The positive inotropic effect of the ethyl acetate fraction from Erythrina velutina leaves on the mammalian myocardium: the role of adrenergic receptors.

OBJECTIVES: We studied the effects of ethyl acetate fraction (EAcF) obtained from Erythrina velutina leaves on mammalian myocardium. METHODS: The effect of EAcF on the contractility was studied using guinea-pig left atria mounted in a tissue bath (Tyrode's solution, 29°C, 95% CO2 , 5% O2 ) and electrically stimulated (1 Hz). Concentration-response curves of EAcF were obtained in the presence of propranolol (1 µm), nifedipine (1 µm) and in reserpinized animals (5 mg/kg). The involvement of l-type calcium current (ICa,L ) on the EAcF effect was observed in cardiomyocytes of mice assessed using patch-clamp technique. KEY FINDINGS: EAcF (550 µg/ml) had a positive inotropic effect, increasing the atrial force by 164% (EC50 = 157 ± 44 µg/ml, n = 6), but it was less potent than isoproterenol (EC50 = 0.0036 ± 0.0019 µg/ml, n = 8). The response evoked by EAcF was abolished by propranolol or nifedipine. Reserpine did not alter the inotropic response of EAcF. Furthermore, an enhancement of the ICa,L peak (31.2%) with EAcF was observed. Chemical analysis of EAcF revealed the presence of at least 10 different flavonoid glycoside derivatives. Two were identified as vicenin II and isorhoifolin. CONCLUSIONS: We conclude that EAcF increases the cardiac contractile force by increasing the l-type calcium current and activating the adrenergic receptor pathway.

Aqueous fraction from Costus spiralis (Jacq.) Roscoe leaf reduces contractility by impairing the calcium inward current in the mammalian myocardium.

ETHNOPHARMACOLOGICAL RELEVANCE: Brazilian folk medicine uses infusion of Costus spiralis leaf to help people to treat arterial hypertension and syndromes of cardiac hyperexcitability. AIM OF THE STUDY: Evaluate the aqueous fraction (AqF) effect on atrial contractility and investigate its mechanism of action. MATERIALS AND METHODS: The AqF effect on the cardiac contractility was studied on isolated electrically driven guinea pig left atria. Atropine and tetraethylammonium (TEA) were employed to investigate whether potassium contributes for the inotropic mechanism of the AqF. The role of calcium in this effect was also studied. This was done by analysing the AqF effect on the Bowditch's phenomenon, as well as by studying whether it could interfere with the concentration-effect curve for CaCl(2), isoproterenol, and BAY K8644. Mice isolated cardiomyocytes were submitted to a whole-cell patch-clamp technique in order to evaluate whether the L-type calcium current participates on the AqF effect. Furthermore, the intracellular calcium transient was studied by confocal fluorescence microscopy. RESULTS: AqF depressed the atrial contractile force. It was the most potent fraction from C. spiralis leaf (EC(50)=305 ± 41 mg/l) (crude extract: EC(50)=712 ± 41; ethyl acetate: EC(50)=788 ± 121; chloroform: EC(50)=8,948 ± 1,346 mg/l). Sodium and potassium content in the AqF was 0.15 mM and 1.91 mM, respectively. Phytochemical analysis revealed phenols, tannins, flavones, xanthones, flavonoids, flavonols, flavononols, flavonones, and saponins. Experiments with atropine and TEA showed that potassium does not participate of the inotropic mechanism of AqF. However, this fraction decreased the force overshoot characteristic of the Bowditch's phenomenon, and shifted the concentration-response curve for CaCl(2) (EC(50) from 1.12 ± 0.07 to 7.23 ± 0.47 mM) indicating that calcium currents participate on its mechanism of action. Results obtained with isoproterenol (1-1,000 pM) and BAY K8644 (5-2000nM) showed that AqF abolished the inotropic effect of these substances. On cardiomyocytes, 48mg/l AqF reduced (∼23%) the L-type calcium current density from -6.3 ± 0.3 to -4.9 ± 0.2 A/F (n=5 cells, p<0.05) and reduced the intracellular calcium transient (∼20%, 4.7 ± 1.2 a.u., n=42 cells to 3.7 ± 1.00 a.u., n=35 cells, p<0.05). However, the decay time of the fluorescence was not changed (control: 860 ± 32 ms, n=42 cells; AqF: 876 ± 26 ms, n=35 cells, p>0.05). CONCLUSIONS: The AqF of C. spiralis leaf depresses myocardial contractility by reducing the L-type calcium current and by decreasing the intracellular calcium transient. Despite the lack of data on the therapeutic dose of AqF used in folk medicine, our results support, at least in part, the traditional use of this plant to treat cardiac disorders.

Extract from leaf of Psidium guajava L depresses the guinea pig atrial contractility by interfering with potassium and calcium channels / Extrato de folha de Psidium guajava L deprime a contratilidade atrial de cobaia, interferindo com os canais de potássio e cálcio

The negative inotropic effect of aqueous fraction (AqF) obtained from the acetic extract of Psidium guajava L leaf was investigated on the guinea pig left atrium. Myocardial force was measured isometrically (27 ± 0.1 ºC, 2 Hz). AqF (100 μg/ml) reduced contractility of about 85 ± 9.4 percent (n = 4, p < 0.001, Fcalc = 51.70, F(0.01; 4; 21) = 5.09, EC50 = 14.28 ± 3 μg/mL) in a concentration-dependent fashion. This effect was reduced by 20 mM of tetraethylammonium (TEA), increasing EC50 to 50 ± 7 μg/ml (n = 4, p < 0.001, Fcalc = 282.13; F(0.01; 21; 66) = 2.36). AqF (100 μg/ml) shifted to the right the CaCl2 concentration-effect curve, increasing the EC50 from 2170 ± 112 to 2690 ± 132 μM (n = 3, p < 0.001, Fcalc = 220.80 ; F(0.01; 29; 60) = 2.19). L-NAME (100 μM) did not modify the AqF inotropic effect (n = 3, p > 0.05) sugesting that the oxide nitric pathway did not participate of the action mechanism of AqF. We can conclude that AqF depresses the atrial contractile by reducing the calcium entry in myocardial cells and also by openenig potassium channels of cardiac tissue.

O efeito inotrópico da fração aquosa (AqF) do extrato acético das folhas de Psidium guajava L. foi investigado em átrio esquerdo de cobaia. A força miocárdica foi medida isometricamente (27 ± 0,1 ºC; 2 Hz). A AqF (100 μg/mL) reduziu a contratilidade em até 85 ± 9,4 por cento (n = 4; p < 0,001; Fcalc = 51,70; F(0,01; 4; 21) = 5,09; CE50 = 14,28 ± 3 μg/mL) de forma dependente da concentração. Este efeito foi reduzido pelo tetraetilamônio (TEA, 20 mM) que também aumentou a CE50 de 14,28 ± 3 μg/mL para 50 ± 7 μg/mL (n = 4; p < 0,001; Fcalc = 282,13; F(0,01; 21; 66) = 2,36). A AqF (100 μg/mL) deslocou para a direita a curva concentração-efeito do CaCl2, aumentando a CE50 de 2170 ± 112 para 2690 ± 132 μM (n = 3; p < 0,001; Fcalc = 220,80 ; F(0,01; 29; 60) = 2,19). Por outro lado, o L-NAME (100 μM) não alterou o efeito inotrópico da AqF (n = 3; p > 0,05), sugerindo que a via do óxido nítrico não participa do mecanismo de ação da AqF. Conclui-se que a AqF deprime a contratilidade atrial por reduzir a entrada de cálcio nas células miocárdicas e por abrir canais de potássio deste tecido.