Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 38, Num. 1, 2006, pp. 33-37

Indian Journal of Pharmacology, Vol. 38, No. 1, January-February, 2006, pp. 33-37

Research Paper

Effects of Mondia whitei extracts on the contractile responses of isolated rat vas deferens to potassium chloride and adrenaline

Watcho¹ P, Fotsing¹ D, Zelefack² F, Nguelefack¹ T B, Kamtchouing³ P, Tsamo² E, Kamanyi¹ A

¹Animal Physiology and Phytopharmacology Laboratory, Faculty of Science, University of Dschang, Box 67 Dschang, Cameroon. ²Department of Chemistry, Faculty of Science, University of Yaounde I, Box 812, Yaounde, Cameroon. ³Animal Phsyiology Labratory, Faculty of Science, University of Yaounde I, Box 812, Yaounde, Cameroon
Correspondence Address: Pierre Watcho, E-mail: pwatcho@yahoo.fr

Code Number: ph06006

Abstract

Keywords: Epinephrine, in vitro preparation, KCl.

Introduction

Materials and Methods

Animals
Healthy male albino rats (150-250 g,> 90 days) of the Wistar strain were used in the present study. The animals were raised at room temperature with a natural light-dark cycle and maintained at standard rat diet and tap water given ad libitum .

Plant collection and extraction
Fresh roots of M. whitei were obtained from the local market and authenticated by Dr. Pinta Jonas of the Botany Department, Faculty of Science, University of Dschang, Cameroon. The roots were air-dried and ground using a mixer (Moulinex). The powdered roots (700 g) were soaked in 6 litres of CH₂Cl₂ : MeOH (1:1) mixture at room temperature for 72 h and filtered. The solvent was removed by vacuum distillation and dried to obtain a black paste (76 g), referred to as the CH₂Cl₂ : MeOH extract; 66 g of this mass were exhausted for 30 min in 2 l of hexane and filtered. The solvent was removed as previously to obtain 10 g of hexane fraction. It was so proceeded to obtain the methanol (10 g) fraction of M. whitei . For each sample, the working solution (100 mg/ml) was prepared extemporarily by dissolving 1 g of paste in 2 ml of 0.3% Tween 20 and 8 ml of distilled water.

Phytochemical tests
The hexane and methanol fractions of M. whitei were treated with several reagents and spectroscopy and physical analysis (RMN, [1]H and [13]C; mass spectrometry (SM) RMN, etc.) were performed. Positive results were obtained with the following constituents: steroids, triterpenes (mixture of amyrine α- and β-acetate, lupeol, β-sitosterol, and β-sitosterol glucoside) and aromatic (2-hydroxy-4-methoxybenzaldehyde, 3-hydroxy-4-methoxy-benzaldehyde (vanillin), and 4-hydroxy-3-methoxy-benzaldehyde) compounds in the hexane fraction, sugar (glucose), and polyholosides [α-d-glucopyranosyl (6-1)-β-d-glucopyranose and 1-methoxy-β-d-glucopyranosyl (6-1)-β-d-gluco-pyranose] in the methanol fraction.

Contractions of the isolated vas deferens

Effects on agonist-induced contractile responses
The rats were sacrificed by a blow on the head and bled to death by cutting the neck vessels. The vas deferens were promptly removed, cleansed of the connective tissue, and cut into two parts as to obtain a proximal portion nearer to the epididymis and a distal portion nearer to the sex accessory complex. The proximal part, which is more sensitive than the distal section,[6] was used in the study and mounted in an organ bath of 20 ml capacity containing fresh Krebs solution of the following composition (mM/l): NaCl 115, NaHCO₃ 25, CaCl₂ 2.5, KCl 4.7, MgCl₂ 1.2, KH₂PO₄ 1.2, and ρ-glucose, 10. The PSS was maintained at 37 ± 0.5°C and continuously bubbled with air. The preparation was allowed to equilibrate for 45 min during which the bathing solution was changed every 15 min. Cumulative concentration-response curves to agonists such as KCl (1.0-7.0 x 10-2 M) and adrenaline (1.21-8.45 x 10-7 M) were recorded in the absence and presence of M. whitei samples (50-400 µg/ml). The contractile responses were expressed as a percentage of the maximal contractile response to KCl or Adr.

Effects on KCl-induced maximal contraction
In separate experiment, after having obtained a stable plateau of contractions [usually 15-20 min after challenging the preparation with supramaximal concentration of KCl (60 mM)], M. whitei extract and its fractions (50-400 µg/ml) were used to relax the preparation in a cumulative manner. The relaxant effects were expressed as the percentage of inhibition of the plateau of contraction to KCl.

Effects of the hexane fraction on calcium activity
In order to determine the effect of M. whitei extracts on calcium activity, the vas deferens was contracted with KCl and relaxed with PSS, and the bath solution replaced with a high K⁺(70 mM), Ca²⁺-free Krebs solution containing EGTA (1 mM). Among the three M. whitei solutions, the hexane fraction showed maximum changes in the two previous experiments and was thus used in this investigation. Cumulative concentration-contraction curve was obtained by a stepwise increase in CaCl₂ (2-14 x 10-2 M) in the absence and presence of the hexane fraction of M. whitei (50-400 µg/ml). The contractile response was expressed as a percentage of the maximal contractile response to CaCl₂.

In the above studies, contraction and inhibition (relaxation) of the vas deferens were recorded by means of an isometric transducer connected to a single-channel recorder (Ugo Basile, Italy), which was calibrated to record change in the tension generated on g Vs. cm displacement basis. The tension applied on the preparation was 0.8 g.

One-way ANOVA with post hoc Dunnett′s or Newman-Keuls multiple comparison test were performed. The value of P < 0.05 was considered to be statistically significant.

Results

Effects on agonist-induced contractile responses
In rat vas deferens, KCl (1-7 x 10-2M ) and adrenaline (1.21-8.45 x 10-7M ) produced concentration-dependent contractions. In the presence of M. whitei (50-400 µg/ml), there were rightward shifts of the concentration-response curves to KCl and adrenaline with significant decrease in the maximal response. The hexane fraction of M. whitei produced comparatively more rightward shift and significant inhibition of the maximal response to KCl and Adr at highest concentration (400 µg/ml) as compared with the other fractions. [Figure - 1][Figure - 2][Table - 1]

Effects on the plateau of contraction induced by KCl
On the plateau of contraction induced by KCl (60 m M ), M. whitei extract and its fractions induced a concentration-dependent relaxant effects with the hexane fraction producing more relaxation with equivalent concentration [Table - 2].

Effects of the hexane fraction on calcium activity
In calcium-free medium, the hexane fraction of M. whitei produced concentration-dependent rightward shift of the concentration-response curve of CaCl₂ (2-14 x 10-2 M ) with significant decrease in the maximal response. [Figure - 3][Table - 1]

Discussion

Smooth muscle contraction is dependent on the concentration of the cytosolic-free calcium, which activates the contractile elements.[7],[8] In the present study, KCl and Adr induced contractions of isolated vas deferens of rat. The contractile effect of KCl is owing to a mechanism related to membrane depolarization and subsequent influx of external calcium through voltage-operated channels.[9] Receptors for adrenaline are abundant in the proximal part of the vas deferens compared with the distal section. It contracts the vas deferens by interacting with either α₁ or α₂ receptors, whereas relaxation is generally associated with β-receptors.[10] Stimulation of α₁ receptors leads to the hydrolysis of phosphatidyl inositol into inositol triphosphate (IP₃) and diacylglycerol (DAG). IP₃ causes the liberation of the intracellular calcium ion stockage, whereas DAG depolarizes the cell membrane inducing the calcium ions reflux.[11] The stimulation of α₂ adrenoceptors activates the adenylcyclase responsible of the increase of cAMP that induces the influx of calcium ions which cause the contraction.[12],[13],[14],[15] The rightward shift curves produced by all M. whitei extracts against KCl and adrenaline-induced contraction of the isolated vas deferens denotes noncompetitive antagonism and indicates reduced entry of Ca²⁺ through membrane Ca²⁺ channels.[16] This inhibitory effect was prominent at high concentration of hexane fraction (400 µg/ml) of the plant, as compared with that obtained with the CH₂Cl₂ : MeOH extract and its methanol fraction. This could be a contributing factor in reducing the continuous transport of sperms at rest and during ejaculation by the vas deferens. Alterations in contractility of rat-isolated vas deferens by many drugs have been reported.[17],[18] Our findings also revealed a concentration-dependent relaxing effect of different extracts on the plateau of contraction induced by a supramaximal dose of KCl, and the hexane fraction was found to be more potent than the other two extracts in relaxing the preparation. This ability of M. whitei to relax KCl-induced sustained contractions suggests the involvement of voltage-operated calcium channels.

From these observations, the hexane fraction of M. whitei appeared to be the most active. Steroids and aromatic compounds revealed in this extract may be responsible for its high activity,[19],[20] compared with the other samples in which these substances are either in less quantity (CH₂Cl₂ : MeOH extract) or absent (methanol fraction). In order to study the action of M. whitei on the movement of external Ca2+, the effect of hexane fraction was examined on the CaCl₂-induced contractions in potassium-depolarized preparation. Since this extract was able to abolish the contractions induced by CaCl₂ in K⁺-depolarized preparation in a concentration-dependent manner, it is likely that the hexane fraction of M. whitei inhibits the calcium influx through voltage-operated channels.

In conclusion, the results of the present investigation suggest that the hexane fraction of M. whitei extracts contains bioactive molecules that block both the receptor-operated and voltage-operated calcium channels, thus preventing the entry of calcium during depolarization of vas deferens by KCl and adrenaline. However, in vivo studies using the hexane fraction of M. whitei are needed to justify and confirm the above assertion.

Acknowledgments

References

Copyright 2006 - Indian Journal of Pharmacology

The following images related to this document are available:

Photo images