INTRODUCTION

Lavandula stoechas (Fam. Labiatae) is an evergreen plant locally known as Ustu-khuddoos which is indigenous from Arabic and Mediterranean coasts to Asia Minor (Nadkarni, 1976). The flowers and aerial parts of the plant possess medicinal value. Traditionally, Lavandula stoechas has been used for the treatment of various diseases such as flatulence, colic, chest affections, nervous headaches, (Usmanghani et al., 1997). Lavandula stoechas has been found to possess diaphoretic, expectorant, antispasmodic, antiphlogistic and emmenagogue properties (Nadkarni, 1976). The oil is extensively used in aromatherapy as a holistic relaxant (Evans, 1996). The plant is also being used traditionally for mild constipation.

L. stoechas has been extensively studied phytochemically, with limited work on pharmacological aspects. It contains 0.5-1.2% of volatile oil (Sharma et al., 1983), oleanolic acid, ursolic acid, vergatic acid, longipinene derivatives (Ulubelen et al., 1988), sterols, β-sitosterol, α-amyrine, α-amyrine acetate, lupeol, erythroidiol and flavonoids luteolin, acacetin and vitexin (Ulubelen and Olcay, 1989). The essential oil also known as lavender oil, is obtained by distillation from the flowers and contains more than 51 compounds, the important of which are borneol, linalol, linalyl acetate, geraniol, cineol, fenchone, α-pinene, pinocarvyl acetate, camphor, eucalyptol, myrthenol (Kokkalou, 1988) and myrthenyl acetate (Evans, 1996). A smooth muscle relaxant principle, 7-methoxycoumarin, has also been isolated (Manzoor-I-Khuda and Khan, 1969).

Lavandula oil has been reported to have antispasmodic activities in guinea pig ileum (Izzo et al., 1996) but the exact mechanism of action was not established. In another study, it was shown that the spasmolytic activity of Lavandula oil in gut preparations is often preceded by an initial spasmogenesis (Lis-Balchin et al., 1996). Similarly, the in vivo experiments in dogs showed that the oil diluted in water, when infused through the gut, caused an increase in tone and rhythmic contractions as well as peristalsis (Plant, 1920; Plant and Miller, 1926). In our preliminary study on gut preparation (Gilani et al., 2000), we observed that the methanolic extract possessed antispasmodic and anticonvulsant activities. However, there is no other study available in the literature explaining the exact nature of spasmogenic and/or spasmolytic activities. In this study we report that the spasmogenic and spasmolytic activities of Lavandula stoechas in isolated gut preparations are mediated through stimulation of mucarinic receptors and calcium channel blockade, respectively. The activity-directed fractionation revealed that the spasmogenic activity is separated in the aqueous fraction while the spasmolytic activity is concentrated in the petroleum fraction.

MATERIALS AND METHODS

Plant material and preparation of crude extract: The dried flowers of Lavandula stoechas (500 g) were purchased from a local market in Judia Bazar, Karachi and were cleaned off from soil and adulterant materials like flower stalks. A sample of the Lavandula stoechas was deposited at the herbarium of the Natural Products Research Unit at the Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan.

The plant material was ground to course powder, soaked in 70% aqueous methanol at room temperature for three days with occasional shaking and filtered. The procedure of soaking and filtration was repeated twice. The combined filtrate was dried through evaporation on Rotary Evaporator under reduced pressure to a thick, semi-solid pasty mass of dark brown color; i.e., the crude extract (Ls.Cr) with approximately 19% yield. Ls.Cr was completely solubilized in both saline and distilled water for use in in vivo and in vitro experiments, respectively.

Bioassay guided fractionation of LS.Cr: The crude extract of Lavandula stoechas (Ls.Cr, 64.45 g) was dissolved in 800 mL of distilled water, and successively extracted with petroleum spirit, chloroform and ethyl acetate using solvent-solvent extraction method (Williamson et al., 1998). The procedure is shown in Fig. 1. In summary, the aqueous solution of crude extract was shaken vigorously with petroleum spirit in a separating funnel. The mixture was allowed to separate into two layers. The Petroleum spirit layer (upper) was removed. The extraction with Petroleum spirit was repeated twice. All the Petroleum spirit fractions were combined and evaporated on Rotary Evaporator i.e., the Petroleum fraction (LS.Pet).

Similarly the procedure was successively repeated with two other solvents i.e., Cholorofom and ethyl acetate.

Fig. 1:	Schematic diagram for the preparation of 70% aqueous- methanolic crude extract of Lavandula stoechas (Ls.Cr) and its fractionation into Petroleum (LS.Pet), Chloroform (LS.CHCl3), Ethyl acetate (LS. EtAc) and Aqueous (LS.Aq.) fractions

The resultant fractions were named as LS. CHCl₃ and LS. EtAc fractions, respectively. While the residue left over named as LS.Aq. fraction. The yields of individual fractions are given in the Fig. 1.

Drugs, standards and animals: The following reference chemicals were obtained from the sources specified: acetylcholine chloride, atropine sulphate, histamine dihydrochloride, pyrilamine maleate, verapamil hydrochloride (Sigma Chemical Company, St. Louis, MO, USA).

The following chemicals were used to make the physiological salt solutions: potassium chloride (Sigma Chemical Company, St. Louis, MO, USA), calcium chloride, glucose, magnesium chloride, magnesium sulphate, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride, sodium dihydrogen phosphate, (E. Merck, Darmstadt, Germany) and ethylenediaminetetra-acetic acid (BDH Laboratory Supplies, Poole, England). Buffers and other chemicals were of extra pure analytical grade. All chemicals used were of the highest purity grade. Stock solutions of all the chemicals were made in distilled water and the dilutions were made fresh on the day of the experiment.

Isolated tissue experiments: The isolated tissue experiments were carried out according to the procedures laid down earlier (Gilani et al., 2005a).

Guinea-pig Ileum and Rabbit Jejunum: Animal were starved for 24 h with free access to water and were sacrificed by cervical dislocation. The abdomen was cut open and a piece of ileum 10-20 cm long, about 15 cm proximal to the ileo-cecal end removed. The segment of ileum about 2 cm long was hanged in a 10 mL tissue bath containing Tyrode’s solution, bubbled with a mixture of 95% oxygen and 5% carbon dioxide (carbogen gas) and maintained at 37°C. The composition of Tyrode’s, in mM, was KCl 2.7, NaCl 136.9, MgCl₂ 1.1, NaHCO₃ 11.9, NaH₂PO₄ 0.4, Glucose 5.6 and CaCl₂ 1.8 (pH 7.4). One end of the segment was attached to the metal tissue hook and the other was attached by a cotton thread to an isotonic transducer and connected to a Harvard Student Oscillograph. Under these conditions, guinea-pig ileum, behaves as a quiescent preparation and is considered more suitable to test spasmogenics (Gilani et al., 2005b). A preload of 1 g was applied to each tissue and kept constant throughout the experiment. The tissue was washed several times within 5 min interval and was allowed to equilibrate for 30 min before isotonic contractions to a sub-maximal concentration (0.3 μM) of Ach were recorded. An agonist contact time of 20 sec, was used together with a 3 min interval between doses. Once the tissue was stabilized with reproducible effects from the doses of the standard, test materials were tested. This same protocol was used for rabbit jejunum. The spontaneously contracting rabbit jejunum was mainly used to assess for spasmolytic activity. The plant materials were added in a cumulative fashion (Van Rossum, 1963) to obtain dose-response curves.

Determination of calcium antagonist activity: The calcium antagonist activity was assessed using isolated rabbit jejunum as described earlier (Taqvi et al., 2006). The tissue was pre-contracted with a high dose of K⁺, 80 mM (Farre et al., 1991). Following the plateau, the test material was introduced in a cumulative manner to obtain dose-dependent inhibitory curve (Van Rossum, 1963). This relaxant effect was expressed as per cent of the control response exhibited by K⁺. The Ca⁺⁺ antagonist activity of the plant material was confirmed by the protocol described below. After stabilization of a tissue in normal Tyrode’s solution, Ca⁺⁺ was removed from the tissue by replacing this solution with a Ca⁺⁺-free Tyrode’s solution containing ethylenediaminetetra-acetic acid, EDTA, (0.1 mM, 30 min) with the following composition: NaCl 136.9, KCl 2.68, MgCl₂ 0.49, NaH₂PO₄ 0.32, Glucose 5.05, NaHCO₃ 11.9 and EDTA 0.1. Later, this was also replaced by Ca⁺⁺-free and K⁺-rich Tyrode’s solution with composition (mM): KCl 50, NaCl 91.04, MgCl₂ 1.05, NaHCO₃ 11.87, NaH₂PO₄ 0.41, Glucose 5.55 and EDTA 0.1. Following an incubation period of 30 min, control Concentration-Response Curves (CRCs) of Ca⁺⁺ were obtained. When the control CRCs of Ca⁺⁺ were found super-impossible (usually after two cycles), the tissue was pretreated with the plant extract or verapamil for 60 min to test CCB effect. The CRCs of Ca⁺⁺ were constructed in the presence of different concentrations of the test material.

Data analysis: All data expressed are mean±standard error of the Mean. The significant differences between two groups and more than two groups were analyzed using student’s t-test and one-way ANOVA, respectively. The p-values<0.05 noted as significantly different.