Plant material

 

Indigenous medicinal plant R. vesicarius L. was collected from the sandy fields of Mondka Shahjamal District, Muzaffar Garh, Pakistan. The plant material was authenticated by expert taxonomist, Professor Dr. A. H. Dasti at the Department of Botany, Bahauddin Zakariya University, Multan, Pakistan (voucher F.P.ST-215). The plant material was made free from foreign adulterants and vegetative debris by hand picking and leaves were detached from the plant, washed and shade dried. Within 8 days, leaves became crispy. Special electrical herbal grinder was used to form coarse powder. Uniform dark green powder was obtained with characteristic smell.

 

Crude extract

 

The powdered plant material (1 kg) was subjected to maceration in 70% methanol in amber coloured glass bottle at room temperature (25°C) for 8 days with occasional shaking (Aziz et al., 2013a). The soaked material was passed through muslin cloth to remove the vegetative material and the fluid obtained was filtered through Whattman-1 Filter paper. The filtrate was evaporated on a rotary evaporator (Rotavapor, BUCHI Labrotechnik AG, Model 9230, Switzerland) at 37°C under reduced pressure. Approximate yield was 11% and the extract obtained was stored at -4°C in air tight jars in lab refrigerator.

 

Preliminary phytochemical screening

 

Vital phytochemical classes were screened by the method described by Aziz et al. (2013b).

 

Chemicals and drugs

 

All the chemicals, solvents, and drugs used were of analytical grade. Carbacholine was purchased from Ethical Laboratories Pvt. (Ltd) Pakistan. Dimethylsulfoxide, ethylenediamine tetraacetic acid, glucose, magnesium chloride, magnesium sulfate, potassium chloride, potassium dihydrogenophosphate, sodium chloride, sodium bicarbonate, and sodium dihydrogenophosphate were purchased from Sigma Chemical Company, St. Louis, MO, USA. Calcium chloride was purchased from Merck (Merck, Darmstadt, Germany).

 

Animals and housing condition

 

Fifteen adult albino rabbits (1.0 to 1.5 kg) of either sex, purchased from the animal market Hussain Agahi Multan, Pakistan with age limit between 6 to 7 months were used for the experiments. Animals were provided with fresh green fodder and tap water ad libitum and maintained in air conditioned room (23 to 25°C) at the Faculty of Pharmacy, Bahauddin Zakariya University, Multan. All rabbits were kept in fasting condition for at least 24 h before the commencement of experiments, but had free access to water. The experiments were approved by the Ethical Committee of the Bahauddin Zakariya University, Multan with reference number EC/12/2012 dated 07 December, 2012.

 

Plant extract solution

 

The plant extract (0.3 g) was dissolved in 1 ml of methanol to produce stock solution from this stock solution further dilutions were made. Solutions were freshly prepared on the day of experiment.

 

Isolated rabbit tracheal preparation

 

The trachea was dissected out and cut into rings of 3 to 4 mm in width, each ring contains about two cartilages for the formation of tracheal strip, and each ring was opened by a longitudinal cut on ventral side opposite to the smooth muscle layer with a central part of smooth muscle sandwiched between cartilaginous portions on the edges. The formed tissue preparation was then suspended in a 10 mL tissue bath containing Krebs physiological salt solution at 37°C and aerated with carbogen. For calibration about 1 g tension was applied to each of tracheal strips; this tension remained constant throughout the experiment. The isolated rabbit tracheal preparation was equilibrated for 45 min prior to recording isometric contractions via force displacement transducers connected to a Powerlab Data Acquisition System (AD Instruments, Sydney, Australia) which was displayed on a computer running Lab Chart version 6. The relaxant effect of the test material was assessed on carbachol-(1 μM) and K⁺-(80 mM) induced contractions in isolated rabbit tracheal preparations as the cumulative addition of the test material to the isolated tissue bath may relax the isolated rabbit tracheal preparation. The isolated rabbit trachea preparations were equilibrated for 45 min prior to the addition of any test substance. Carbachol (1 μM) and K⁺ (80 mM) were used to produce sustained contractions in isolated rabbit trachea preparations on which the possible tracheal relaxant activity of the extract was studied following addition to the tissue baths in a cumulative manner in comparison to control drugs. The cumulative concentration response curves for carbachol were constructed through cumulative increase in concentration of agonist in tissue bath till a 3-fold increase in cumulative concentration did not produce further increase in response. The tissues were washed to re-establish the base-line tension, and concentration response curves (CRCs) for CCh were prepared in the presence of different concentrations of the aqueous-methanol extract and the standard drug dicyclomine (Gilani et al., 1997).