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Year : 2013  |  Volume : 45  |  Issue : 1  |  Page : 13--17

The effect of hydro-ethanolic extract of Achillea millefolium on muscarinic receptors of guinea pig tracheal smooth muscle

Azadeh Feizpour, Mohammad Hossein Boskabady, Goltaj Byrami, Zahra Golamnezhad, Mohammad Naser Shafei 
 Applied Physiology Research Centre and Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Correspondence Address:
Mohammad Hossein Boskabady
Applied Physiology Research Centre and Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad
Iran

Abstract

Objective: To investigate one possible mechanism for the observed relaxant effect of A. millefolium (Achillea millefolium), in the present study the inhibitory effect of the extract of this plant on muscarinic receptors was examined. Materials and Methods: The effects of three concentrations of aqueous-ethanolic extract, 10 nM atropine, and saline on muscarinic receptors were tested in three conditions: In non incubated tracheal smooth muscle (group 1), tracheal chain incubated with propranolol and chlorpheniramine (group 2), and the one incubated with propranolol (group 3). Results: The EC 50 obtained in the presence of all three concentrations of the extract were significantly higher compared to saline in groups 2 and 3 (P < 0.001and P < 0.01 in group 2 and 3 respectively). The EC 50 obtained in the presence of all concentrations of the extract in group 2 were significantly improved compared to groups 1 and 3 (P < 0.05 to P < 0.001). The maximum responses to methacholine in presence of only the higher concentration of the extract (0.8mg/ml) was significantly lower than that of saline in groups 1 (P < 0.05). There was neither significant difference between slopes of methacholine-response curves obtained in the presence of different concentrations of the extract and that of saline nor between the three groups. The values of (CR-1), obtained in the presence of all concentrations of the extract, were significantly lower compared to atropine in the first group but were not significantly different in other groups. The values of (CR-1) obtained in the presence of all concentrations of the extract were significantly improved in groups 2, compared to groups 1 and 3 (P < 0.05 to P < 0.001). Conclusion: These results showed an inhibitory effect for the extract of A. millefolium on muscarinic receptors of tracheal smooth muscle. A histamine (H 1 ) receptor blockade was also suggested for the extract.



How to cite this article:
Feizpour A, Boskabady MH, Byrami G, Golamnezhad Z, Shafei MN. The effect of hydro-ethanolic extract of Achillea millefolium on muscarinic receptors of guinea pig tracheal smooth muscle.Indian J Pharmacol 2013;45:13-17


How to cite this URL:
Feizpour A, Boskabady MH, Byrami G, Golamnezhad Z, Shafei MN. The effect of hydro-ethanolic extract of Achillea millefolium on muscarinic receptors of guinea pig tracheal smooth muscle. Indian J Pharmacol [serial online] 2013 [cited 2022 May 27 ];45:13-17
Available from: https://www.ijp-online.com/text.asp?2013/45/1/13/106428


Full Text

 Introduction



The genus Achillea is a widespread plant from the Asteraceae family with more than 100 species in the world. [1] These plants are native to Europe, Asia, Australia, New Zealand, North America [2],[3] and Iran. [3] The A. millefolium, commonly named yarrow, is an important species that is known for many years in the folk Medicine and has been used in a number of ailments. [4],[5] Due to potential medicinal uses, this plant is included in the national pharmacopoeias of countries such as Germany, France and Switzerland. [6] A. millefolium is also included in the list of medicinal plants of Iranian traditional medicine supported by the Ministry of Health and Medicinal Education (MHME). [6]

The main components of A. millefolium are: flavonoids, phenolic acids, alkaloids, terpens (Cineol, borneol, pinens, camphor, azulen), tannins, cis-Carveol, achillin and leucosis. [6],[7],[8] Flavonoids and phenol carbonic acids of yarrow constitute the most important groups of pharmacologically active substances. [6],[7],[8]

A. millefolium is widely used for remedy of several disorders. The most noticeable effects of this plant are: Antioxidant and antimicrobial activity, [9] anti-inflammatory, [10] antihypertensive, bronchodilatory, [11] gastrointestinal antispasmodic (cramps, indigestion, epigastric distension, flatulence, and intestinal colic), [12],[13],[14] diuretic, urinary antiseptic [13] and astringent and antihemorrhagic effects. [15]

Previous studies have shown that the A. millefolium extract reduces the tonicity of guinea-pig isolated trachea, ileum and pulmonary artery. [8],[16] Additionally, our previous study showed the relaxant effect of this plant on tracheal smooth muscle. [17] An antitussive effect for the plant is also reported. [8] Base on these evidences, in the present study, the effect of hydro alcoholic extract of A. millefolium on muscarinic receptors of guinea pig trachea was examined to evaluate the possible mechanisms for the relaxant effect of the plant on smooth muscle.

 Materials and Methods



Plant and Extraction

Achillea millefollium was collected from Nishabour city (Khorasan province, Iran) and identified by Ferdowsi University Herbarium (voucher No. 1357-2216-6). The plant was dried in the absence of sunlight. The aqueous-ethanolic extract of the plant was prepared as follows: 50 grams of the plant were grinded and added to 700 mL of ethanol 50% (350 mL distilled water and 350 mL ethanol) using the Soxhlet apparatus. The solvent was then removed under reduced pressure. The plant ingredient concentration in the final extract was adjusted to 0.1 g/ml by adding distilled water to the dried extract.

Tissue Preparation

Guinea pigs (400-700 g, both sexes) were sacrificed by a blow on the neck and their tracheas were removed. Each trachea was cut into 8 ring shaped pieces (each containing 2-3 cartilaginous rings). All the rings were then cut open opposite the trachealis muscle, and sutured together to form a tracheal chain. [18] The tissue was then suspended in a 10 ml organ bath (organ bath 61300, Bio Science Palmer-Washington, Sheerness, Kent U.K.) containing Krebs-Henseliet solution with this composition (mM): NaCl 120, NaHCO 3 25, MgSO 4 0.5, KH 2 PO 4 1.2, KCl 4.72, CaCl 2 2.5 and dextrose 11. The Krebs solution was maintained at 37 o C and gassed with 95% O 2 and 5% CO 2 . The tissue was suspended under an isometric tension of 1 g and allowed to equilibrate for at least 1hr while it was washed with Krebs solution every 15 min.

Protocols

The anticholinergic effect of A.millefolium was examined by producing the cumulative log concentration-response curve of methacholine hydrochloride (Sigma Chemical Ltd, Ltd UK) induced contraction of tracheal chains 10 min after exposure of the tissue to one solution of 10 nM atropine maleate (Sigma Chemical Ltd UK, Catalogue No. C4915) as well as three concentrations of aqueous-ethanolic extract of A.millefolium (0.2, 0.4 and 0.8 mg/mL). The consecutive concentrations of methacholine were added every 2 min (range 0.1 – 1000 μM); and the percentage of contraction (each concentration in proportion to the maximum contraction) obtained in the presence of saline, was plotted against log concentration of methacholine. The effective concentration of methacholine causing 50% of maximum response (EC 50 ) in each experiment was measured using the log concentration-response curve of the corresponding experiment. [19]

The shift of cumulative log concentration-response curves obtained in the presence of different concentrations of extract and atropine were examined by comparing the EC 50 obtained in the presence of each solution with that of saline. In addition, the maximum responses to methacholine obtained in the presence of different concentrations of the extract and atropine was compared with that of saline. To examine the parallel rightward shift, the slope of the methacholine-response curve of each experiment was measured and was compared with that of saline. In experiments with parallel shift in methacholine-response curve, the concentration-ratio minus one (CR-1) as an index of the competitive antagonism effect was calculated by the following equation:

EC 50 obtained in the presence of effective solutions

[INLINE:1]

The study was performed with three different experimental designs as follows:

Non incubated tracheal chains (group 1 experiments), (n = 6)Tissues incubated with 1 μM chlorpheniramine (Sigma Chemical Ltd UK) and 1 μM propranolol hydrochloride (Sigma Chemical Ltd UK), (group 2 experiments), (n = 9)Tissues incubated with 1 μM propranolol hydrochloride (group 3 experiments)

All of the experiments were performed randomly. The tissue had a 1 hour rest between each two experiment while being washed every 15 min with Krebs solution. In all experiments the contractions were measured using an isotonic transducer (MLT0202, AD Instruments, Australia) which was connected to a power lab system (PowerLab 8/30, ML870, AD Instruments, Australia).

The study was approved by Ethical Committee of Mashhad University of Medical Sceiences.

Statistical Analysis

All data were expressed as mean΁SEM. The EC 50 , slope, and maximum response obtained in the presence of extract and atropine were compared with those obtained in the presence of saline and the (CR-1) obtained in the presence of extract with those obtained in the presence of atropine using paired t test. The values of EC 50 , slope, (CR-1), and maximum response obtained in three groups were compared using One-way Analysis of Variance (ANOVA) with Tukey- Kramer multiple pot test. Significance was accepted at p < 0.05.

 Results



Concentration-Response Curves and the Values of EC50

The methacholine concentration response curves obtained in the presence of different concentrations of the extract as well as atropine showed right ward shift compared to the curve obtained in the presence of saline [Figure 1].{Figure 1}

The EC 50 of methacholine obtained in the presence of atropine in all experimental conditions was significantly higher than that of saline (p < 0.001 for all groups). The EC 50 obtained in the presence of all concentrations of the extract in group1 was not significantly different compare to saline. However, in group 2 and group 3, the EC 50 obtained in the presence of all concentrations of the extract was significantly greater than those of saline (p < 0.001 and p < 0.01 for all cases in group 2 and 3 respectively) [Figure 2]. The EC 50 methacholine obtained in the presence of all concentration of the extract (0.2, 0.4 and 0.8 mg/ml) in group 2 and 3 were significantly higher than those of group 1 (p < 0.001 and p < 0.05 to p < 0. 01 for group 2 and 3 respectively) [Figure 2]. In addition, the EC 50 methcholine in the presence of last concentration in group 3 was also significantly higher than those of group 1 (p < 0. 05) [Figure 2].{Figure 2}

Maximum Response to Methacholine

The maximum responses to methacholine obtained in the presence of only higher concentration of the extract in groups 1 (0.8 mg/ml) was significantly lower than that of saline (p < 0.05) [Table 1]. There was not statistical difference in maximum response between three groups [Table 1].{Table 1}

Slope of Methcholine-Response Curves

The slopes of methacholine-response curves obtained in the presence of different concentrations of the extract in three groups were not significantly different with those of saline [Table 2]. There was also no significant difference in the slopes obtained, between three groups [Table 2].{Table 2}

Shift in Methacholine Concentration-Response Curves (CR-1)

The values of (CR-1) obtained in the presence of all concentrations of the extract in group 1 were significantly lower than that of atropine (p < 0.01 for all concentrations) [Figure 3]. In group 2 and 3, the value of (CR-1) obtained in the presence all concentration of the extract were not significantly different compared to atropine [Figure 3]. The values of (CR-1) obtained in group 2 and 3 were significantly higher than those of group 1 (p < 0.05 to p < 0.001) [Figure 3]. However, the values of (CR-1) obtained in group 3 were not statistically different with those of group 1 [Figure 3].{Figure 3}

Schild Plot

The slopes of Schild plot [log (CR-1)] against log extract concentrations) for the extract was -0.568, -0.837 and -0.761 for group 1, 2 and 3 respectively [Figure 4].{Figure 4}

 Discussion



The relaxant effect of A. millefolium extract on different types of smooth muscle including tracheal chains has been shown. [8],[17] The relaxant effect on smooth muscle seen for the extract of A. millefolium might be produced due to several different mechanisms including inhibitory effect on muscarinic receptors because relaxant effect of inhibition of this receptors has been shown. [20] To clarify one possible mechanism responsible for the observed relaxant effect seen for the extract of A. millefolium on smooth muscles, in this study, the inhibitory effect of the aqueous-ethanolic extract of the plant was examined on muscarinic receptors of isolated guinea pig tracheal smooth muscle.

The parallel rightward shifts in methacholine log concentration-response curves, obtained in the presence of the different concentrations of aqueous-ethanolic extract compared to that of saline in group 1 experiments (non-incubated trachea) suggested a possible competitive antagonistic effect of the extract at muscarinic receptors of guinea pig trachea. [21] However, the EC 50 obtained in the presence of different concentrations of the extract was not different with that of saline. In addition, maximum contraction effect to methacholine obtained in the presence of higher concentration of the extant was lower than that of saline. These findings indicated a functional antagonistic effect for the extract in this group of experiment. [21]

To evaluate the contribution of beta-adrenergic stimulatory and/or histamine (H 1 ) blocking effect on functional antagonism seen for the extract at muscarinic receptors seen in group 1, the inhibitory effects of the extract from this plant on these receptors were also examined on incubated tracheal preparation with propranolol and chlorpheniramine in group 2. The parallel rightward shift in methacholine-response curves obtained in the presence of different concentrations of the extract compared to that of saline, non-significant difference in maximum responses to methacholine compared to saline and increase in EC 50 obtained in this group of the experiments, relative to those of group 1, showed possible competitive antagonistic effects of the hydro-ethanolic extract of the plant on muscarinic receptors. The data of the group 2 may also indicates inhibitory effect on histamine (H 1 ) receptors and/or adrenergic stimulatory effects for the extract.

The similar values of (CR-1) obtained in the presence of the concentrations of the extract in group 2 compared to that of atropine indicates comparable antagonistic effect of the extract with that of atropine at used concentrations.

These results supported by those of our previous study indicating significant decrease in the relaxant effect of the extract of another species of this plant family in tracheal smooth muscle incubated with propranolol and cholrpheniramine, contracted with methacholine. [22] There was a significant positive correlation between the effects of the extract and concentration indicated concentration-dependent effect of the extract.

To clarify the contribution of beta-adrenergic stimulatory or histamine (H 1 ) blocking effect on functional antagonism seen for the extract at muscarinic receptors seen in group 1, the inhibitory effect of the extract from this plant on muscarinic receptors was also examined on incubated tissues with only propranolol in group 3. The results of this part of the study were almost similar to those of group 2. These findings showed that the functional antagonistic effect of the extract on muscarinic receptors seen in group 1 is mainly due to its stimulatory effect on b-adrenergic receptors. However, the values of EC 50 and (CR-1) obtained in group 3 were significantly lower than those of group 2. These results indicate that the inhibitory effect on histamine (H 1 ) receptors also may have a contribution on functional antagonistic effect of the extract on muscarinic receptors seen in group 1. However, further studies needed to examine the effect of the plant on beta-adrenergic and histamine (H 1 ) receptors by performing concentration response curves to isoprenaline and histamine in the presence of respective competitive antagonists and the extract.

In conclusion, the results of this study suggested a competitive antagonistic effect of A. millefolium at muscarinic receptors. The results also indicated a stimulatory effect for the extract at beta-adrenergic receptors and suggested a small inhibitory effect on histamine (H 1 ) receptors.

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