Carvacrol

Animal sensitisation and animal groupsAnimals were sensitized to OA according the method described previously (17 (link), 18 (link)). Briefly, guinea pigs were sensitized to 10 mg OA (Sigma Chemical Ltd, UK) and 100 mg Al (OH)₃ dissolved in 1 ml saline i.p. One week later they were given 2 mg OA and 100 mg Al (OH)₃ dissolved in 1 ml saline i.p. as a booster dose. From day 14 sensitized animals were exposed to an aerosol of 4% OA for 18±1 days, 5 min daily. The aerosol was administered in a closed chamber, dimensions 30 x 20 x 20 cm. Control animals were treated similarly but saline was used instead of OA solution. The study was approved by the ethical committee of the ashhad University of Medical Sciences.
The study was performed in control animals (group C, treated the same as sensitized group, but normal saline was used instead of OA and they were given pure drinking water) and five different groups of sensitized animals which were pure given drinking water (group S, an animal model of asthma) or drinking water containing treatment agents during sensitization period as follows (n=6 for each group);
1. 50 µg/ml dexamethasone (group S+D)
2. 40 µg/ml carvacrol (group S+C1)
3. 80 µg/ml carvacrol (group S+C2)
4. 160 µg/ml carvacrol (group S+C3)
Measurement of blood IL-4 and IFN- γlevelsFive ml of peripheral blood was obtained immediately after sacrificing the animals and placed at room temperature for 1 hr. The samples were then centrifuged at 2500 rpm at 4°C for 10 min. The supernatant was collected and immediately stored at 70° C until analyzed. Finally, blood IL-4 and IFN- γ levels were measured using Elisa sandwich (Ab Sandwich) method. The Elisa kits were purchased from Bender Medsystem Sakhte, Austria. The ratio of IFN-γ/IL4 as an index of Th1/Th2 was also calculated.
Measurement of blood endothelin levels Five ml peripheral blood was obtained immediately after sacrificing the animals and placed at room temperature for 1 hr. The samples were then centrifuged at 2500 rpm at 4°C for 10 min. The supernatant was collected and immediately stored at 70°C until analyzed. Blood endothelin was measured using the enzyme-linked immunosorbent assay (ELISA) Sandwich method according to the manufacturer’s instructions, (IBL’s ET-1 Assay Kit, Code No. 27165).
Statistical analysisThe data were quoted as mean±SEM. According to the Kolmogorov Smirnov test, the data had normal distribution. The data of sensitized group were compared with the control guinea pigs using unpaired t- test. The data of treated groups was also compared with sensitized guinea pigs using unpaired “t” test. The data of three groups of animals treated with carvacrol were compared with each other using one way ANOVA through Tukey Kermar post hoc test. Significance was accepted at P<0.05.

Nanocapsules containing carvacrol were produced with chia mucilage, according to the methodology described by Campo et al. [16 (link)]. The amount of each component to be added was determined in previous studies [31 (link)]. Briefly, an organic phase and an aqueous phase were prepared for encapsulation. The organic phase containing Tween 80 (13.5 mg), carvacrol (40 mg), and ethanol (4 mL) was maintained under magnetic stirring for 15 min. The aqueous phase was prepared with chia mucilage hydrated in distilled water (0.1% w/v), mixed with a magnetic stirrer for 2 h at room temperature (25 °C), and autoclaved (121 °C for 15 min). The organic phase was added dropwise to 20 mL of the aqueous phase during homogenization in Ultra-Turrax (digital model T25; IKA, Staufen, Germany), thus forming the nanoparticles (final carvacrol concentration of 1.67 mg/mL). Previous work conducted by our research group revealed that the diameter size of CMNC was approximately 179 nm, with a zeta potential of −11.4 mV and an encapsulation efficiency of 98.65% [31 (link)].
Additionally, a carvacrol emulsion, referred to as free carvacrol in this work, was prepared by vortex homogenizing 10 mL of sterile distilled water, 0.106 g of carvacrol, and 0.1 g of Tween 80 [29 (link)].

Carvacrol (5 g, 33 mmol), manganese sulphate (4.98 g, 33 mmol), and conc. hydrochloric acid (6.69 mL, 80 mmol) were added to water in a three-neck flask equipped with a reflux condenser(Merck, Darmstadt, Germany). The mixture was heated and stirred in an oil bath. Then, H₂O₂ (10.47 mL, 92 mmol of a 30% aqueous solution) was added dropwise during the reaction. After the reaction was complete, the mixture was allowed to stir for 1.5 h at room temperature, resulting in the formation of a distinct organic phase separated from the aqueous solution at the bottom. After that, the reaction mixture was extracted three times with diethyl ether. The combined organic extracts were dried over anhydrous MgSO₄ and concentrated in vacuo. The crude chlorination products were purified via gradient flash dry column chromatography (eluent: n-hexane–diethyl ether (v/v)) to give the desired compound 1.