Abstract
2,3-dehydrosilybin (DHS) is a minor flavonolignan component of Silybum marianum seed extract known for its hepatoprotective activity. Recently we identified DHS as a potentially cardioprotective substance during hypoxia/reoxygenation in isolated neonatal rat cardiomyocytes. This is the first report of positive inotropic effect of DHS on perfused adult rat heart. When applied to perfused adult rat heart, DHS caused a dose-dependent inotropic effect resembling that of catecholamines. The effect was apparent with DHS concentration as low as 10 nM. Suspecting direct interaction with β-adrenergic receptors, we tested whether DHS can trigger β agonist-dependent gene transcription in a model cell line. While DHS alone was unable to trigger β agonist-dependent gene transcription, it enhanced the effect of isoproterenol, a known unspecific β agonist. Further tests confirmed that DHS could not induce cAMP accumulation in isolated neonatal rat cardiomyocytes even though high concentrations (≥ 10 μM) of DHS were capable of decreasing phosphodiesterase activity. Pre-treatment of rats with reserpine, an indole alkaloid which depletes catecholamines from peripheral sympathetic nerve endings, abolished the DHS inotropic effect in perfused hearts. Our data suggest that DHS causes the inotropic effect without acting as a β agonist. Hence we identify DHS as a novel inotropic agent.
Highlights
We have shown that DHS displays uncoupler-like activity in isolated mitochondria [11] and attenuates reactive oxygen species formation during hypoxia/ reoxygenation in primary neonatal rat cardiomyocytes [12]
Basing the initial concentration on our previous work in isolated cardiomyocytes [11,12], we used this perfusion model to observe the effect of 10 μM DHS, on the whole organ as a prerequisite for cardioprotective work
The inotropic effect was less pronounced at lower concentrations of DHS, where, following the 15 min of substance perfusion, biomechanical parameters gradually returned to values observed in control hearts
Summary
Dulbecco’s modified Eagle’s medium (DMEM), heat-inactivated fetal bovine serum (FBS), stabilized penicillin-streptomycin solution (PenStrep), sterile dimethylsulfoxide (DMSO), norepinephrine (NE), isoproterenol (ISO), propranolol (PRO) and reserpine (RES) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Brief summary of the preparation: silybin (2.5 g, 5.183 mmol) and NaHCO3 (1.74 g, 20.798 mmol) were dissolved in methanol (100 ml) and the mixture was heated under reflux for 16 h. The precipitate formed was filtered off, washed with H2O, dissolved in a mixture of ethyl acetate/acetone (1:1), and evaporated to give 2.17 g of dry residue. The mother liquor was filtered through a silica gel pad (CHCl3/acetone/HCOOH 90:10:1–70:30:1) to obtain, after concentration, another portion of the product, which after recrystallization from methanol yielded pure DHS (270 mg, 11%). Stock solution of DHS (10 mM) was prepared in DMSO. The final concentration of DMSO in the medium was 0.5% (v/v)
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