*2.8. In Vivo Studies*

2.8.1. Pharmacokinetic Study

### Study Design

The study was conducted on female Wistar rats (200 ± 20 g) that were kept under standard conditions of light, temperature, and humidity, with free access to food and water during the study.

Rats were randomly divided into 6 groups (each of 6 animals). Animals were fasted overnight prior to the experiment. A single dose of FS suspension, FS-NS, and LF-FS-NS equivalent to 30 mg FS/kg was administered either orally by gastric gavage or by IP injection. FS suspension was prepared in 0.5% *w*/*v* carboxymethyl cellulose sodium for oral administration and in 10% PEG 400 saline solution for IP injection. Blood samples were collected via the orbital plexus under anesthesia at predetermined time points (0.25, 0.5, 1, 3, 5, 7, and 24 h) in EDTA-containing tubes. Blood samples were centrifuged at 4000 rpm for 10 min. Plasma samples were frozen and maintained at −80 ◦C pending analysis.

### Quantification of FS in Plasma Samples

FS in plasma samples was analyzed using a reported HPLC-UV method for the quantification of FS in plasma samples, with slight modification [39]. Plasma samples (300 μL) were vortex mixed with 40 μL quercetin (1 μg/mL) as an internal standard and an equal volume of methanol acidified with 0.5% *v*/*v* formic acid for plasma protein precipitation for 1 min, and then centrifuged at 10,000 rpm for 10 min. The obtained supernatants were filtered through 0.22 μm syringe PTFE filters. The HPLC instrument (Agilent 1260 infinity, Germany) equipped with a quaternary pump (G1311C) and a UV variable wavelength detector (G1314F) was used. Samples (100 μL) were manually injected in a reversed-phase C18 column (Agilent HC-C18(2), 4.6 × 150 mm, 5 μm). The mobile phase was a mixture of acetonitrile and water acidified with 0.5% *v*/*v* formic acid in the ratio of 40:60. The flow rate was maintained at 1 mL/min, and detection was performed at 360 nm. Quantification was achieved using a calibration curve for peak area ratios of FS/quercetin in spiked plasma obtained under the same conditions.

## Pharmacokinetic Data Analysis

The plasma concentrations vs. time data were analyzed by a non-compartmental pharmacokinetic model using the Excel pharmacokinetic solver add-in [40], and pharmacokinetic parameters were calculated. Results were expressed as mean ± standard deviation (SD) (n = 6).
