Plant material and extraction
Gentiana floribunda whole plant material was collected from Kurram valley, Pakistan in 2009 and identified with help of botanical authority at Kohat University of Science and Technology, Kohat, Pakistan and voucher specimen was deposited at Herbarium of the same University. The plant material was cleaned, shade dried and coarsely ground. The powdered material (1.5 kg) was soaked in 70% aqueous-methanol solution in a large container for 3 days with occasional shaking. It was filtered through a muslin cloth and then through filter paper . This procedure was repeated twice more and the combined filtrate was evaporated on a rotary evaporator, under reduced pressure to a thick semi-solid mass of dark brown color i.e. the crude extract of Gentiana floribunda (Gf.Cr, 250 g), yielding 16.7%. Gf.Cr was solubilized both in saline and distilled water.
The following reference chemicals were obtained from the sources specified: acetylcholine chloride (ACh), atropine, indomethacin, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), norepinephrine hydrochloride (NE), phentolamine hydrochloride, phenylephrine hydrochloride (PE), urethane and verapamil hydrochloride (Sigma Chemical Company, St. Louis, MO, USA). The following chemicals were used to make physiological salt solutions: potassium chloride (Sigma Chemical Company, St. Louis, MO, USA), calcium chloride, glucose, magnesium sulphate, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride (Merck, Darmstadt, Germany) and ethylenediaminetetra-acetic acid (EDTA) from BDH Laboratory Supplies, Poole, England. The chemicals used in phytochemical analysis include: acetic anhydride, aluminum chloride, ammonium hydroxide, ferric chloride (Sigma Chemical Co, St Louis, MO, USA), benzene, chloroform, hydrochloric acid and petroleum ether (BDH Laboratory supplies, Poole, England). All chemicals used were of the highest analytical grade available.
Preliminary phytochemical screening of the extract was carried out for the presence of anthraquinones, coumarins, flavonoids, saponins, sterols, tannins and terpenes in accordance to the reported procedures . Presence of saponins was detected based on the appearance of froth upon vigorous shaking of diluted samples. The observation of yellow florescence under ultraviolet light on examination of filter paper previously exposed to the vapors from boiling plant material indicated the presence of coumarins. For the detection of sterols and terpenes, plant material was treated with petroleum ether and subsequently extracted with CHCl3. The gradual appearance of green to pink (for sterols) and pink to purple color (for terpenes) was then noted after treatment of CHCl3 layer with acetic anhydride and concentrated H2SO4 in succession. Plant material was detected as positive for flavonoids when it gave yellow color with AlCl3 reagent and for tannins, when green or black color was produced with aqueous FeCl3. Lastly, for detecting anthraquinones, the extract was dissolved in 1% HCl, then in benzene and later if extract showed pink, violet or red color with NH4OH, that indicate the presence of anthraquinones.
Male Sprague–Dawley rats (240–260 g) of local breed were housed at animal house of the Department of Pharmacology, University of Malaya, under controlled environment (23-25 °C). Animals were given tap water ad libitum and standard diet. Experiments performed complied with rulings of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council , approved by University of Malaya Animal Experimentation Ethics Committee.
Measurement of blood pressure
These experiments were performed according to method described previously . Rats were anaesthetized with urethane (1.2-1.5 g/kg, i.p.). Animal was fixed in supine position on a dissecting table. A small mid-tracheal incision (approx. 1 cm) was made to expose trachea, right jugular vein and left carotid artery. The trachea was cannulated with a polyethylene tubing Pe-20 to maintain the spontaneous respiration and cleaned from time to time. The right jugular vein was cannulated with polyethylene tubing Pe-50 to facilitate the intravenous administration of 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 PowerLab ML 4/30 data acquisition system (AD Instruments, Sydney, Australia) for BP recording. The exposed surface for cannulation was covered with a piece of gauze moistened in warm saline. Rats were injected with heparinized 0.1 mL saline (0.9% NaCl) to prevent blood clotting. Body temperature of the animal was maintained by using overhead lamp. Following 20 min period of equilibrium, rats were injected intravenously with test substance. Arterial BP was allowed to return to resting level between injections. Changes in BP were recognized as difference between the steady state values before and the peak readings after injection. Mean arterial blood pressure (MABP) was calculated as the diastolic BP plus one-third of the pulse width (systolic BP - diastolic BP). The ACh (1 μg/kg) and NE (1 μg/kg) control responses were obtained before the administration of any test material.
Isolated rat aorta preparations
Rats were sacrificed by cervical dislocation. After abodominal opening, the thoracic aorta was dissected out, cleaned of fat and adipose tissues and cut into 3–5 mm long rings and individually mounted in 5 mL tissue bath containing Kreb’s solution composed of mM): NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4 1.3, MgSO4 1.2 and glucose 11.7 (pH 7.4). The bath solution was maintained at 37 °C and continuously aerated with carbogen (95% O2 in 5% CO2). A 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 . In experiments using endothelium-denuded tissues, endothelium lining of the aortic rings was removed mechanically by gentle rubbing with blunted forceps. Denudation of endothelium was confirmed by the absence of relaxation to ACh, 0.1-0.3 μM . The test drug was tested for its ability to relax the contractions, induced with high K+ (80 mM) and PE, 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 PE-induced contractions, would signify blockade of the Ca++ influx through receptor-operated calcium channels [17, 18]. To confirm CCB activity, concentration-response curves (CRCS) of Ca++ were constructed . For this purpose tissue was stabilized in normal Kreb’s solution and then placed in Ca++-free Kreb’s solution, containing EDTA (0.1 mM) for 30 min to remove calcium from the tissues. This solution was further replaced with K+-rich and Ca++-free Kreb’s solution, having the following composition (mM): KCl 50, NaCl 50.58, MgSO4 3.10., NaHCO3 23.8, KH2PO4 1.26, glucose 11.1 and EDTA 0.1. Following an incubation period of 1 hr, control CRCS of Ca++ were obtained. When the control CRCS of Ca++ were found super-imposable (usually after two cycles), the tissue was pre-treated with test drug for 50–60 min for the possible CCB effect. The Ca++-CRCS were reconstructed in presence of different concentrations of the test material. In order to determine if the extract was inhibiting Ca++ release from intracellular stores, the effect of increasing concentrations of extract was observed on PE (1 μM) peaks obtained in the Ca++-free environment (Ca++ omitted and EDTA (0.1 mM) added) to ensure total elimination of extracellular Ca++ without harmful effects on Ca++ inside the cell . In Ca++-free medium, PE acts through stimulation of α1-adrenergic receptors. Consequent conversion of phosphatidylinositol to inositol triphosphate (IP3), which in turn releases Ca++ from the intracellular stores, brings about the contraction . To assess the presence of any competitive adrenergic antagonism, cumulative curves to PE were constructed using increasing concentration of agonist. When 3-fold increase in concentration produced no further increment in response, the tissue was washed to re-establish the base-line tension (within 30–35 min). The PE-curves were then re-determined in the presence of test material. To study whether or not the vasodilator effect of test substances is endothelium-dependent, endothelium-intact aortic rings were preincubated with L-NAME (0.1 mM), atropine (1 μM) and indomethacin (1 μM) for 60 min prior to PE (1 μM)-induced contractions . The endothelial integrity of aortic ring was indicated by administration of ACh (0.1 μM) on PE-induced contraction, resulting in vasorelaxation . Changes in tension were recorded and analyzed isometrically, using force transducers of Multi Wire Myograph system-Model 610 M-version 2.2 (DMT A/S, Skejbyparken152, 8200 Aarhus N., Denmark) coupled to PowerLab ML 8/30 data acquisition system (AD Instruments, Sydney, Australia).
Acute toxicity test
Mice were divided in groups of five mice each. The test was performed using increasing doses of the plant extract, given orally in 10 mL/kg volume to different groups serving as test groups. Another group of mice was administered saline (10 mL/kg, p.o.) as negative control. The mice were allowed food ad libitum and kept under regular observation for lethality recorded after 24 hrs .
All the data is expressed as mean ± SEM and the median effective concentrations (EC50) values are given as geometric mean with 95% confidence intervals (CI). CRCs were analyzed by nonlinear regression (Sigmoidal dose–response curve variable slop). The statistical parameter applied was one way analysis of variance (ANOVA). Difference of p < 0.05 was considered statistically significant. All the graphs, calculation and statistical analyses were performed using GraphPad Prism software version 4.00 for Windows (GraphPad Software, San Diego California USA).