3.9. In Vivo Pharmacokinetic Study

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Hallym University (Permit number: Hallym2020-48) and performed in accordance with their guidelines as well as ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) (https://www.nc3rs.org.uk/arriveguidelines; accessed on 15 February 2021). Eight-week-old Sprague–Dawley (SD) female rats, weighing about 230–250 g, were purchased from DBL Ltd. (Eumseong, Korea) and used for the pharmacokinetic study. All experimental rats were housed in individual cages at the Hallym University Laboratory Animal Resources Center under specific pathogen-free (SPF) conditions with a controlled consistent temperature (23 ± 2 °C) and lighting environment (12 h/12 h light/dark cycle). At the end of the study, the experimental mice were sacrificed by CO₂ inhalation. A gradual fill rate of 20% chamber volume per minute of displacement was used for CO₂ euthanasia. All efforts were made to minimize the number and suffering of any animals used in these experiments. Animals were randomly divided into two groups after an acclimation period of 1 week. SFN dissolved in water was administered as a single oral dose of either 10 mg/kg or 20 mg/kg. Blood samples (300 μL) were collected in lithium-heparinized tubes from the tail vein before dosing and subsequently at 0.25, 0.5, 1, 2, 3, 6, 8, and 12 h after administration. Blood samples were then centrifuged at 10,000× g for 3 min at 4 °C to separate plasma. The SFN pharmacokinetic parameters processed by non-compartmental analysis of plasma concentration versus time data using the computer program Winnonlin Ver. 5.1 (Pharsight Corporation, Mountain View, CA, USA) were as follows: the area under the plasma concentration–time curve to the last measurable plasma concentration (AUC_0–t); the area under the plasma concentration–time curve to time infinity (AUC_0–∞); the maximum plasma concentration (C_max); the time to reach the maximum concentrations (T_max); the elimination half-life (t_1/2); the mean residence time (MRT); and total plasma clearance (CL). Both C_max and T_max were obtained directly from the generated curve.