Licochalcones extracted from Glycyrrhiza inflata inhibit platelet aggregation accompanied by inhibition of COX-1 activity.

Licochalcones extracted from Glycyrrhiza inflata are known to have a variety of biological properties such as anti-inflammatory, anti-bacterial, and anti-tumor activities, but their action on platelet aggregation has not yet been reported. Therefore, in this study we investigated the effects of licochalcones on platelet aggregation. Collagen and U46619, a thromboxane A2 receptor agonist, caused rabbit platelet aggregation, which was reversed by pretreatment with licochalcones A, C and D in concentration-dependent manners. Among these compounds, licochalcone A caused the most potent inhibitory effect on collagen-induced platelet aggregation. However, the licochalcones showed marginal inhibitory effects on thrombin or ADP-induced platelet aggregation. In addition to rabbit platelets, licochalcone A attenuated collagen-induced aggregation in human platelets. Because licochalcone A also inhibited arachidonic acid-induced platelet aggregation and production of thromboxane A2 induced by collagen in intact platelets, we further examined the direct interaction of licochalcone A with cyclooxygenase (COX)-1. As expected, licochalcone A caused an inhibitory effect on both COX-1 and COX-2 in vitro. Regarding the effect of licochalcone A on COX-1 enzyme reaction kinetics, although licochalcone A showed a stronger inhibition of prostaglandin E2 synthesis induced by lower concentrations of arachidonic acid, Vmax values in the presence or absence of licochalcone A were comparable, suggesting that it competes with arachidonic acid at the same binding site on COX-1. These results suggest that licochalcones inhibit collagen-induced platelet aggregation accompanied by inhibition of COX-1 activity

Licochalcones extracted from <i>Glycyrrhiza inflata</i> inhibit platelet aggregation accompanied by inhibition of COX-1 activity - Fig 1

<p>Chemical structures of licochalcones A, C and D.</p

Concentration-dependent inhibition of U46619- and collagen-induced platelet aggregation by licochalcones.

<p><b>(A)</b> Licochalcone A (Lico A, 100 μM), licochalcone C (Lico C, 100 μM), licochalcone D (Lico D, 100 μM) or DMSO (-) was preincubated for 5 min before addition of U46619 (3 μM), collagen (3 μg/ml) or thrombin (0.03 U/ml) in the presence of 1 mM CaCl₂. Results are shown as mean±S.E.M. (*<i>P</i><0.05 compared with control, n = 3, Tukey–Kramer’s method). <b>(B)</b> Licochalcones (2–100 μM) or DMSO (control) were preincubated for 5 min before addition of U46619 (3 μM) or collagen (3 μg/ml) in the presence of 1 mM CaCl₂. Results are shown as mean±S.E.M. (*<i>P</i><0.05 compared with control, n = 3–8, Dunnett’s method). <b>(C)</b> Licochalcone A (10 or 100 μM) or DMSO (control) were preincubated for 5 min before addition of collagen (3 μg/ml) in the presence of 1 mM CaCl₂. Representative traces of the collagen-induced platelet aggregation with or without licochalcone A are shown.</p

Scanning electron microscopy images of platelets stimulated by collagen in the presence of licochalcones.

<p>Platelets were incubated with collagen (3 μg/ml) for 3 min in the presence or absence of licochalcones (100 μM), then fixed overnight with 1% glutaraldehyde. The samples were washed twice for 5 min with PBS. The fixed platelets were dehydrated with ethanol and t-butyl alcohol, and after the samples were freeze-dried and coated with Au/Pd, they were observed under a scanning electron microscope. <b>(A)</b> Unstimulated platelets, <b>(B)</b> collagen (3 μg/ml), <b>(C)</b> licochalcone A (100 μM) +collagen, <b>(D)</b> licochalcone C (100 μM) +collagen and <b>(E)</b> licochalcone D (100 μM) +collagen. (Magnification: 7000×, bar = 5 μm).</p

Competitive inhibition of COX-1 and COX-2 activity by licochalcone A <i>in vitro</i>.

<p><b>(A)</b> Hematin (2 μM) and L-tryptophan (5 mM) were added to 100 mM Tris-HCl buffer (pH 8.0), then two units of COX-1 or COX-2 and each concentration of licochalcone A (0.5–100 μM) were added and incubated at 37°C for 10 min. The reaction was initiated by adding arachidonic acid (0.5 μM). After 2 min, the reaction was terminated by adding 1.0 M HCl, and then the amount of PGE₂ was measured by LC-MS/MS. Results are shown as mean±S.E.M. (*<i>P</i><0.05 compared with control, n = 3, Dunnett’s method). <b>(B)</b> Two units of COX-1 and licochalcone A (2.5 μM) were incubated at 37°C for 10 min as described above. The reaction was initiated by adding each concentration of arachidonic acid (0.1–10 μM). After 2 min, the reaction was terminated by adding 1.0 M HCl, and then the amount of PGE₂ was measured by LC-MS/MS. Results are shown as mean±S.E.M. (*<i>P</i><0.05 compared with control, n = 3, Student’s t-test).</p

Concentration-dependent inhibition of arachidonic acid metabolism by licochalcone A.

<p><b>(A)</b> Licochalcone A (2–50 μM) or DMSO (−) was preincubated for 5 min before addition of arachidonic acid (30 μM) in the presence of 1 mM CaCl₂. Results are shown as mean±S.E.M. (*<i>P</i><0.05 compared with control, n = 3, Dunnett’s method). <b>(B)</b> Licochalcone A (5–50 μM) or DMSO (−) was preincubated for 5 min before addition of collagen (3 μg/ml) in the presence of 1 mM CaCl₂, and terminated by EDTA/indomethacin after incubating for 5 min. Samples were diluted 1/1000 and TXB₂ amount was measured by EIA. Results are shown as mean±S.E.M. (*<i>P</i><0.05 compared with control, n = 3, Dunnett’s method).</p

Concentration-dependent inhibition of collagen-induced human platelet aggregation by licochalcone A.

<p>Licochalcone A (10–100 μM) or DMSO (control) were preincubated for 5 min before addition of collagen (5 μg/ml) in the presence of 1 mM CaCl₂. Results are shown as mean±S.E.M. (*<i>P</i><0.05 compared with control, n = 3, Dunnett’s method).</p

Licochalcones extracted from <i>Glycyrrhiza inflata</i> inhibit platelet aggregation accompanied by inhibition of COX-1 activity

<div><p>Licochalcones extracted from <i>Glycyrrhiza inflata</i> are known to have a variety of biological properties such as anti-inflammatory, anti-bacterial, and anti-tumor activities, but their action on platelet aggregation has not yet been reported. Therefore, in this study we investigated the effects of licochalcones on platelet aggregation. Collagen and U46619, a thromboxane A₂ receptor agonist, caused rabbit platelet aggregation, which was reversed by pretreatment with licochalcones A, C and D in concentration-dependent manners. Among these compounds, licochalcone A caused the most potent inhibitory effect on collagen-induced platelet aggregation. However, the licochalcones showed marginal inhibitory effects on thrombin or ADP-induced platelet aggregation. In addition to rabbit platelets, licochalcone A attenuated collagen-induced aggregation in human platelets. Because licochalcone A also inhibited arachidonic acid-induced platelet aggregation and production of thromboxane A₂ induced by collagen in intact platelets, we further examined the direct interaction of licochalcone A with cyclooxygenase (COX)-1. As expected, licochalcone A caused an inhibitory effect on both COX-1 and COX-2 <i>in vitro</i>. Regarding the effect of licochalcone A on COX-1 enzyme reaction kinetics, although licochalcone A showed a stronger inhibition of prostaglandin E₂ synthesis induced by lower concentrations of arachidonic acid, Vmax values in the presence or absence of licochalcone A were comparable, suggesting that it competes with arachidonic acid at the same binding site on COX-1. These results suggest that licochalcones inhibit collagen-induced platelet aggregation accompanied by inhibition of COX-1 activity.</p></div