The crude extract of Lavandula stoechas (Ls.Cr) and its fractions were studied in vitro for the possible presence of spasmogenic and spasmolytic constituents to rationalize some of the traditional uses. Ls.Cr (1-10 mg mL-1) caused atropine-sensitive spasmogenic effect in guinea pig ileum. In spontaneously contracting rabbit jejunum, Ls.Cr (0.03-1 mg mL-1) caused a transient spasmogenicity followed by relaxation at higher doses. Ls.Cr also relaxed high K+- induced contractions at the similar dose range (0.03-1 mg mL-1), which suggests that the spasmolytic effect is mediated through Calcium Channel Blockade (CCB). The CCB effect was confirmed when pretreatment of the tissue with Ls.Cr produced a dose-dependent shift in the Ca++ dose-response curves to the right, similar to that produced by verapamil. Activity-directed fractionation revealed that the spasmolytic effect is concentrated in the petroleum fraction while the spasmogenic effect is more evident in the aqueous fraction. These data indicate the presence of both spasmogenic and spasmolytic components mediated through muscarinic receptor activation and calcium channel blockade, respectively. This study also resolves the controversial results obtained from earlier studies and may explain some of its medicinal uses in gut disorders, like constipation and spasm.
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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 Tyrodes solution, bubbled with a mixture of 95% oxygen and 5% carbon dioxide (carbogen gas) and maintained at 37°C. The composition of Tyrodes, 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 Tyrodes solution, Ca++ was removed from the tissue by replacing this solution with a Ca++-free Tyrodes 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 Tyrodes 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 students t-test and one-way ANOVA, respectively. The p-values<0.05 noted as significantly different.
RESULTS AND DISCUSSION
In isolated guinea pig ileum Ls.Cr caused a concentration-dependent spasmogenic effect at the dose range of 1-10 mg mL-1 (Fig. 2). The maximum response achieved (57.1±3.57, n = 3) was significantly less than that of acetylcholine maximum (p<0.01) (Fig. 2), which indicates that either the plant extract has a partial agonistic activity or the spasmogenic effect is accompanied by a spasmolytic effect.
We previously observed that the spasmogenic effect in the plant is usually mediated through acetylcholine like activity (Bashir et al., 2006; Gilani and Attau-Rehman 2005; Gilani et al., 2000, 2006) To see whether the contractile effect of the crude extract was also mediated through an acetylcholine-like mechanism, the tissue was pretreated with atropine (0.1 μM), an antimuscarinic agent (Arunlakhshana and Schild, 1959). This treatment abolished the spasmogenic effect of the crude extract, similar to that of acetylcholine, while the stimulatory effect of histamine remained unchanged, suggesting that the stimulatory effect is specifically mediated through acetylcholine like mechanism. This cholinergic effect was further confirmed when pretreatment of the tissues with pyrilamine, a histamine (H1) receptor blocker (Sharif et al., 1994), did not alter the response of the plant extract or Ach, while completely blocked the effect of histamine as expected (Fig. 2).
|Fig. 2:||Effect of Ls. Cr on Guinea pig ileum the absence A and presence of atropine B and Pyrilamine C|
Acetylcholine is a neurotransmitter released by the parasympathetic nervous system, mediates its action in the gut by stimulation of M3 receptor subtypes and atropine blocks all muscarinic receptor sites (Brown and Taylor, 2001). Through this mechanism, acetylcholine plays important physiological role to regulate the peristaltic movements of the gut (Brown and Taylor, 1996).
When tested on hyperactive smooth muscle preparation (spontaneously contracting rabbit jejunum), the spasmogenic effect was less marked, followed by a spasmolytic effect at higher concentrations. The spasmogenic component was not studied further in this preparation as the effect was neither dose-dependent, nor reproducible and disappeared after repeated administration of the plant extract. In our previous study we reported that the spasmolytic effect of Ls.Cr was usually mediated through calcium channel blocker(s)-like activity (Gilani et al., 2000). When Ls.Cr was tested for its possible Calicum antagonist effect, it caused dose-dependent relaxation of high K+(80 mM)-induced contractions in rabbit jejunum (Fig. 3).
|Fig. 3:||Dose-dependent inhibitory effect of the crude extract of Lavandula stoechas (Ls.Cr) on spontaneous and K+- induced contractions in rabbit jejunum. The values shown are mean±SEM of four determinations|
Contractions induced by high K+ depends upon ingress of Ca++ into the cells through voltage-dependent calcium channels (Bolton, 1979; Van Breeman et al., 1979). Sufficient calcium ions enter through these membrane channels to activate intracellular contractile proteins and cause smooth muscle contraction (Adelstein and Eisenberg, 1980) and high K+ depolarization-induced contractions only utilize Ca++ influx (Loutzenhiser et al., 1985; Chiu et al., 1986). Calcium Channel Blockers (CCBs) such as diltiazem, nifedipine and verapamil interfere with smooth muscle contractions by binding to voltage-sensitive calcium channels in a way that prevents depolarization-induced Ca++ influx (Fleckenstein, 1977). Thus the inhibition of high K+-induced contraction of smooth muscle by the plant extract may be visualized as an outcome of restricted Ca++ entry through voltage-dependent calcium channels. The CCB-like activity was confirmed when pretreatment of the tissue with Ls.Cr caused right-ward shift in the Ca++ curves in a dose-dependent manner (Fig. 4). Verapamil, a standard calcium channel blocker (Hamilton et al., 1986) also produced similar shift in the Ca++ curves as expected (Fig. 4).
In order to separate constituents responsible for spasmogenic and spasmolytic activities, activity-directed fractionation was carried out using different solvents (Fig. 1). Following fractions were obtained, petroleum fraction (LS.Pet), chloroform fraction (LS.CHCl3) and ethyl acetate fraction (LS.EtAc) and aqueous fraction (LS.Aq.).
|Fig. 4:||Effect of increasing doses of (A) Ls.Cr (n = 4 ), (B) LS.Pet (n = 3) or (C) Verapamil (n = 7) on Ca2+ dose-response curves constructed in Ca2+-free and K+-rich (80 mM) medium in isolated rabbit jejunum (values shown are mean±SEM)|
|Fig. 5:||Comparison of dose-dependent inhibitory effects of Lavandula stoechas crude extract (LS. Cr.) and its petroleum (LS.Pet) and ethyl acetate (LS.EtAc) fractions on K+- induced contractions in rabbit jejunum. The values shown are mean±SEM of four determinations|
The chloroform fraction was insoluble in water and other permitted solvents for assays hence excluded from further studies.
The CCB-like activity was separated in the petroleum and ethyl acetate fractions (LS.Pet and LS.EtAc) as tested against high K+-induced contractions in rabbit jejunum. The comparative dose-response curves of different fractions are shown in Fig. 5. Both LS.Pet and LS.EtAc were found to relax spontaneous and K+-induced contractions in rabbit jejunum. The comparative IC50 values of the crude plant extract (Ls.Cr) and its resultant petroleum ether (LS.Pet) and ethyl acetate (LS.EtAc) fractions were found to be 0.19±0.04; 0.1±0.03; 0.36±0.08 and against high K+-induced contractions (0.18±0.02; 0.07±0.01; 0.92±0.14), respectively. LS.Pet was the most potent fraction of the crude extract. LS.Pet caused dose-dependent (0.01-0.03 mg mL-1) relaxation of spontaneous contractions as well as high K+ -induced contractions. The comparative calcium curves of Ls.Cr and LS.Pet are shown in Fig. 3. Furthermore, this fraction was devoid of acetylcholine like activity (data not shown). However, the cholinergic constituents were separated in the aqueous fraction. This fraction was more potent and efficacious in its cholinergic activity than the parent crude extract and was devoid of CCB like activity. The comparative curves are shown in Fig. 6.
|Fig. 6:||The comparative stimulatory effects of the crude extract of Lavandula stoechas (Ls.Cr) and its aqueous fraction (LS.Aq) in guinea-pig ileum. The values shown are mean±SEM of 3-6 determinations (*** p<0.001)|
In summary, Lavandula stoechas (Ls. Cr) exhibits both spasmogenic and spasmolytic activities, mediated through cholinergic and CCB like mechanisms, respectively. In bioassay-guided fractionation, CCB like activity was concentrated in LS.Pet fraction while cholinergic activity in the aqueous fraction. This study resolves some contradictory results reported earlier and may explain the medicinal use of the plant in the gut disorders like, constipation and spasm.
This study was supported in part by Higher Education Commission, Government of Pakistan through Ph.D. scholarship to Ms. Qaiser Jbeen was on study leave from Islamia University Bahawalpur.
- Bashir, S., K.H. Janbaz, Q. Jabeen and A.H. Gilani, 2006. Studies on spasmogenic and spasmolytic activities of Calendula officinalis flowers. Phytother. Res., 20: 906-910.
- Bolton, T.B., 1979. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol. Rev., 59: 606-718.
- Farre, A.J., M. Colombo, M. Fort and B. Gutierrez, 1991. Differential effects of various Ca2+ antagonists. Gen. Pharmacol.: Vasc. Syst., 22: 177-181.
- Fleckenstein, A., 1977. Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Annu. Rev. Pharmacol. Toxicol., 17: 149-166.
- Gilani, A.H., N. Aziz, M.A. Khan, F. Shaheen, Q. Jabeen, B.S. Siddiqui and J.W. Herzig, 2000. Ethnopharmacological evaluation of the anticonvulsant, sedative and antispasmodic activities of Lavandula stoechas L. J. Ethnopharmacol., 71: 161-167.
- Gilani, A.H. and Atta-ur-Rahman, 2005. Trends in ethnopharmacology. J. Ethnopharmacol., 100: 43-49.
- Gilani, A.H., M.N. Ghayur, P.J. Houghton, Q. Jabeen, S.F. Kazim, M.I. Jumani and S.A. Saeed, 2006. Studies on the hypotensive, cardio-suppressant, vasodilator and antiplatelet activities of betel nut crude extract and its constituents. Int. J. Pharmacol., 2: 33-41.
- Gilani, A.H., S. Bashir, K.H. Janbaz and A.J. Shah, 2005. Presence of cholinergic and calcium channel blocking activities explains the traditional use of Hibiscus rosasinensis in constipation and diarrhoea. J. Ethnopharmacol., 102: 289-294.
- Gilani, A.H., A.J. Shah, M.N. Ghayur and K. Majeed, 2005. Pharmacological basis for the use of turmeric in gastrointestinal and respiratory disorders. Life Sci., 76: 3089-3105.
- Izzo, A.A., R. Capasso, F. Senatore, S. Seccia and P. Morrica, 1996. Spasmolytic activity of medicinal plants used for the treatment of disorders involving smooth muscles. Phytother. Res., 10: 107-108.
- Lis-Balchin, M., S. Hart and S.G. Deans, 1996. Comparison of the pharmacological and antimicrobial action of commercial plant essential oils. J. Herbs Spices Med. Plant, 4: 69-86.
- Taqvi, S.I.H., T.M. Aftab, N.M. Ghayur, H.A. Gilani and S.Z. Saify, 2006. Synthesis and pharmacological screening of 1-(2', b s4'-dimethoxyphenacyl)-4-hydroxy-4-phenylpiperidinium bromide. Int. J. Pharmacol., 2: 146-151.
- Ulubelen, A., N. Goren and Y. Olcay, 1988. Longipinene derivatives from Lavandula stoechas subsp. stoechas. Phytochem., 27: 3966-3967.
- Van Rossum, J.M., 1963. Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch. Pharmacodyn. Therapie, 143: 299-330.