Cardiovascular inhibitory properties of Lens culinaris

In present study, we report the blood pressure lowering, vasodilatory and cardio-depressant activities of Lens culinaris . The crude extract of L. culinaris induced dose-dependent (3-30 mg/kg) fall in the arterial pressure of rats under anesthesia. When tested in rat aortic ring preparations, L. culinaris at concentration range of 0.03-5.0 mg/mL relaxed high K + (80 mM) and phenylephrine (1 μM)-induced contractions, like that caused by verapamil. In isolated guinea-pig atria, L. culinaris caused inhibition of atrial force and rate of spontaneous contractions, similar to that exhibited by verapamil. These data indicate that L. culinaris exhibits blood pressure lowering potential, mediated possibly through Ca ++ channel blockade mechanism. DOI: http://dx.doi.org/10.3329/bjp.v9i3.19090


Introduction
We recently observed that Lens culinaris Moench (Leguminosae) exhibited gut and airways relaxant activities (Khan et al., 2014).In the current era, lot research is going on for exploration of alternative therapy for hypertension management (Ghayur and Janssen, 2010) because they are safe, economical and possesses synergistic and side-effect neutralizing properties (Gilani and Atta-ur-Rahman, 2005).In the present study, we evaluated the effects of L. culinaris on cardiovascular parameters and reported that it exhibits blood pressure lowering, cardio-depressant and vasodilator activities, mediated possibly via inhibition of Ca ++ entry via membranous Ca ++ channels.

Plant material and extraction
The seeds were cleaned and approximately 1 kg of the material was soaked in the aqueous-methanol.All the filtrates were combined and evaporated to dryness on a Rotary Evaporator under reduced pressure (-760 mm Hg) at 35-40°C to a thick and dark brown mass, the crude extract of Lens culinaris (Lc.Cr), yielding approximately 3.6%.

Animals
Experimentsl animals used in this study include adult Sprague-Dawley rats (180-200 g) and guinea-pigs (450-500 g) of local breed and either sex housed in the animal facility of The Aga Khan University under controlled environment (23-25ºC).

Measurement of blood pressure
Rats were anaesthetized with thiopental sodium (Pentothal ® , 70-90 mg/kg, i.p.) Trachea was cannulated with a polyethylene tubing Pe-20 to maintain the spontaneous respiration (Consolini et al., 1999;Gilani et al., 2005).The right jugular vein was cannulated with polyethylene tubing Pe-50 to facilitate the intravenous (i.v) administration of different drugs.The left carotid artery was cannulated with similar tubing filled with heparinized saline 60 IU/mL and connected to a pressure transducer (MLT 0380/D Reusable BP-Transducer) coupled to ML 224 Quad Bridge Amplifier and Power-Lab ML 4/25 data acquisition system (AD Instruments, Australia) for blood pressure recording.Following 20 min period of equilibrium, rats were injected intravenously with the test substance.Arterial blood pressure was allowed to return to resting level between injections.Mean arterial pressure (MAP) was calculated as the diastolic BP plus one-third of the pulse width (systolic blood pressure -diastolic blood pressure).

Rat aortic tissues
Rats were sacrificed by cervical dislocation.After abdominal opening, the thoracic aorta was dissected out, cleaned of fat and adipose tissues and cut into 3-5 mm long rings and mounted individually in a 5 mL tissue bath containing Kreb's solution.Resting tension of 1 g was applied to each tissue and an equilibrium period of 30 min was allowed before any experimentation.The tissues were then stabilized with repeated exposure (usually 3 times) to high KCl solution (Gilani et al., 2006;Khan et al., 2012).The test drug was applied for its ability to relax the contractions induced with high K + (80 mM) and phenylephrine (1 μM).The ability of extract to relax K + (80 mM)-induced contractions would indicate L-type voltage-operated calcium channel blocking (CCB) mode of vasodilation, while inhibition of the phenylephrine-induced contractions, would suggest the blockade of the Ca ++ influx through receptor-operated Ca ++ channels (Godfraind et al., 1986;Wang et al., 2001).Changes in tension were recorded and analyzed isometrically through a force transducer (Fort-10, WPI, UK) coupled to a bridge amplifier (Transbridge TBM 4M, WPI) and PowerLab ML 845 data acquisition system (AD Instruments, Australia).

Guinea-pig atrial preparations
In the in vitro assays, right atria isolated from the guinea pigs were mounted in 20 mL tissue baths containing Kreb's solution, at 32°C and aerated with carbogen.Each tissue was allowed to beat spontaneously (due to pacemaker) under the resting tension of 1 g (Khan and Gilani, 2008;Khan and Gilani, 2011;Khan et al., 2012).An equilibrium period of 45 min was allowed before the application of any drug.The druginduced changes in force of atrial contractions were measured as the percent change in base-line values.Tension changes in the tissue were recorded via forcedisplacement transducer (FT-03) using Grass Model 7 Polygraph.

Discussion
The aqueous-methanolic L. culinaris extract in dosedependent fashion caused fall in the arterial BP of rats under anesthesia.As the blood pressure is considered the product of cardiac output and peripheral vascular resistance (Johansen, 1992), the plant extract was  studied in heart and vascular preparations for the possible cardio-depressant and vasodilator actions.To see effect on vascular resistance, L. culinaris was studied in rat thoracic aorta, which is routinely used for elucidating underlying mechanism(s) of blood pressure -lowering effect (Ghayur and Gilani, 2005;Ajay et al., 2007).In isolated rat aortic tissues, L. culinaris extract was screened against high K + and phenylephrineinduced contractions.L. culinaris relaxed high K + and phenylephrine-induced contractions in aortic rings, indicating that it was acting through blockade of voltage-and receptor-operated Ca ++ channels (Okmura et al., 1993;Musha et al., 2005;Khan et al., 2013).As L.
culinaris was found relatively more potent against K + (having 10 times lower EC50) as compared to phenylephrine-induced contractions, thus showing greater potency to block the voltage-sensitive Ca ++ channels as comparison to the receptor-operated Ca ++ channels, a pattern of activity similar to verapamil, a reference Ca ++ antagonist (Fleckenstein, 1977).L. culinaris was more potent in its inhibitory effect on vascular tissues than cardiac.There is evidence of heterogeneity of Ca ++ channels and different Ca ++ antagonists exhibit selectivity for different organ systems (Farre et al., 1991).For example, dihydropyridine antagonists are considered selective for vascular tissues and are more commonly used to lower blood pressure (Joseph and Barry, 1999).
In guinea-pig atria, L. culinaris exhibited negative inotropic and chronotropic effects.The cardiac inhibitory action of the L. culinaris may be due to Ca ++ antagonist effect leading to decrease in cardiac output and thus falling blood pressure.