Fig 1.
Chemical structures of licochalcones A, C and D.
Fig 2.
Concentration-dependent inhibition of U46619- and collagen-induced platelet aggregation by licochalcones.
(A) 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. (*P<0.05 compared with control, n = 3, Tukey–Kramer’s method). (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. (*P<0.05 compared with control, n = 3–8, Dunnett’s method). (C) 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.
Fig 3.
Scanning electron microscopy images of platelets stimulated by collagen in the presence of licochalcones.
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. (A) Unstimulated platelets, (B) collagen (3 μg/ml), (C) licochalcone A (100 μM) +collagen, (D) licochalcone C (100 μM) +collagen and (E) licochalcone D (100 μM) +collagen. (Magnification: 7000×, bar = 5 μm).
Fig 4.
Concentration-dependent inhibition of collagen-induced human platelet aggregation by licochalcone A.
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. (*P<0.05 compared with control, n = 3, Dunnett’s method).
Fig 5.
Concentration-dependent inhibition of arachidonic acid metabolism by licochalcone A.
(A) 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. (*P<0.05 compared with control, n = 3, Dunnett’s method). (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. (*P<0.05 compared with control, n = 3, Dunnett’s method).
Fig 6.
Competitive inhibition of COX-1 and COX-2 activity by licochalcone A in vitro.
(A) 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. (*P<0.05 compared with control, n = 3, Dunnett’s method). (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. (*P<0.05 compared with control, n = 3, Student’s t-test).