4.5.2. Arachidonic Acid (AA)-Induced Rat Ear Inflammation Model

The anti-inflammatory effects of the FS1, FS1a–FS1d were assessed using the AA-induced rat ear edema model as performed in previous studies [34,62] by analyzing histological scores and the cytokine expression by RT-qPCR. In summary, the study protocol using adult male Sprague Dawley^® rats was approved by the Ethics Committee of Animal Experimentation of the University of Barcelona (the guidelines for the experiments followed are stated in the protocol “Principles of Laboratory Animal Care” publication 214/97 of 30 July). At first, 5 mg of AA were dissolved in 1 mL of phosphate buffered saline solution. Then, 60 µL of AA solution was applied on both sides of all the ears of the animals (n = 5 for each treatment, 200–240 g), except the negative control group (Control -), to induce the inflammatory process with 20 min of exposure. The animals in the positive control (Control +) were treated only with AA solution. Moreover, the other groups were treated with 50 µL of the respective flavanone solution (FS1, FS1a, FS1b, FS1c, and FS1d) for 20 min. A solution of diclofenac sodium (5 mg/mL, 50 µL) in EtOH/H₂O (7:3) was used as a reference drug solution (RS). The ear thickness was verified with a digital micrometer (Wisamic Digital Thickness Gauge 0–12.7 mm) in basal state, then measured again after inducing inflammation with AA and, thirdly, after the different treatments with the FS. The edema reduction was calculated by the following Equation (2) [25]:

In the same way, the stratum corneum hydration (SCH, arbitrary units AU) of the rat ears was calculated by the difference between SCH value in basal state and the SCH value measured after the AA application and treatments. The measurement was performed with a corneometer CM825 (Courage & Khazaka electronics GmbH, Köln, Germany).

In addition, the left ears of the rats treated with FS1, FS1a–FS1d and the corresponding controls were cut off, rinsed with PBS pH 7.4 and set for 24 h in 4% buffered formaldehyde. Finally, the tissues were dehydrated and embedded in paraffin wax and the ear inflammation was then analyzed under microscope (BX41 microscope and XC50 camera, Olympus Hamburg, Germany) in 5 µm transversal sections stained with hematoxylin and eosin on blind-coded samples.