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

Double seismic zone and seismicity in the mantle wedge beneath the Ogasawara Islands identified by an ocean bottom seismometer observation

Abstract Around the Ogasawara Islands, only a few seismic stations in the area can be used to determine the hypocenters of regional earthquakes; thus, hypocenter location precision tends to be low. To more precisely determine hypocenter locations, we deployed a temporary seismic observation network of pop-up ocean bottom seismometers around the Ogasawara Islands from July to October 2015. We identified a double seismic zone in the 70–200 km depth range associated with the subducting Pacific slab. The slab-normal distance between the two planes of the double seismic zone is about 30–35 km, similar to such distances observed along the Japan and Mariana trenches. Furthermore, we found unusual seismicity in the mantle wedge at 20–50 km depth beneath the Ogasawara trough that might be related to structure formed at the onset of the oceanic slab subduction. The hypocenters determined from the ocean bottom seismometer observation were horizontally separated by a few tens of kilometers from hypocenters published by the Seismological Bulletin of Japan. USGS locations (Preliminary Determination of Epicenters) seem to be offset westward about 30 km compared with the locations determined in this study

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

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

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