein kinase Cα (PKCα) and COX-1 accompanied by an inhibition of PKCα activity in resting and AA-activated platelets. However, the above effects of ALA on platelets were markedly reversed by simultaneous addition of selective PPARα antagonist (GW6471) or PPARγ antagonist (GW9662). Taken together, the present study provides a novel mechanism by which ALA inhibition of platelet aggregation is mediated by PPARα/γ-dependent processes, which involve interaction with PKCα and COX-1, increase of cyclic AMP formation, and inhibition of intracellular Ca(2+) mobilization.     

Antioxidative and antiplatelet effects of aqueous inflorescence Piper betle extract

Piper betle, belonging to the Piperaceae family, is a tropical plant, and its leaf and inflorescence are popularly consumed by betel quid (BQ) chewers in Taiwan and many other South and Southeast Asian countries. However, little is known about the biochemical properties of inflorescence Piper betle (IPB) toward reactive oxygen species (ROS) and platelet functions. In the present work, aqueous IPB extract was shown to be a scavenger of H(2)O(2), superoxide radical, and hydroxyl radical with a 50% inhibitory concentration (IC(50)) of about 80, 28, and 73 microg/mL, respectively. IPB extract also prevented the hydroxyl radical induced PUC18 plasmid DNA breaks at concentrations higher than 40 microg/mL. Since ROS are crucial for platelet aggregation, we further found that IPB extract also inhibited the arachidonic acid (AA) induced and collagen-induced platelet aggregation, with an IC(50) of 207 and 335 microg/mL, respectively. IPB extract also inhibited the AA-, collagen- (>100 microg/mL of IPB), and thrombin (>250 microg/mL of IPB)-induced thromboxane B(2) (TXB(2)) production by more than 90%. However, IPB extract showed little effect on thrombin-induced aggregation. These results indicated that aqueous components of IPB are potential ROS scavengers and may prevent the platelet aggregation possibly via scavenging ROS or inhibition of TXB(2) production.     

Grape-seed procyanidins act as antiinflammatory agents in endotoxin-stimulated RAW 264.7 macrophages by inhibiting NFkB signaling pathway

Procyanindin extract (PE) is a mixture of polyphenols, mainly procyanidins, obtained from grape seed with putative antiinflammatory activity. We evaluated the PE effect on RAW 264.7 macrophages stimulated with lipopolysaccharide plus interferon-gamma that show a rapid enhanced production of prostaglandin E2 (PGE2) and nitric oxide (NO). Our results demonstrated that PE significantly inhibited the overproduction of NO, dose and time dependently. PE caused a marked inhibition of PGE2 synthesis when administered during activation. Moreover, PE pretreatment diminished iNOS mRNA and protein amount dose dependently (10-65 microg/mL). PE (65 microg/mL) pretreatment inhibited NFkappaB (p65) translocation to nucleus by nearly 40%. Trimeric and longer oligomeric-rich procyanidin fractions from PE (5-30 microg/mL) inhibited iNOS expression but not the monomeric forms catechin and epicatechin. Thus, we show that the degree of polymerization is important in determining procyanidin effects. PE was considerably a more effective inhibitor of NO biosynthesis (IC50 = 50 microg/mL) in comparison to other antiinflammatories, such as aspirin (3 mM), indomethacin (20 microM), and dexamethasone (9 nM). In conclusion, PE modulates inflammatory response in activated macrophages by the inhibition of NO and PGE2 production, suppression of iNOS expression, and NFkB translocation. These results demonstrate an immunomodulatory role of grape seed procyanidins and thus a potential health-benefit in inflammatory conditions that exert an overproduction of NO and PGE2.     

Antiplatelet activity of soy sauce as functional seasoning.

In seeking the functionality of foodstuffs applicable to medicine, soy sauce was found to show antiplatelet activity. Therefore, the active components in soy sauce were purified, structurally identified, and studied for their inhibitory effects on the aggregation of human platelets. Aqueous 2-fold diluents of soy sauce inhibited platelet aggregation induced by collagen and epinephrine depending on the dilution factor. Since a basic extract with diethyl ether completely inhibited collagen-induced aggregation, it was subjected to serial extractions and multistep HPLC fractionations for purifying antiplatelet components. The finally obtained isolates were identified as 1-methyl-1,2,3,4-tetrahydro-beta-carboline and 1-methyl-beta-carboline on the basis of EI-MS, (1)H NMR, diode array, and fluorescence spectra. Their spectral data and chromatographic behaviors were the same as those of synthetic ones. 1-Methyl-1,2,3, 4-tetrahydro-beta-carboline showed mean concentrations (n = 5-6) of 4.6, 4.2, 28.6, 11.6, and 65.8 microgram/mL to produce 50% inhibition of the maximal aggregation response induced by epinephrine, platelet-activating factor, collagen, adenosine 5'-diphosphate, and thrombin, respectively. Its inhibitory effect was much greater than that of 1-methyl-beta-carboline on platelet aggregation by all the tested inducers. The quantitative HPLC analysis revealed that the significant amounts of both antiplatelet compounds were uniformly contained in commercially available soy sauce. From these results, soy sauce may be referred to as functional seasoning containing alkaloidal components with the potent preventive effect on thrombus formation.     

Involvement of Ca2+ in the inhibition by crocetin of platelet activity and thrombosis formation

Crocetin, a unique carotenoid with potent antioxidative and anti-inflammatory activities, is a major ingredient of saffron used as an important spice and food colorant in various parts of the world. In the present study, the effects of crocetin on platelet activity and thrombosis formation were systematically investigated. Crocetin showed a dose-dependent inhibition of platelet aggregation induced by ADP, collagen, but not by arachidonic acid (AA). Crocetin significantly attenuated dense granule release, while neither platelets adhesion to collagen nor cyclic AMP level was altered by crocetin. Pretreatment with crocetin was confirmed to partially inhibit Ca (2+) mobilization via reducing both intracellular Ca (2+) release and extracellular Ca (2+) influx. Besides that, crocetin prolonged the occlusive time in electrical stimulation-induced carotid arterial thrombosis. These findings suggest that the favorable impacts of crocetin on platelet activity and thrombosis formation may be related to the inhibition of Ca (2+) elevation in stimulated platelets.     

Inhibition of human cAMP-phosphodiesterase as a mechanism of the spasmolytic effect of Matricaria recutita L

Mechanisms underlying the spasmolytic activity of chamomile still remain unclear. Inhibition of cAMP- and cGMP-phosphodiesterases (PDE) is one of the mechanisms operated by spasmolytic drugs. In this study, the effect of chamomile on PDE was investigated. Human platelet cAMP-PDE and recombinant PDE5A1 were assayed in the presence of infusions prepared from sifted flowers and capitula. LC-ESI-MS/MS analysis showed different compositions in infusions made with sifted flowers and capitula. Chamomile inhibited cAMP-PDE activity (IC50 = 17.9-40.5 microg/mL), while cGMP-PDE5 was less affected (-15% at 50 microg/mL). Among the individual compounds tested, only flavonoids showed an inhibitory effect (IC50 = 1.3-14.9 microM), contributing to around 39% of the infusion inhibition; other compounds responsible for cAMP-PDE inhibition still remain unknown. Although experimental evidence supporting the use of chamomile for gastrointestinal minor spasms dates back to the fifties, cAMP-PDE inhibition as a likely mechanism underlying the spasmolytic activity is reported for the first time.     

Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclooxygenase enzyme, and lipid peroxidation

The antioxidant ferulic and caffeic acid phenolics are ubiquitous in plants and abundant in fruits and vegetables. We have synthesized a series of ferulic and caffeic acid esters and tested for tumor cell proliferation, cyclooxygenase enzymes (COX-1 and -2) and lipid peroxidation inhibitory activities in vitro. In the tumor cell proliferation assay, some of these esters showed excellent growth inhibition of colon cancer cells. Among the phenolics esters assayed, compounds 10 (C12-caffeate), 11 (C16-caffeate), 21 (C8-ferulate), and 23 (C12-ferulate) showed strong growth inhibition with IC50 values of 16.55, 13.46, 18.67, and 7.57 microg/mL in a breast cancer cell line; 9.65, 7.45, 17.05, and 4.35 microg/ mL in a lung cancer cell line; 5.78, 3.5, 4.29, and 2.46 microg/mL in a colon cancer cell line; 12.04, 12.21, 14.63, and 8.09 microg/ mL in a central nervous system cancer cell line; and 8.62, 7.76, 11.0, and 5.37 in a gastric cancer cell line. In COX enzyme inhibitory assays, ferulic and caffeic acid esters significantly inhibited both COX-1 and COX-2 enzymes. Caffeates 5-10 (C4-C12), inhibited COX-1 enzyme between 50% and 90% and COX-2 enzyme by about 70%, whereas ferulates 15-21 (C3-C8) inhibited COX-1 and COX-2 enzymes by 85-95% 25 microg/mL. Long-chain caffeates 11-14 (C16-C22) and short-chain ferulates 15-20 (C3-C5) were the most active in lipid peroxidation inhibition and showed 60-70% activity at 5 microg/mL concentration.     

Free radical scavengers and antioxidants from Lemongrass (Cymbopogon citratus (DC.) Stapf.)

Methanol, MeOH/water extracts, infusion, and decoction of Cymbopogon citratus were assessed for free radical scavenging effects measured by the bleaching of the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical, scavenging of the superoxide anion, and inhibition of the enzyme xanthine oxidase (XO) and lipid peroxidation in human erythrocytes. The extracts presented effect in the DPPH and superoxide anion assay, with values ranging between 40 and 68% and 15-32% at 33 and 50 microg/mL, respectively, inhibited lipid peroxidation in erythrocytes by 19-71% at 500 microg/mL and were inactive toward the XO at 50 microg/mL. Isoorientin, isoscoparin, swertiajaponin, isoorientin 2' '-O-rhamnoside, orientin, chlorogenic acid, and caffeic acid were isolated and identified by spectroscopic methods. Isoorientin and orientin presented similar activities toward the DPPH (IC(50): 9-10 microM) and inhibited lipid peroxidation by 70% at 100 microg/mL. Caffeic and chlorogenic acid were active superoxide anion scavengers with IC(50) values of 68.8 and 54.2 microM, respectively, and a strong effect toward DPPH. Caffeic acid inhibited lipid peroxidation by 85% at 100 microg/mL.     

Anti type I allergic property of Japanese butterbur extract and its mast cell degranulation inhibitory ingredients

Pollenosis is a disease that affects 1 in 10 of the Japanese population. During the season of cedar pollen dispersal, many patients suffer from symptoms such as sniffling, sternutation, and itching of the eyes. Japanese butterbur is a popular vegetable and is one of the few domestic vegetables in Japan. The anti type I allergic effects of an aqueous ethanol extract from aerial parts of Japanese butterbur (JBE) were evaluated in rats and RBL-2H3 mast cells. In the passive cutaneous anaphylaxis reaction in rats, a single oral treatment of JBE (1000 mg/kg) was found to suppress the reaction. In IgE-sensitized RBL-2H3 cells, JBE (10-100 microg/mL) inhibited beta-hexosaminidase release, leukotriene C(4)/D(4)/E(4) synthesis, and TNF-alpha production. Moreover, a high concentration of JBE (1000 microg/mL) suppressed smooth muscle constriction induced by histamine (10 microM) and leukotriene D(4) (10 nM) in a guinea pig trachea strip. The search for components in JBE with an inhibitory activity on mast cell degranulation was guided by inhibition of beta-hexsosaminidase release. Two eremophilane-type sesquiterpenes, six polyphenolic compounds, and two triterpene glycosides were isolated. Of these compounds, fukinolic acid, a principal polyphenol constituent, showed potent inhibitory activity (IC(50) value = 2.1 microg/mL). Consequently, On the basis of its inhibition of mast cell activation and direct smooth muscle reaction induced by released mediators, JBE was found to suppress the type I allergic reaction.     

Effects of hydroxytyrosol and hydroxytyrosol acetate administration to rats on platelet function compared to acetylsalicylic acid

Virgin olive oil (VOO) contains the polyphenols hydroxytyrosol (HT) and hydroxytyrosol acetate (HT-AC). This study investigated the antiplatelet effect of HT and HT-AC in healthy rats and compared their effects to acetylsalicylic acid (ASA). All compounds were administered orally for 7 days. HT and HT-AC inhibited platelet aggregation in whole blood, with a 50% inhibitory dose (ID50) of 48.25 mg/kg per day for HT, 16.05 mg/kg per day for HT-AC, and 2.42 mg/kg per day for ASA. Platelet synthesis of thromboxane B2 was inhibited by up to 30% by HT and 37% by HT-AC; the ID50 of this effect for ASA was 1.09 mg/kg per day. Vascular prostacyclin production was inhibited by up to 27.5% by HT and 32% by HT-AC; the ID50 of this effect for ASA was 6.75 mg/kg per day. Vascular nitric oxide production was increased by up to 34.2% by HT, 66% by HT-AC, and 64% by ASA. We conclude that HT and HT-AC administered orally inhibited platelet aggregation in rats and that a decrease in thromboxane synthesis along with an increase in nitric oxide production contributed to this effect.     

Effect of 2alpha-hydroxyursolic acid on NF-kappaB activation induced by TNF-alpha in human breast cancer MCF-7 cells

Apples are one of the largest contributors of fruit phenolics of all fruits consumed by Americans and contain a variety of bioactive compounds, which have health benefits. Consumption of apples has been linked to reduced risk of chronic diseases such as cancer and cardiovascular disease. Apple extracts have been shown to have the capabilities of inhibiting NF-kappaB activation in human breast cancer MCF-7 cells. 2Alpha-hydroxyursolic acid is one of the major triterpenoids isolated from apple peels, and its effects on cell proliferation and TNF-alpha-induced NF-kappaB activation in MCF-7 cells were examined. 2Alpha-hydroxyursolic acid significantly inhibited MCF-7 cell proliferation at doses of 20 microM (p < 0.05). Preincubation with 2alpha-hydroxyursolic acid suppressed TNF-alpha-induced NF-kappaB activation in a dose-dependent manner and significantly inhibited the activation at a dose of 20 microM of 2alpha-hydroxyursolic acid (p < 0.05). 2Alpha-hydroxyursolic acid treatment did not affect the phosphorylation level of NF-kappaB inhibitor (IkappaB-alpha), but proteasome activity in MCF-7 cells was inhibited significantly at doses of 10 and 20 microM ( p < 0.05). These results suggest that 2alpha-hydroxyursolic acid has antiproliferative activities against MCF-7 cells and capabilities inhibiting NF-kappaB activation induced by TNF-alpha partially by suppressing proteasome activities.     

Evaluation of the dietetic and therapeutic potential of a high molecular weight hydroxycinnamate-derived polymer from Symphytum asperum Lepech. Regarding its antioxidant, antilipoperoxidant, antiinflammatory, and cytotoxic properties

A water-soluble hydroxycinnamate-derived polymer (>1000 kDa) from Symphytum asperum Lepech. (Boraginaceae) strongly reduced the diphenylpicrylhydrazyl radical (IC(50) approximately 0.7 microg/mL) and inhibited the nonenzymatic lipid peroxidation of bovine brain extracts (IC(50) approximately 10 ng). This polymer exhibited only a low hydroxyl radical scavenging effect in the Fe(3+)-EDTA-H(2)O(2) deoxyribose system (IC(50) > 100 microg/mL) but strongly decreased superoxide anion generation in either the reaction of phenazine methosulfate with NADH and molecular oxygen (IC(50) approximately 13.4 microg/mL) or in rat PMA-activated leukocytes (IC(50) approximately 5 microg/mL). The ability to inhibit both degranulation of azurophil granules and superoxide generation in primed leukocytes indicates that the NADPH oxidase responsible for this later effect is inhibited, pointing to the Symphytum asperum polymer as a potent antiinflammatory and vasoprotective agent. At all concentrations tested (0-200 microg/mL), we observed no cytotoxicity on normal human fibroblasts and neither antiproliferative effects nor proliferation activation on neoplastic cells.     

Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro

Prenylated flavonoids found in hops and beer, i.e., prenylchalcones and prenylflavanones, were examined for their ability to inhibit in vitro oxidation of human low-density lipoprotein (LDL). The oxidation of LDL was assessed by the formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS) and the loss of tryptophan fluorescence. At concentrations of 5 and 25 microM, all of the prenylchalcones tested inhibited the oxidation of LDL (50 microg protein/ml) induced by 2 microM copper sulfate. The prenylflavanones showed less antioxidant activity than the prenylchalcones, both at 5 and 25 microM. At 25 microM, the nonprenylated chalcone, chalconaringenin (CN), and the nonprenylated flavanone, naringenin (NG), exerted prooxidant effects on LDL oxidation, based on TBARS formation. Xanthohumol (XN), the major prenylchalcone in hops and beer, showed high antioxidant activity in inhibiting LDL oxidation, higher than alpha-tocopherol and the isoflavone genistein but lower than the flavonol quercetin. When combined, XN and alpha-tocopherol completely inhibited copper-mediated LDL oxidation. These findings suggest that prenylchalcones and prenylflavanones found in hops and beer protect human LDL from oxidation and that prenylation antagonizes the prooxidant effects of the chalcone, CN, and the flavanone, NG.     

Attenuation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced gap junctional intercellular communication (GJIC) inhibition in MCF-10A cells by c9,t11-conjugated linoleic acid

The protective effect of c9,t11-conjugated linoleic acid (CLA) on the inhibition of gap junctional intercellular communication (GJIC) was examined in a human mammary epithelial cell line (MCF-10A) treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), relative to t10,c12-CLA isomer. TPA inhibited GJIC in a dose-dependent and reversible manner and was associated with connexin 43 phosphorylation. Pretreatment of 20 μM c9,t11-CLA for 24 h prior to 60 nM TPA for 1 h prevented the inhibition of GJIC by reducing the phosphorylation of connexin 43 via suppressing extracellular signal-regulated kinases (ERK1/2) activation. Reactive oxygen species (ROS) accumulation by TPA was attenuated by c9,t11-CLA. The efficacy of c9,t11-CLA in protecting inhibition of GJIC, connexin 43 phosphorylation, and ROS production was superior to that of t10,c12-CLA. These results suggest that c9,t11-CLA, including t10,c12-CLA, prevents the carcinogenesis of MCF-10A cells by protecting down-regulation of GJIC during the cancer promotion stage, and lack of their toxicities could be an excellent indicator for the chemoprevention of breast cancer.     

Phytotoxins from the leaves of Ruta graveolens

Bioassay-guided fractionation of the ethyl acetate extract of Ruta graveolens (common rue) leaves led to the isolation of the furanocoumarins 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and the quinolone alkaloid graveoline as phytotoxic constituents. Graveoline and 8-MOP substantially inhibited growth of Lactuca sativa (lettuce) seedlings and reduced chlorophyll content at 100 microM; this effect was not due to a direct effect on chlorophyll synthesis. Radical growth of L. sativa was inhibited by 10 microM 8-MOP. Graveoline inhibited growth of Lemna paucicostata (duckweed) at 100 microM. This is the first report of the phytotoxic activity of graveoline. Growth of Agrostis stolonifera (bentgrass) was inhibited by 5-MOP at 30 microM. All three compounds substantially reduced cell division in Allium cepa (onion) at or below 100 microM. None of the compounds caused significant cellular leakage of Cucumis sativus (cucumber) cotyledon disks at 100 microM. All three compounds inhibit plant growth, at least partially through inhibition of cell division.     

Caffedymine from cocoa has COX inhibitory activity suppressing the expression of a platelet activation marker, P-selectin

Caffedymine (N-caffeoyldopamine) is a clovamide-type phenylpropenoic acid amide found in plants. Previous studies indicate that caffedymine inhibits P-selectin expression via increasing cAMP through beta-2 adrenoceptors, but the inhibition was only partially repressed by beta-2 adrenoceptor antagonists, suggesting additional mechanisms underlying the inhibitory effect. Therefore, in this study, the effect of caffedymine and its analogues (N-caffeoyltyramine, N-feruloyltyramine, N-coumaroyltyramine, N-cinnamoyltyramine) on COX enzymes (I and II) was investigated, because COX enzymes are deeply involved in regulating P-selectin expression on human platelets. The decreasing order of COX-I inhibitory activity was caffedymine>N-caffeoyltyramine>N-feruloyltyramine>N-coumaroyltyramine>N-cinnamoyltyramine. Caffedymine was the most potent compound tested, able to inhibit COX-I enzyme activity by 43% (P<0.013) at the concentration of 0.01 microM. At the same concentration, caffedymine was also able to inhibit COX-II enzyme activity by 36% (P<0.015), and the decreasing order of COX-II inhibitory activity was similar as that of COX-I. As a result of the COX inhibition, the production of thromboxane B2 (thromboxane A2 derivative) also decreased significantly in mouse blood treated with caffedymine and its analogues (0.05 microM). Caffedymine and N-caffeoyltyramine, both with potent COX inhibitory activity, were also able to inhibit P-selectin expression and platelet-leukocyte interactions. These data indicate that COX inhibition is likely to be another mechanism for caffedymine to inhibit P-selectin expression on platelets.     

Apigenin inhibits platelet adhesion and thrombus formation and synergizes with aspirin in the suppression of the arachidonic acid pathway

Previous studies using washed platelets demonstrated that certain flavonoids inhibit platelet function through several mechanisms including blockade of TxA(2) receptors (TPs). We aimed to analyze the binding capacity of flavonoids to TPs in platelet rich plasma (PRP), investigated their effect in flowing blood, and evaluated the ability of apigenin to improve the efficacy of aspirin in the inhibition of platelet aggregation. The binding of flavonoids to TPs in PRP was explored using binding assays and the TP antagonist [ (3)H]SQ29548. Effects of flavonoids on platelet adhesion were assessed using arterial subendothelium with annular plate perfusion chambers, and global evaluation of apigenin on high-shear-dependent platelet function was determined by the PFA-100. To evaluate the ability of apigenin to potentiate the effect of aspirin, arachidonic acid-induced platelet aggregation was measured prior to and after consumption of subaggregatory doses of aspirin in the presence or absence of apigenin. Binding assays revealed that apigenin was an efficient competitor of [ (3)H]SQ29548 binding to PRP ( K i = 155.3 +/- 65.4 microM), and perfusion studies showed that apigenin, genistein, and catechin significantly diminished thrombus formation when compared to control (26.2 +/- 3.8, 33.1 +/- 5.2, and 26.2 +/- 5.2 vs 76.6 +/- 2.6%, respectively; p < 0.05). Apigenin, similarly to the TP antagonist SQ29548, significantly prolonged collagen epinephrine-induced PFA-100 closure time in comparison to the control and, when added to platelets that had been exposed in vivo to aspirin, potentiated its inhibitory effect on platelet aggregation. The inhibitory effect of some flavonoids in the presence of plasma, particularly apigenin, might in part rely on TxA(2) receptor antagonism. There is a clear increase in the ex vivo antiplatelet effect of aspirin in the presence of apigenin, which encourages the idea of the combined use of aspirin and certain flavonoids in patients in which aspirin fails to properly suppress the TxA(2) pathway.