Zataria multiflora Boiss and Carvacrol Affect β 2-Adrenoceptors of Guinea Pig Trachea

The stimulatory effect of Zataria multiflora Boiss (Labiatae) and carvacrol on β-adrenoceptors was examined on guinea pig trachea. The effects of three concentrations of aqueous-ethanolic extract, carvacrol, and propranolol (β-receptor antagonist) on β-adrenoceptors were tested in nonincubated (group 1, n = 8) and incubated tracheal chains with 1 μM chlorpheniramine (histamine H1 receptor antagonist) (group 2, n = 5). Isoprenaline (β-receptor agonist) curves obtained in the presence of all concentrations of the extract and carvacrol showed leftward shifts compared with that of saline in both groups. In both groups, the EC50 (the effective concentration of isoprenaline, causing 50% of maximum response) obtained in the presence of all concentrations of the extract and carvacrol was significantly lower compared to that of saline (P < .01 to P < .001). All values of (CR-1: (EC50 in the presence of active substances/EC50 obtained in the presence of saline)-1) obtained in the presence of concentrations of the extract and carvacrol in both groups were negative and significantly different from that of propranolol (P < .001 for all cases). The results indicated a stimulatory effect of Zataria multiflora Boiss extract on β 2-adrenoceptors which is perhaps due to its constituent, carvacrol.


Introduction
Zataria multiflora Boiss L is a perennial plant with a woody, fibrous root, and its leaves are small, narrow, and elliptical, greenish-grey in colors. Identified constituents of this plant belong to the classes like terpenes, phenols, aliphatic alcohols, flavonoids, saponins, and tannins. Among the compounds, some classes have been previously identified as bioactive chemicals, particularly terpenes such as thymol and carvacrol. Zataria multiflora also contains apigenin, luteolin, and 6-hydroxyluteolin glycosides, as well as di-, tri-, and tetramethoxylated [1,2].
The extract of this plant has been used to treat coughs due to colds, bronchitis, and pertussis, laryngitis and tonsillitis (as a gargle), the common cold, and disorders of the oral cavity and as an antibacterial agent in oral hygiene by traditional healers in Iran [3][4][5]. Both the essential oil and thymol are ingredients of a number of proprietary drugs including antiseptic and healing ointments, syrups for the treatment of respiratory disorders, and preparations for inhalation [1]. It has also been used to treat pertussis, stomatitis, and halitosis [2].
Our previous study showed a potent bronchodilatory effect for carvacrol [12] while our other study did not show any bronchodilatory effect for thymol [27]. Carvacrol is a terpene with phenolic structure. It is a volatile constituent of 2  Zataria multiflora essential oil. Figure 1 illustrates chemical structure of carvacrol, its chemical formula, and molecular weight.
To examine the possible mechanism for the relaxant effect of the plant on smooth muscle, in the present study, the stimulatory effect of aqueous-ethanolic extracts of Zataria multiflora Boiss, and its constituent, carvacrol on β-adrenoceptors was examined on tracheal chains of guinea pigs. In fact, if Zataria multiflora Boiss and its constituents have stimulatory effect on β-adrenoceptors, they could have a potential therapeutic effect on obstructive pulmonary diseases such as asthma and chronic obstructive pulmonary diseases (COPD).

Plant and Extracts. Zataria multiflora Boiss was collected
From a mountain in the region between Tabas and Yazd, (centre east region of Iran), Fleurine mine, and identified by M. R. Joharghi. A voucher specimen was preserved in the Herbarium of the School of Agriculture, Ferdowsi University (Herbarium no: 35314, FUMH). The aqueous-ethanolic extract of the plant was prepared as follows: fifty grams of Zataria multiflora seeds 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 extract concentration in the final extract was adjusted to 0.1 g/mL by adding distilled water to the dried extract.

Characterization of the Extract of Zataria multiflora by
HPLC. The quality of the extract of Zataria multiflora was characterized by HPLC (Waters 474, Waters Corporation, MA, USA) fingerprint. The extract was dissolved in mobile phase and filtered through 0.22 μm membrane filter. An aliquot (20 μL) of sample (500 μg/mL) was injected to the reverse phase HPLC column (C18). The mobile phase consisted of phosphate buffer (pH = 4.8) : methanol : acetonitrile (40 : 30 : 30) with an isocratic elution at the flow rate of 1 mL/minute. The peaks were monitored at 280 nm ( Figure 1). All solvents used were HPLC grade and supplied by Caledon Laboratories, Georgetown Ltd, Canada.

Tissue Preparations.
Male Dunkin-Hartley guinea pigs (400-700 g) were sacrificed by a blow on the neck, and the tracheas were removed. Each trachea was cut into 10 rings (each containing 2-3 cartilaginous rings). All the rings were then cut open opposite the trachealis muscle and sutured together to form tracheal chain [28]. Tissue was then suspended in a 10 mL organ bath (organ bath 61300, Bio Science Palmer-Washington, Sheerness, Kent UK) containing Krebs-Henseleit solution with the following 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.
Krebs solution was maintained at 37 • C and gassed with 95% O 2 and 5% CO 2 . Tissue was suspended under isotonic tension (1 g) and allowed to equilibrate for at least 1 hr while it was washed with Krebs solution every 15 min. This study was approved by the University's Ethics Committee. The allowance number of the relevant ethical committee for the animal experiments is 85301.

Protocols.
The stimulatory effect of of Zataria multiflora Boiss and carvacrol on β 2 -adrenoceptors was examined by producing the cumulative log concentration-response curve of isoprenaline sulphate-induced (Sigma Chemical Ltd., UK) relaxation of precontracted tracheal chains by 10 μm methacholine hydrochloride (Sigma Chemical Ltd., UK) 10 min after the exposure of tissue to one solution. Different tested solutions were included: 10 nM propranolol (0.1 mL of propranolol hydrochloride with 0.1 μm concentration, Sigma Chemical Ltd., UK), three concentrations of aqueousethanolic extract from Zataria multiflora Boiss (0.5, 1, and 2 μg/mL), and carvacrol (Fluka, Italy, Catalogue no. C4915, purity 75%) (0.1, 0.2, and 0.4 μg/mL) or 0.2 mL saline. The consecutive concentrations of isoprenaline were added every 2 min (including 5 nM-1000 μm); and the percentage of relaxation due to each concentration in proportion to the maximum relaxation obtained in the presence of saline was plotted against log concentration of isoprenaline. The effective concentration of isoprenaline causing 50% of maximum response (EC 50 ) in each experiment was measured using the log concentration-response curve of the corresponding experiment.
The shift of cumulative log concentration-response curves obtained in the presence of different concentrations Evidence-Based Complementary and Alternative Medicine 3 of extract, carvacrol, and propranolol was examined by comparing the EC 50 obtained in the presence of each solution with that of saline. In addition, the maximum responses to isoprenaline obtained in the presence of different concentrations of extract, carvacrol, and propranolol in all sets of experiments were compared with that of saline. To examine the parallel rightward shift, the slope of the isoprenalineresponse curve of each experiment was measured and was compared with that of saline. In experiments with parallel shift in isoprenaline-response curve, the concentration-ratio minus one (CR-1) as an index of the competitive antagonism effect was calculated by the following equation: (1) The stimulatory effect of Zataria multiflora on β 2 -adrenoceptors was tested on two different experimental conditions as follows: (a) nonincubated tracheal chains (group 1, n = 8), (b) incubated tracheal chains 30 min prior to the beginning and while obtaining the isoprenaline curve with 1 μm chlorpheniramine maleate (Sigma Chemical Ltd., UK) (group 2, n = 5).
Isoprenaline is β-receptor agonist which causes relaxation of airway smooth muscle. Propranolol is a β-receptor competitive antagonist, and chlorpheniramine is a histamine H 1 receptor competitive antagonist. Competitive antagonists bind to their specific receptor and prevent agonist binding to the receptor, causing a parallel shift in agonist concentrationresponse curve.
All of the experiments were performed randomly with 1-hour resting period of tracheal chains between each two experiments while washing the tissues every 15 min with Krebs solution. In all experiments contractions were measured using an isotonic transducer (Harvard APP LTD, 50-6360 SINO 0210) and measured using a software by computer (Acer model no.: G781) recording.

Statistical Analysis.
All data were expressed as mean ± SEM. The EC 50 , slope, and maximum response obtained in the presence of extract, carvacrol, and propranolol were compared with those obtained in the presence of saline and (CR-1) obtained in the presence of extract and carvacrol, with those obtained in the presence of propranolol using the paired t-test. The comparison of the data of different concentrations of extract and carvacrol was performed using one-way analysis of variance (ANOVA) with Tukey-Kramer multiple posttest. The values of EC 50 , the slope, (CR-1), and maximum response obtained in group 2 experiments were compared with those of group 1 using unpaired "t" test. Correlations between the concentrations of the extract and carvacrol with the values of EC 50 and (CR-1) were examined using least square regression. Significance was accepted at P < .05.

Characterization of the Extract.
Carvacrol of the extract from Zataria multiflora was identified using HPLC method in the Department of Pharmacology, Medical School, Mashhad University of Medical Sciences (Figure 1).

The Extract and Carvacrol Stimulate β-Adrenoceptors.
Cumulative log concentration-response curves to isoprenaline obtained in the presence of all concentrations of the extract and carvacrol showed clear leftward shift while the curve of propranolol showed clear rightward shift compared to isoprenaline curves produced in the presence of saline in both groups 1 and 2 (Figures 2(a)-2(d)).
Isoprenaline EC 50 obtained in the presence of propranolol was significantly higher than that of saline in both groups of experiments (P < .001). However, the EC 50 obtained in the presence of all concentrations of the extract and carvacrol was significantly lower than that of saline in both groups 1 and 2 (P < .01 to P < .001), (Figures 3(a) and 3(b)).
Maximum responses to isoprenaline obtained in the presence of all concentrations of the extract and carvacrol were not significantly different compared to that of saline in both groups 1 and 2 (Figure 4(a)). Slopes of isoprenalineresponse curves obtained in the presence of all three concentrations of the extract and carvacrol were not significantly different from that of saline in both groups 1 and 2 ( Figure 4(b)).
Values of (CR-1) obtained in the presence of all concentrations of the extract and carvacrol were negative and significantly different from those of propranolol in both groups 1 and 2 (P < .001 for all cases) (Figures 5(a) and  5(b)). There were significant negative correlations between the concentrations of the extract and carvacrol with the values of EC 50 and (CR-1) in both groups (P < .005 to P < .001) ( Table 1).

Differences between the Extract of Zataria multiflora and Carvacrol.
Values of isoprenaline EC 50 obtained in the presence of two higher concentrations of the extract were significantly lower than those of carvacrol in group 1 (P < .05 to P < .001) (Figure 3(a)). The values of (CR-1) obtained in the presence of two higher concentrations of the extract were significantly greater than those of carvacrol in group 1 (P < .05 to P < .01) (Figure 5(a)). In group 2 experiment, the values of isoprenaline EC 50 obtained in the presence of all concentrations of the extract were significantly greater than those of carvacrol (P < .05 to P < .001) (Figure 3(b)). The values of (CR-1) obtained in the presence of two higher concentrations of the extract were significantly lower than those of carvacrol (P < .05 for both cases) ( Figure 5(b)). However, there was no significant difference in maximum response to isoprenaline and slope between the extract and carvacrol in both groups (Figures 4(a) and 4(b)).
Values of isoprenaline EC 50 obtained in the presence of all concentrations of the extract and only in the highest concentration of carvacrol in group 1 were significantly lower      Statistical comparison between chlorpheniramine and other solutions. NS: nonsignificant difference, * * * P < .001. Statistical comparison between different concentrations of the extract and carvacrol + P < .05, ++ P < .01. Values of (CR-1) obtained in the presence of two higher concentrations of the extract in group 1 were higher than those in group 2 (P < .001 for two higher concentrations). The comparison of (CR-1) obtained in the presence of extract and carvacrol with those of propranolol and data between two groups was done using the paired t-test. The comparison of the data of different concentrations of extract and carvacrol was performed using one-way analysis of variance (ANOVA) with Tukey-Kramer multiple post test.
than those in group 2 (P < .05 to P < .001) (Figure 3). The values of (CR-1) obtained in the presence of two higher concentrations of the extract in group 1 were significantly greater than those of group 2 (P < .001 for both cases) ( Figure 5). However, there was no significant difference in maximum response to isoprenaline and slope between the two groups (Figures 4(a) and 4(b)).

Discussion
In the present study the stimulatory effect of the aqueousethanolic extract of the plant on β-adrenoceptors (as one possible mechanism responsible for the observed relaxant effect seen for the extract of Zataria multiflora Boiss and its constituent, carvacrol) on tracheal and other smooth muscles [6][7][8][9][10] was examined. In fact, the relaxant effect of tracheal smooth muscle due to stimulation of βadrenoceptors is a well-known phenomenon [29]. The parallel rightward shifts in isoprenaline log concentrationresponse curves obtained in the presence of the different concentrations of aqueous-ethanolic extract and carvacrol and the achievement of maximum relaxation effect to isoprenaline compared to that of saline showed possible stimulatory effects of the hydroethanolic extract of the plant and carvacrol on β-adrenoceptors of guinea pig's trachea [29][30][31]. Lower values of EC 50 obtained in the presence of the different concentrations of aqueous-ethanolic extract and carvacrol compared to that of saline confirmed this effect. In addition the negative values of (CR-1) obtained in the presence of the extract and carvacrol are another indicator of stimulatory effect of the plant and its constituent on β-adrenoceptors of trachea smooth muscle. The stimulatory effect of extract from Zataria multiflora and carvacrol on β 2 -adrenoceptors was also examined on incubated tracheal preparation with chlorpheniramine to block histamine H 1 receptors, (group 2 experiments). The group 2 experiment was done in order to evaluate the effect of the extract and carvacrol on β 2 -adrenoceptors more precisely. The results of group 2 experiments were very similar to those obtained in group 1, confirming the stimulatory effect of the extract and carvacrol on β 2 -adrenoceptors [29][30][31]. The dose response curves obtained in the presence of extract in group 2 (incubated with chlorpheniramine) showed smaller leftward shift compared to group 1, but for carvacrol the shift in the two groups was similar. In addition the values of EC 50 obtained in the presence of concentrations of extract in group 1 were significantly lower than those of group 2. These results indicated an inhibitory effect for the extract on histamine (H1) receptors in addition to stimulatory effect of the plant on β 2 -adrenoceptors.
The significant negative correlations between both values of EC 50 and (CR-1) and the concentrations of the extract  Evidence-Based Complementary and Alternative Medicine concentration of carvacrol in group 1 were significantly lower than those in group 2, and there was not significant difference in the values of (CR-1) obtained in the presence of concentrations of carvacrol between the two groups which is an indicator of the absence of inhibitory effect of carvacrol on histamine (H 1 ) receptors. In fact, in our previous study the relaxant effect of macerated and aqueous extract of other species plant from this family was almost completely abolished in incubated tracheal preparations incubated with propranolol and chlorpheniramine [10] which confirms the result of the present study. However, the effect of both the extract and carvacrol on histamine (H 1 ) receptors should be examined in further studies more precisely.
In addition, the values of EC 50 isoprenaline obtained in the presence of two higher concentrations of the extract in group 1 were significantly lower and those of its all concentrations in group 2 were greater than those of carvacrol. The values of (CR-1) obtained in the presence of two higher concentrations of the extract in group 1 were significantly greater and in group 2 were lower than those of carvacrol. These results also support the suggestion of an inhibitory effect on histamine (H 1 ) receptors for the extract in addition to its stimulatory effect on β 2 -adrenoceptors. These results indicated the differences between the effects of hydroethanolic extract of Zataria multiflora and its constituent carvacrol. Therefore, the pharmacological properties of the extract are not solely due to its constituent, carvacrol, which is a very important finding of this study; because in phytotherapy it is the extract that defines the efficacy not some analytical lead substance, even if it contributes to the overall effect.
The concentration of carvacrol in the essential oil of Zataria multiflora is reported between 17% and 40%, with a mean value of 30% [32][33][34] which should be lesser in the hydroalcoholic extract. Therefore, the concentrations of carvacrol used in the present study are equal to those of extract (the concentrations of carvacrol were one fifth of the extract). Thus the stimulatory effect of the extract of Zataria multiflora on β 2 -adrenoceptors could be due to its constituent carvacrol. Different suggested mechanism for the relaxant effect of the extract from Zataria multiflora on tracheal smooth muscle and the contribution of its constituent carvacrol was illustrated in Figures 5 and 6. The major limitation of the present study is that the selectivity of the stimulatory effect of Zataria multiflora Boiss and its constituent carvacrol on β 2 -adrenoceptors can not estimated from the present results precisely, and further studies needed to find out their selectivity on these receptors. In addition, the bronchodilatory and β 2 -receptor stimulatory effect should be examined in further studies.

Conclusions
In conclusion, these results indicated a relatively potent stimulatory effect for the extract from Zataria multiflora Boiss on β 2 -adrenoceptors which is perhaps due to its constituent, carvacrol. A possible inhibitory effect of the plant on histamine (H 1 ) receptors was also suggested.