2.1 Chemicals
The chemicals used in the study were of analytical grade and in good quality. Carrageenan, NaCl, NH4OH, and phenol red were procured from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Ibuprofene syrup (Laboratory Roemmers S.A.I.C.F, Argentina), codeine syrup (Laboratory Andromaco, Argentina), meprednisone oral solution (Laboratory Biotenk S.A., Argentina), and bromhexine hydrochloride syrup (Laboratory Boehringer Ingelheim, Germany) were obtained from the local pharmacy.
2.2 Geopropolis sample
In this work, we used propolis of S. jujuyensis and T. fiebrigi. The hives are located in the Famaillá Agricultural Experiment Station of INTA, in the province of Tucumán, Argentina. Both species of bees were identified and deposited in the Museo de La Plata, La Plata, Argentina (MLP; Lanteri,A.). Codes of material studied: S. jujuyensis (Schrottky) 30-III-2012, Alvarez-Lucia-Gennari (MLP) and T. fiebrigi (Schwarz), 26III-2012, Alvarez-Lucia (MLP).
2.3 Preparation of extracts
The extraction of the propolis of both species was performed by maceration, using ethanol as the extraction solvent, for 5 days in the dark. The extracts were filtered through Whatman paper N° 1, and the supernatant was evaporated to dryness. The dry extract was stored in sterile Eppendorf at 4 °C until used. In this way, the ethanol extracts of S. jujuyensis (EPS) and T. fiebrigi (EPT) propolis were obtained.
2.4 Animals
Wistar male rats (weighing 190–240 g) were used for this study and were obtained from the Bioterio de la Facultad de Bioquímica, Química y Farmacia, Instituto de Biología (INSIBIO), Universidad Nacional de Tucumán. The rats were first left for 7 days to acclimatize to laboratory conditions. All animals were kept under normal laboratory conditions of humidity, temperature (25 ± 1 °C), and light (12hs dark/light cycle) and allowed free access to food and water ad libitum. The studies were conducted in accordance with the internationally accepted principles for laboratory animal use and care (EEC Directive of 1986; 86/609/EEC). The study protocol for antitussive, expectorant, anti-inflammatory, and acute toxicity evaluation of test propolis was approved by the Institutional Committee for the Care and Use of Laboratory Animals CICUAL), approval number: No. CICUAL 012/2018, dated July 14, 2019.
2.5 Carrageenan-induced hind paw edema in rats
Paw edema was induced in rats by carrageenan injection 0.1 mL of 1.5 % (w/v) into the sub plantar region of the right hind paw of the rats according to the method described by Winter et al. [19]. All rats (six per group) were given free access to food and water after the sub plantar injections. Control group rats received saline solution [0.9 % (w/v) NaCl] (2 mL/kg), and the reference group received 100 mg/kg ibuprofen, orally. The test groups of rats were treated orally with 250, 500, and 1000 mg/kg b.w. of the ethanol extracts of S. jujuyensis and T. fiebrigi propolis 30 min before the carrageenan injection. The paw volume was measured before administering carrageenan (Vo) and 1, 2, 3, 4, and 6 h after (Vt) with the help of digital vernier caliper (Wembley 5940). Inflammation was calculated as the increase in volume (mL) of the paw after treatment subtracted of the basal volume. Results were expressed as percentage of inhibition of edema, calculated according to the following formula [(Vt−Vo)/Vo] × 100 [20].
2.6 Cotton pellet-induced granuloma formation
Male rats weighing 180–200 g were randomly divided into seven groups of six rats each. Two sterilized cotton pellets (20 ± 1 mg) were implanted subcutaneously, one on each side of the abdomen in all groups, under light ether anesthesia. Rats in group I (control group) received saline solution [0.9% (w/v) NaCl] (2 mL/kg), orally. Rats in groups II and III received ibuprofen and meprednisone, at the dose of 100 and 5 mg/kg b.w./day, respectively. Rats in groups IV to VII received ethanol extracts of S. jujuyensis and T. fiebrigi propolis at the dose of 500 and 1000 mg/kg b.w./day, respectively. Each test substance was administered for 7 days. On the eighth day, each rat was anesthetized. The rat was then sacrificed, and the implanted pellets as well as the thymus were dissected out and determined for their wet and dry weights (dried at 60 ± 1 °C for 18 h). The granuloma and transudative weights and the percent inhibition of granuloma formulation of the test compounds were calculated [21].
2.7 Antitussive effects
Male rats weighing 210–240 g were divided randomly, 6 rats per group. The negative control of animals was treated with saline solution [0.9% (w/v) NaCl] (2 mL/kg) orally, and other groups received single daily dose of extracts (125, 250, 500, and 1000 mg/kg b.w.) and codeine phosphate syrup (3 mg/kg b.w.) orally for 3 days, respectively. Antitussive activity was investigated on a classical cough model induced by ammonia liquor [22, 23], 30 min after oral administration of the test compounds, and each rat was placed in a 1000-mL special glass chamber and exposed to 0.3 mL 25% NH4OH produced by a nebulizer for 45 s. During ammonia exposure, the animal was continuously monitored by a trained observer. The cough frequency and latent period of cough were recorded for 6 min. The antitussive activity was assessed as the percentage of inhibition of the number of coughs in terms of that in control groups by using the following equation:
% inhibition = [(C0 − Ct)/C0 × 100%], C0: the number of coughs of control, Ct: the number of coughs of the treatment groups.
2.8 Expectorant activity of extracts
Rats (210–240 g) were divided into 6 groups (n = 6). The control group received saline solution [0.9% (w/v) NaCl] (2 mL/kg), and other groups received single daily dose of extracts (125, 250, 500, and 1000 mg/kg b.w.) and bromhexine syrup (12 mg/kg b.w.) oral for 3 days, respectively. One hour after the last drug administration, 5% of phenol red in saline solution (500 mg/kg b.w.) is injected via intraperitoneal. After 30 min, the rats were killed. The trachea was dissected free from adjacent organs and removed from the thyroid cartilage to the main stem bronchi and then put into 4.0 mL of saline solution, and 1 mL of this wash solution was measured and mixed with 0.5 mL NaOH (1 mol/L). The optical density (OD) values were measured on a spectrophotometer with the wavelength of 546 nm. The excretion of phenol red was determined according to the standard curve [23].
2.9 Acute toxicity study in rats
The animals were divided into three groups, with six animals each. They were treated orally with a single dose of the ESP and ETP dissolved in distilled water and at supra-therapeutic doses of 2000 and 5000 mg/kg in 10 mL/kg volume. The control group received distilled water as a single dose, orally, in 10 mL/kg volume. All animals were observed after treatment. The parameters evaluated were death, alertness, sedation, ptosis, dyspnea, urination, diarrhea, seizures, spontaneous motor activity, postural reflex, piloerection, and response to touch. Body weight, food, and water consumption were also monitored for 2 weeks. At the end of the experimental period, all animals were weighed and sacrificed, and organs were removed for necropsy [24, 25].
2.10 Euthanasia and Anesthesia
At the end of the evaluations, the death of the animals is induced humanely. Euthanasia is performed using (a) injection of chemical anesthetics (e.g., pentobarbital 120–210 mg/kg) or (b) inhalant anesthetics, e.g., CO2 or isoflurane from a vaporizer.
2.11 Statistical analysis
Data obtained from animal experiments were expressed as the mean and standard error of the mean (mean ± S.E.M.). Statistical differences between the treated and the control groups were evaluated by ANOVA and Dunnett’s tests. The criterion for statistical significance was p < 0.05.
