2.5. In Vivo Study

The in vivo study has been carried out following the “Guide for the care and use of laboratory animals” of the National Institute of Health (USA), and the protocols have been approved by the Committee of Ethics of the University of Granada (Reference No. 28/03/2016/030). Adult female Sprague Dawley rats (240 g), provided by Charles River Laboratories (Les Oncins, France), were maintained individually in metabolic cages and in an air-conditioned atmosphere with a 12 h light–dark cycle and provided with food and tap water ad libitum. Rats were randomly assigned to different experimental groups: a control group (n = 10) and a treated group (n = 25), which received by oral gavage a single dose of PTSO (55 mg kg⁻¹), suspended in sterile water. A single dose of 55 mg kg⁻¹ was selected according to the previously described NOAEL value [8]. The control group was administered with the vehicle. Rats from the treated (n = 5) and control groups (n = 2) were anaesthetized with isoflurane; the blood was collected by cardiac puncture, and then the rats were sacrificed to obtain liver and faeces. This was performed at different times from the PTSO administration: (30 min, 2 h, 5 h, 24 h, and 48 h). For plasma collection, the blood was centrifuged at 3000× g for 10 min at 4 °C. All samples were stored at −20 °C until use. One day prior to the administration of PTSO and 24 and 48 h after, the urine was collected from animals from the different experimental groups.