Components of Volatile Fractions from Eucalyptus camaldulensis Leaves from Iraqi–Kurdistan and Their Potent Spasmolytic Effects

Inhalation of vapors from a hot tea of Eucalyptus camaldulensis Dehnh. leaves is considered by Iraqi–Kurdistan people an effective spasmolytic and antipyretic remedy for the treatment of respiratory diseases. The constituents of volatile fractions isolated by hydrodistillation from dried leaves of the plant collected in Kurdistan were determined by GC-FID and GC-MS analyses. More than 90% components were identified. The most abundant constituents were 1,8-cineole, p-cymene, α-pinene, terpinen-4-ol, aromadendrene, and α-terpineol. The different volatile fractions induced relaxation on rat isolated aortic and tracheal rings in concentration-dependent manner. These effects appeared to be due to a complex interaction between various terpenoid components rather than being only due to the main oil constituent, 1,8-cineole. The KCa channel and the NO pathway were not significantly involved in the relaxation mechanism, while Ca2+ channels played a major role in the spasmolytic effects.


Introduction
The Kurdistan region of Iraq has a rich biodiversity and many medicinal herbs, including Eucalyptus species, are used by local people in traditional medicine since time immemorial [1]. A common antispasmodic and antipyretic remedy for treating respiratory tract diseases is prepared with the leaves of Eucalyptus camaldulensis Dehnh. (family Myrtaceae), also known as river red gum or Murrey red gum tree [2]. A hot tea is made and then the vapors from the aromatic infusion are inhaled by sick people. The empirical knowledge of this traditional medicine is passed on by oral tradition. No scientific rationale has been reported so far and the mechanism of action remains largely uninvestigated.
Our data clearly showed that a significant quantity of oxygenated components remained dispersed in the aqueous phase collected in the condenser during leaf hydrodistillation. Therefore, we considered that a more accurate analysis of E. camaldulensis volatiles should include not only the components of the oil separated from the hydrosol, i.e., EO W , but also the mixture of polar compounds dispersed in the water condensed during the distillation process, i.e., EO A and EO Ar . To this purpose, the column EO Tot in Table 1 shows the sum of the percentages of each compound occurring in EO W , EO A , and EO Ar , averaged by the weight of each fraction. It thus resulted that the percentage of oxygenated monoterpenoids in the total volatile fraction EO Tot was significantly higher than in the fraction EO W alone, while that of hydrocarbons was lower. Only twelve components of EO Tot had an abundance > 1%, namely, 1,8-cineole (1, 55.83%), p-cymene (2, 4.80%), terpinen-4-ol (4, 4.56%), α-terpineol , and p-menth-1-en-7-al (1.02%), which together accounted for 86.32% EO Tot .
Phytochemical results indicate that three chemotypes of E. camaldulensis can be distinguished on the basis of the main components of the oil obtained by hydrodistillation under analogous conditions: one rich in 1,8-cineole (28-84%), one rich in p-cymene (20-30%) and one rich in spathulenol (18%) [8,22]. For example, the essential oil from E. camaldulensis leaves collected in Pakistan contained high concentration of oxygenated sesquiterpenes [11], while the main components of the leaf oil hydrodistilled from E. camaldulensis collected in Iran were p-cymene (68.43%), 1,8-cineole (13.92%), α-pinene (3.45%), and limonene (2.84%) [4]. Therefore, the compositions of EOs and EO W clearly indicate that the variety of E. camaldulensis growing in Kurdistan can be classified as a chemotype with high 1,8-cineole and low p-cymene contents.

Relaxant Effects of E. camaldulensis Volatile Fractions on Aortic Rings
In a preliminary study we showed that the essential oil of E. camaldulensis produced a potent dilation in aortic force development probably acting as a Ca 2+ channel antagonism and partially via NO and cyclooxygenase pathways [17]. To investigate the relationship between oil composition and in vivo spasmolytic effects, the vasorelaxant effects of volatile fractions EO S , EO W , and EO Ar on isolated rat aortic rings were determined separately, following the same procedure described previously [17]. The concentration-response curves for the oil-induced relaxation against phenylephrine (PE)-induced contractions are shown in Figure 2, while the percentages of relaxation, the values of Log EC 50 , and 95% CI (Confidence Interval) for Log EC 50 are shown in Table 2. The curves show that the cumulative addition of the three samples at the plateau of contraction caused a relaxant effect in a concentration-dependent manner, with a strong potent dilation. Relaxation caused by EO W did not differ significantly from EO S , as it could be anticipated by the comparable chemical compositions of the two oils; instead, significantly minor relaxation effects were observed for EO Ar . This finding seems to indicate that the reduced relaxant properties of EO Ar , compared to EO S and EO W , is caused by the minor amount of 1,8-cineole (1) in EO Ar (10.14%) than in EO S (62.70%) and EO W (59.09%). However, the physiological effects of EO Ar were not totally suppressed, due to the significant presence of other  (Table 1), such as α-terpineol (6, 17.26%) [23], spathulenol (5.74%) [24], carvacrol (2.73%), and thymol (2.52%) [25]. relaxant effect in a concentration-dependent manner, with a strong potent dilation. Relaxation caused by EOW did not differ significantly from EOS, as it could be anticipated by the comparable chemical compositions of the two oils; instead, significantly minor relaxation effects were observed for EOAr. This finding seems to indicate that the reduced relaxant properties of EOAr, compared to EOS and EOW, is caused by the minor amount of 1,8-cineole (1) in EOAr (10.14%) than in EOS (62.70%) and EOW (59.09%). However, the physiological effects of EOAr were not totally suppressed, due to the significant presence of other active terpenoids (Table 1), such as α-terpineol (6, 17.26%) [23], spathulenol (5.74%) [24], carvacrol (2.73%), and thymol (2.52%) [25].

Relaxant Effects of EOS and 1,8-Cineole (1) on Rat Tracheal Rings
Since the oil EOS showed the highest activity on rat aortic rings, compared to EOW and EOAr, we deemed it to be interesting to measure the relaxant effect of this volatile fraction also in rat tracheal rings. Moreover, the major component of EOS, namely the monoterpenoid 1,8-cineole (1), was also tested in parallel experiments. In fact, it is well-known that 1,8-cineole (1) displays a wide range of

Relaxant Effects of EO S and 1,8-Cineole (1) on Rat Tracheal Rings
Since the oil EO S showed the highest activity on rat aortic rings, compared to EO W and EOAr, we deemed it to be interesting to measure the relaxant effect of this volatile fraction also in rat tracheal rings. Moreover, the major component of EO S , namely the monoterpenoid 1,8-cineole (1), was also tested in parallel experiments. In fact, it is well-known that 1,8-cineole (1) displays a wide range of biological effects, including muscle relaxant, bronchodilatatory [26][27][28][29], and anti-inflammatory [30,31] properties. Therefore 1,8-cineole (1) was used as the reference compound and its contribution to the biological activity of the volatile fraction EO S was roughly estimated. The concentration-response curves for 1,8-cineole (1) and EO S relaxant effects against acetyl choline (ACh)-induced contractions are shown in Figure 3. The percentages of relaxation were 33.01% and 79.44% for 1,8-cineole (1) and EO S , respectively. On the other hand, the Log EC 50 were 1.030 and 0.508 (mg/mL), respectively. These results clearly indicated that both 1,8-cineole (1) and EO S produced potent concentration-dependent relaxation in rat tracheal rings precontracted with ACh (10 µM), although the activity of EO S was much higher than that of 1,8-cineole (1) alone. These findings nicely agree with the results of other authors who suggested that the relaxant [26,32] and cardiovascular [33] effects induced by Eucalyptus essential oils appeared to derive from a complex interaction between various terpenoid components rather than being due to a single compound.
concentration-dependent relaxation in rat tracheal rings precontracted with ACh (10 μM), although the activity of EOS was much higher than that of 1,8-cineole (1) alone. These findings nicely agree with the results of other authors who suggested that the relaxant [26,32] and cardiovascular [33] effects induced by Eucalyptus essential oils appeared to derive from a complex interaction between various terpenoid components rather than being due to a single compound.

Relaxation Mechanism
Aimed at clarifying possible molecular mechanisms involved in the relaxation induced by EOS in tracheal rings, we explored the effects of EOS on the roles of the KCa and Ca 2+ channels, and on NO release. Actually, the crucial roles of KCa and L-type Ca 2+ channels, as well as NO release in airway smooth muscle contraction are well known [17,23,[25][26][27][28][29][33][34][35], and they are important therapeutic targets.
The concentration-response curve for the effect exerted by EOS against tracheal rings precontracted with ACh (10 μM) and preincubated with the calcium-activated K + (KCa) channel blocker tetraethyl ammonium chloride (TEA), is shown in Figure 4, in comparison with the curve of the control. The two curves were almost superimposed, indicating that the inhibition of the KCa channel was not a significant signaling pathway.
In another experiment, we measured the relaxation induced by EOS in tracheal rings precontracted with ACh (10 μM) and preincubated with the L-type Ca 2+ channel blocker nifedipine (30 μM). The corresponding concentration-response curve with respect to the control is shown in Figure 5. In tracheal smooth muscle, blockade of voltage dependent Ca 2+ channels (VDCs) by nifedipine at the extracellular surface of the membrane may attenuate bronchoconstriction [34]. Spasm evoked by ACh and the maintenance of spontaneous tone largely depend on mechanisms for increasing the cytoplasmic concentration of free Ca 2+ which are resistant to nifedipine [35]. In the event, the relaxation effect induced by EOS was significantly reduced, suggesting that the presence of active constituents in the EOS can cause tracheal relaxation via the inhibition of Ca 2+ influx through the plasma membrane in tracheal smooth muscle.

Relaxation Mechanism
Aimed at clarifying possible molecular mechanisms involved in the relaxation induced by EO S in tracheal rings, we explored the effects of EO S on the roles of the K Ca and Ca 2+ channels, and on NO release. Actually, the crucial roles of K Ca and l-type Ca 2+ channels, as well as NO release in airway smooth muscle contraction are well known [17,23,[25][26][27][28][29][33][34][35], and they are important therapeutic targets.
The concentration-response curve for the effect exerted by EO S against tracheal rings precontracted with ACh (10 µM) and preincubated with the calcium-activated K + (K Ca ) channel blocker tetraethyl ammonium chloride (TEA), is shown in Figure 4, in comparison with the curve of the control. The two curves were almost superimposed, indicating that the inhibition of the K Ca channel was not a significant signaling pathway.   In another experiment, we measured the relaxation induced by EO S in tracheal rings precontracted with ACh (10 µM) and preincubated with the L-type Ca 2+ channel blocker nifedipine (30 µM). The corresponding concentration-response curve with respect to the control is shown in Figure 5. In tracheal smooth muscle, blockade of voltage dependent Ca 2+ channels (VDCs) by nifedipine at the extracellular surface of the membrane may attenuate bronchoconstriction [34]. Spasm evoked by ACh and the maintenance of spontaneous tone largely depend on mechanisms for increasing the cytoplasmic concentration of free Ca 2+ which are resistant to nifedipine [35]. In the event, the relaxation effect induced by EO S was significantly reduced, suggesting that the presence of active constituents in   In the last experiment, the relaxant effect of EOS in tracheal rings precontracted with ACh and preincubated with NG-nitro-L-arginine methyl ester (L-NAME) was measured. L-NAME is a very well-known endothelial NO synthase inhibitor. The concentration-response curve of EOS, compared to the control, is shown in Figure 6. The graphic shows that the relaxation was attenuated to some extent; however, EOS was still quite effective (45.41% relaxation). Table 3 shows the log EC50, the 95% CI values, and the percentages of tracheal ring relaxation calculated from the concentration-response curves (Figures 4-6) determined for EOS in the three tests, compared to the control. In conclusion, these experiments clearly indicated that the pretreatment of tracheal rings with TEA or with L-NAME was unable to alter significantly the dose-dependent relaxant effects exerted by EOS. Therefore, the KCa channel and NO release and activation of the NO-cGMP pathway were not significantly involved in the relaxation mechanism. Instead, the inhibitory effect of EOS in tracheal rings preincubated with nifedipine blocked the concentration-response relaxation, indicating that L-type Ca 2+ channels were significantly (P < 0.001) involved in the relaxation mechanism. In the last experiment, the relaxant effect of EO S in tracheal rings precontracted with ACh and preincubated with NG-nitro-L-arginine methyl ester (L-NAME) was measured. L-NAME is a very well-known endothelial NO synthase inhibitor. The concentration-response curve of EO S , compared to the control, is shown in Figure 6. The graphic shows that the relaxation was attenuated to some extent; however, EO S was still quite effective (45.41% relaxation).     Table 3 shows the log EC 50 , the 95% CI values, and the percentages of tracheal ring relaxation calculated from the concentration-response curves (Figures 4-6) determined for EO S in the three tests, compared to the control. In conclusion, these experiments clearly indicated that the pretreatment of tracheal rings with TEA or with L-NAME was unable to alter significantly the dose-dependent relaxant effects exerted by EO S . Therefore, the K Ca channel and NO release and activation of the NO-cGMP pathway were not significantly involved in the relaxation mechanism. Instead, the inhibitory effect of EO S in tracheal rings preincubated with nifedipine blocked the concentration-response relaxation, indicating that L-type Ca 2+ channels were significantly (P < 0.001) involved in the relaxation mechanism. Table 3. Relaxant effects of volatile fractions (EO S ) in tracheal rings preincubated with K + and Ca 2+ channel blockers, and with NG-nitro-L-arginine methyl ester (L-NAME), respectively.

Plant Material
Fresh leaves of E. camaldulensis Dehnh. were collected in January 2010 near the Duhok Damp, in the Iraqi Kurdistan region, and were identified by Prof. Dr. Saleem Shahbaz, of the Herbarium of the Department of Biology, Faculty of Science, University of Zakho. The plant name has been checked with http://www.theplantlist.org on 08.24.2019. The plant is neither endangered nor protected in Kurdistan, and no specific permission was required for the collection. A voucher has been deposited at the Herbarium with the accession number AC031. The leaves were rinsed with tap water, air-dried for about twenty days at 20-25 • C in the shade, and ground in an electrical mill (IKA-WERKE, Staufen im Breisgau, Germany) to a fine powder just prior to extraction.

Isolation of Volatile Fractions EO
Five volatile fractions EO were obtained as dense oils by two different procedures: (i) Hydrodistillation provided fractions EO S , EO W , EO A , and EOAr (see Section 3.3); (ii) CO 2 extraction at atmospheric pressure afforded fraction EO D (see Section 3.4). All samples were dissolved in dichloromethane and analyzed by GC-FID and GC-MS.

Hydrodistillation
Powdered air-dried leaves of E. camaldulensis were hydrodistilled in a standard Clevenger-type apparatus, by two different procedures, A and B. In procedure A, named hydrodistillation with circulating-water, condensed water was circulating, while water was not circulating in procedure B. In mode A, leaves (3 lots of 250 g each) were hydrodistilled and volatiles were collected in a cylindrical glass condenser, where an oily layer (labeled EO S ) accumulated at the top of an aqueous phase. The latter could return by gravity to the distillation pot connected to the tip of the collector tube through a plastic tube. Water condensation and dipping in the pot were synchronized, so as to leave about 10 mL of an aqueous layer in the condenser. After 2.5 h of distillation no more oil apparently accumulated at the top of the aqueous phase. EO S was carefully separated from the aqueous layer by a micropipette, dried over anhydrous Na 2 SO 4 , filtered using Whatman filter paper (No.1), and stored at −20 • C in a sealed vial until analysis. The average yield over 3 distillations was 0.98 ± 0.02% (w/w). This oil was considered as the reference essential oil of E. camaldulensis leaves. In mode B. named hydrodistillation with non-circulating-water, the leaves were hydrodistilled (3 repetitions from lots of 250 g each) while the total condensed aqueous phase remained in the collector tube during the entire distillation time (2.5 h). After 1 h from the end of distillation, two phases clearly separated in the condenser. The accumulated oily layer, labeled EO W [average yield 0.88 ± 0.02% (w/w)] was carefully removed from the top of the hydrosol and treated as described for EO S . The condensed aromatic aqueous layer (Aq, about 750 mL) from each hydrodistillation was subjected to multiple solid phase extraction (SPE). To this purpose, a home-made cartridge was prepared by packing 10 g of Lichroprep RP-18 powder inside a glass syringe-like tube; subsequently, the reversed phase was washed with MeOH (50 mL), followed by deionized water (50 mL), and then dried under vacuum. Each fraction Aq was divided in 80 mL portions, which were individually subjected to SPE. Each portion was added to the top of the column, which was then drained out under vacuum, collecting the liquid (F). The column was then washed with MeOH (50 mL), and the eluate (E) was collected. The experiment was then repeated with a new portion of Aq, following an identical procedure. Subsequently, all F and E fractions were separately collected together and carefully evaporated under reduced pressure (200 mmHg) to give two oily residues, EO Ar (average yield 0.05 ± 0.015%) and EO A (average yield 0.28 ± 0.01%), respectively, which were stored under N 2 in sealed vials at −20 • C until analysis.

CO 2 Extraction
A sample of powdered dried leaves (250 g) was submitted to CO 2 extraction at atmospheric pressure for 2 h, in a home-made glass equipment, according to the method described by Honkanen and Karvonen [36]. An oily residue (EO D , yield = 0.004%) was obtained which was stored under N 2 in a sealed vial at −20 • C until analysis. Given the low yield, CO 2 extraction was not repeated.

GC-FID Analysis
GC-FID analyses of the different volatile fractions (EO) were performed on a Perkin Elmer Auto system gas chromatographer equipped with a HP5 capillary column (25 m × 0.32 mm i.d.; 0.52 µm film thickness). Nitrogen was used as the carrier gas at a flow rate of 1.5 mL/min. The injector operated in split mode (split ratio 25:1) and was heated at 220 • C. The GC oven temperature was hold at 60 • C for 1 min, then increased by a gradient of 3 • C/min until 100 • C, hold at 100 • C for 30 min, then increased to 250 • C by a gradient of 10 • C/min, finally hold at 250 • C for 5 min. Each analysis lasted 64.3 min. One microliter of each oil (5% in CH 2 Cl 2 ) was injected in each analysis. Percentages (%) of compounds > 0.01 occurring in each EO (Table 1) were calculated from the corresponding GC peak areas with respect to the total peak area in the GC-FID chromatogram without applying any correction factor. Three GC-FID analyses were performed for EO S and EO W , while EO A , EO Ar , and EO D were analysed only once.

GC-MS Analysis
GC-MS analyses of the different volatile fractions (EO) were performed using an Agilent Bench Top GC-MS equipment, comprising a 6890N network gas-chromatographic system combined with a 5973 Network Mass Selective Detector (Agilent Technologies, Wilmington, DE, USA), and equipped with a DB-5 glass capillary column (30 m × 0.25 mm i.d.; 0.25 µm film thickness). Helium was used as the carrier gas at a flow rate of 1 mL/min. Temperature of the injector: 220 • C; oven temperature program: isotherm at 60 • C for 1 min, then increased (3 • C/min) to 270 • C, followed by an isotherm at 270 • C for 5 min. Sample split ratio: 1:20; 1 µL of each oil (5% in CH 2 Cl 2 ) was injected in each analysis. Mass spectra were acquired at 70 eV within a mass range of 41-350 Daltons (Da) with a scan time of 0.73 scans s −1 ; ion source temperature: 230 • C. Compound identification was at first based on their linear retention indices (LRIs). They were calculated according to Van Den Dool and Kratz [20], with reference to a homologous series of n-alkanes C 6 -C 20 (TPH-6RPM of CHEM SERVICE), which were injected immediately after each oil analysis under the same gas-chromatographic conditions. Calculated LRIs were compared with the retention indices of authentic samples or literature data [18,19]. Comparison of the retention indices was considered reasonable in a range of ±20 units. The identity of each EO constituents was confirmed by comparing the mass spectral fragmentation patterns with those reported in the literature [18,19,21] and, whenever possible, with the GC-MS data of pure standard compounds.

Physiological Activities of Volatile Fractions
Experiments on rats were performed according to the Iraqi and institutional rules considering animal experiments, and in accordance with the internationally accepted principles for laboratory animal use and care as found in the European Community guidelines (EEC Directive of 1986; 86/609/EEC). Stock solutions (0.05% g/L) of tested volatile fractions and 1,8-cineole were prepared by dissolving the desired amount in DMSO in which samples are completely soluble and which helped dissolution in water. They were kept refrigerated until analysis. Desired serial dilutions were then prepared by diluting stock solutions with aqueous NaCl (0.09% g/L) and warmed to 37 • C prior to use.

Measurement of Isometric Force with Isolated Thoracic Aortic Rings
Adult male albino rats, weighting 200-300 g each, were used for all experiments. They were kept in plastic cages at 24 • C and were exposed to a photoperiod cycle of 12 h light followed by 12 h darkness. Rats were fed standard diet and tap water. Access to water was free, while food was removed 24 h prior to experiments. Rats were injected intraperitoneally with heparin (1500 units/Kg body weight), then left for 30 min to avoid blood clotting and possible damage of the aorta endothelium, and finally anaesthetized with racemic ketamine (40 mg/kg) and xylazine (10 mg/kg) intraperitoneally. Chest cavity was opened, and excess tissues and fat were removed. Aorta was isolated and transferred to a beaker containing a Krebs solution aerated with 95% O 2 and 5% CO 2 , that was placed in a water-bath at 37 • C. Subsequently, the aorta was segmented into rings 3-5 mm long. Isolated thoracic aortic rings were used in preparations with intact endothelium. Aortic rings were mounted between two stainless steel hooks, connected by a thread to a force transducer coupled to a transbridge amplifier and a Power Lab Data Acquisition system (model ML 870, Power Lab, AD Instrument, Sydney, Australia), which was connected to a computer running chart software (Version 7). The isometric force produced was monitored and recorded. Experiments were performed in 10 mL organ baths filled with a physiological Krebs solution (pH = 7.4) maintained at 37 • C by means of a thermoregulating system with water continuously circulating throughout a double-walled water-jacketed system, and continuously gassed with 95% O 2 and 5% CO 2 . Tension was set at 2 g for 60 min, and the buffer solution was changed every 15 min until the resting tone became constant. After these numerous washings most xylazine was considered to have been removed from the organ so that its effect on the tissue was negligible. Sample concentration-response curves were then determined, with phenilephrine (1 µM) used as the aorta ring-contraction agonist.

Measurement of Isometric Force with Isolated Tracheal Rings
Trachea was removed from anaesthetized rats, cleaned, and segmented into 3-5 mm long rings (each containing 3-4 cartilaginous rings). Measurements of the isometric force with isolated tracheal rings were then performed according to international standard procedures for in vitro study using organ bath. Rings were suspended in 10 mL organ baths filled with physiological Krebs solution (pH = 7.4), maintained at 37 • C by a thermoregulating system with continuous water circulating throughout a double walled water jacket system, and aerated with 95% O 2 and 5% CO 2 . Tracheal rings were maintained under an isometric tension of 2 g and allowed to stabilize for 60 min, while changing the Krebs solution every 15 min. After these numerous washings most xylazine was considered to have been removed from trachea so that its effect on the tissue was negligible. Concentration-response curves (Figures 3-6 and Table 3) were then determined adding increasing amounts of EO S and 1,8-cineole, in separate experiments. ACh (10 µM) was used as the tracheal ring-contraction agonist. In subsequent experiments, increasing amounts of EO S were added to tracheal rings preincubated with the K + channel blocker TEA (1 mM) for 20 min and then precontracted with ACh (10 µM). Analogous experiments were performed with tracheal rings preincubated with nifedipine (30 µM) for 10 min prior to precontraction with ACh (10 µM). Finally, tracheal rings were preincubated with L-NAME (0.3 mM) for 10 min. prior to precontraction with ACh (10 µM); then, increasing amounts of EO S were added.

Statistical Analysis
The vasorelaxation response, calculated as a percentage of contraction produced by PE or ACh, was expressed as the mean ± standard error of the mean (SEM). The base line tension was expressed as a measure of 100% relaxation, and the tension induced by agonist was taken as a measure of 0% relaxation. All data analyses were fitted with a Hill equation, that the median effective concentration (Log of IC 50 ) value was given as geometric mean with 95% confidence intervals (95% CI), using the statistics program GraphPad Prism ™ software, version 6 (GraphPad Software, Inc., San Diego, CA, USA). Two-way analysis of variance (ANOVA) was performed, supported with Bonferroni test when carrying out pairwise comparison between the same doses of different groups using GraphPad program. p-Values less than 0.05 (p < 0.05) were considered significant. Symbols * mean p < 0.05, ** p < 0.01 and *** p < 0.001 for all graphs.

Conclusions
In this study the chemical profiles of the volatile fractions isolated from air-dried leaves of E. camaldulensis Dehnh. collected in Iraqi Kurdistan, were investigated for the first time. Essential oils EO S and EO W , respectively, were obtained by hydrodistillation with circulating water and with non-circulating-water, respectively. They were compared with an oil (EO D ) obtained by CO 2 extraction. The first two methods were much more efficient in terms of oil yield and number of oil components. A total of 31 and 42 components have been identified and quantified in EO S and EO W , respectively, by GC-FID and GC-MS, and comparison with literature databases. They accounted for more than 98% of the contents of the two oils. In both EO S and EO W , monoterpenoids prevailed over sesquiterpenoids, and oxygenated monoterpenes were more abundant than monoterpene hydrocarbons. The major components of EO S and EO W , accounting for more than 80%, were 1,8-cineole (1, about 60%), followed by p-cymene (2), α-pinene (3), terpinen-4-ol (4), aromadendrene (5), and α-terpineol (6).
In conclusion, the volatile fractions isolated from E. camaldulensis leaves collected in Kurdistan contained important and widely used flavor and fragrance ingredients. Most constituents, mainly 1,8-cineole (1) and other oxygenated monoterpenoids, are biologically active, exhibiting well-known antibacterial, bronchodilatory, anti-inflammatory, and analgesic effects [2,5,[26][27][28][29][30][31][32][33]37,38]. In this regard, we have shown that the relaxant activity of the volatile fractions does not depend only on the most abundant constituent, 1,8-cineole (1), but it is likely due to the synergistic effects of different monoterpenoids. An important pathway for the relaxation effects exerted by E. camaldulensis volatile fractions involves the inhibition of Ca 2+ influx through the plasma membrane in tracheal smooth muscle.
We are all well-aware that the chemical composition of vapors inhaled from a leaf hot tea can be different from that of a volatile fraction isolated by hydrodistillation, such as EO S , and should be analyzed by techniques such as solid phase microextraction (SPME)-GC-MS. However, it appears plausible that most of the bioactive components of the EOs also occur in the vapors, although the relative percentages can be different. Thus, based on our findings, we believe that inhalation of vapors from a hot aqueous infusion of E. camaldulensis leaves for treating the symptoms of respiratory tract diseases, is sustained by scientific evidence. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.