2.3.1. Antitussive Test

We selected 24 rats with body weight of 227.75 ± 14.60 g (mean ± SD) which were randomly divided into four groups (n = 6). The animals were treated for three consecutive days by gavage: group 1 (CN)—distilled water (negative control); group 2 (Cd)—codeine phosphate, 3 mg/kg b.w./day (positive control); group 3 (WSE)—10 mL WSE (containing 134 mg GAE)/kg b.w./day; group 4 (WSE 1:2)—5 mL WSE (containing 67 mg GAE)/kg b.w./day). One hour after the last oral treatment, each rat was placed in an air-tight transparent exposure chamber and exposed to citric acid aerosols (17.5%) using an ultrasonic nebulizer (Laica MD6026, Laica Spa, Italy). The antitussive effect of WSE in rats was assessed by the number and frequency of coughs caused by citric acid aerosols, cough latency after exposure being also evaluated.

The exposure period to citric acid was 4 min, followed by a further observation period of 4 min. The total number of coughs was determined over a total period of 8 min. Individual coughs were recorded with a video data acquisition platform and detected by trained staff. Each of the animals was exposed to citric acid aerosols only once.

The antitussive effect was expressed as the percentage of inhibition of number of coughs and was calculated using the equation:

where C₀ was the number of coughs in the negative control group and Ct was the number of coughs in the treatment group [20].