Inhibitory Effect of Methanolic Extract of Asparagus pubescens Bak Root in Rats and Guinea Pigs Uterine Muscle

INTRODUCTION

Asparagus pubescens (Liliaceae) is an evergreen plant which grows luxuriantly around the rocky hills and valleys of Jos metropolis in Nigeria between the months of April and September. The dried root of Asparagus pubescens locally called Khayan bera, has a long history of traditional medicinal use among the Rukuba community in Jos township and its neighbours where it is used for family planning, treatment of various gastrointestinal disorders and inflammatory pains (Hutchinson and Dalziel, 1968). Nwafor et al. (1998) had reported on the contraceptive and non-estrogenic effect of the methanolic extract of the Bak root. They have also established the antidiarrhoeal and antiulcerogenic effects of the plant as well as its antinoiceptive and anti-inflammatory effects (Nwafor and Okwuasaba, 2003; Nwafor et al., 2000). Recently, they reported on its effects on non-vascular muscle (Nwafor et al., 2005) and its effects on sexual behaviour and pituitary hormone secretion in wistar rats during pregnancy and lactation (Nwafor et al., 2007). Though, they showed that the mechanism of this inhibition was receptor-operated channel dependent, they did not establish the involvement of calcium dependent mechanism in calcium free Physiological Salt Solution (PSS) hence the aim of this investigation is to determine if the involvement of calcium dependent mechanism is through the extracellular or intracellular inhibition or both.

MATERIALS AND METHODS

Preparation of Extract
The plant material used in this study was collected from Rukuba village in Jos metropolis Plateau state, Nigeria between the months of April and September, 1995. The plant was identified and authenticated by Dr. S.S. Sanusi, Department of Botany, University of Maiduguri. Specimen vouchers (FPS. 014) were made and deposited at the herbarium of Department of Pharmacology, Faculty of Pharmaceutical Sciences, University of Jos. The dried root was pulverized by grinding using pestle and mortar. Then, 57 g of the ground root were subjected to exhaustive soxhlet extraction in methanol (250 mL) for 72 h at 60°C. This gave a mean yield 16±0.23 g W/W of extract. The extract was stored in -4°C from where it was used when required.

Animal Stock
Adult female guinea pigs and albino rats (weighing 350-410 and 165-200 g, respectively were used in this study. All animals were housed in a cross-ventilated room (temperature 22±2.5°C, 12 h light/12 h dark cycle) and were fed with standard mash (Feedex Nigeria, Kaduna, Nigeria) and water ad libitum.

Pharmacological Studies
Preparation of Isolated Uterine Horns
The isolation of uterine horn was done according to the methods of Okwuasaba and Otubu (1988) and Nwafor et al. (2005). The animals were killed by cervical dislocation and exsanguinated. The abdomen was opened and the uterine horns were served at the junctions with the fallopian tubes cleared of adhering mesentery and placed in a dish containing De-Jalon’s solution of the following composition in millimole (mM): NaCl, 15.4; KCl, 5.63; CaCl₂, 0.41; NaHCO₃, 5.95 and glucose 2.25. Approximately, 2-3 cm of the uterine preparation was used for each experiment and was mounted in 5 mL organ bath containing De-Jalon’s solution constantly gassed with 95% oxygen and 5% carbon dioxide mixture and the temperature was kept constant at 35±1°C in a thermostatically regulated water circulator (Grant instrument 51963 Cambridge). A tension of 0.5 g was applied to the tissue and it was allowed to equilibrate for at least 30 min before adding drugs or extract. Non-cumulative/cumulative responses were recorded isometrically using Ugo Basile strain guage transducer connected to microdynanometer recorder model 7080 (Uyo Basile Italy, Aqel et al., 1991).

Effect of Extract on Spontaneous Rhythmic Contraction of Rat Uterine Muscle
To determine the effect of extract on spontaneous movement of rat uterine muscle, 1 mg of the extract was added cumulatively into the organ bath at 5 min interval and the results were recorded.

Effect of Extract on Acetylecholine-induced Contraction on Guinea Pig Uterine Muscle
The effect of extract was investigated on non-pregnant guinea pig uterine muscle. The tissue preparation procedure was similar to that of rat uterine muscle as described earlier (Aqel et al., 1991). The tissue was stimulated with acetylcholine (Ach 5.0x10^-4 -3.0x10^-3 g mL^-1). Extract incubation period was 15 min while the agonist contact time was 20 sec.

Effect of Cumulative Concentration of Extract on Ach-induced Contraction on Guinea Pig Uterine Muscle
After an initial equilibration of 30 min, cumulative concentration of A. pubescens extract (1.0x10^-6 -1.28x10^-4 g mL^-1) was investigated on submaximal dose of acetylcholine-induced contraction on guinea pig uterine muscle and the results were recorded.

Effect of Verapamil on Extract-sustained Contraction on Guinea Pig Uterine Muscle
As a result of extract sensitization on Ach-induced contraction, verapamil, an extracellular calcium antagonist was introduced to investigate if the mechanism of this sensitization is calcium dependent. At the end of the equilibration period, 10 sec after the administration of Ach, verapamil (1.0x10^-6 g mL^-1) was introduced while the extract was added cumulatively as described earlier. This procedure was repeated using promethazine, in place of verapamil, the results recorded.

Effect of Extract on Acetylcholine-induced Contraction in Ca²⁺ Free Physiological Salt Solution on Guinea Pig Uterine Muscle
As a result of Asparagus pubescens extract sensitization on Ach-induced contraction, further experimental investigations were carried out to ascertain if this effect is due to extracellular calcium mobilization. Calcium ion was deliberately omitted in physiological salt solution (De-Jalon) preparation and the experiment (2.3.4) was repeated in the absence of calcium. Time dependent contractile responses of Ach in Ca²⁺ free Physiological Salt Solution (PSS) was also recorded (Ebeigbe et al., 1986).

In another series of experiments, the submaximal dose of Ach (8.8x10^-5 M) was used to stimulate the tissue. In the presence of Ach, increasing concentrations of extract (1.0x10^-6-6.4x10^-5 g mL^-1) were added cumulatively. When the depressant effect of the extract was maximal, extracellular calcium (25-100 μm) was injected cumulatively to see if the inhibitory effect of the extract could be reversed.

Statistical Analysis
All the values were expressed as Mean±SEM. The data was analysed using Student’s t-test. A probability level of less than 5% was considered significant.