2.7. Oral bioavailability study

Before the experiment, 12 SD rats were randomly divided into two groups of six animals each for pharmacokinetic analysis. Animals were orally administrated with ENR suspension (ENR powders suspended in 2% HPMC solution (w/v)) and ENR‐Cas with an equivalent dose of 20 mg/kg ENR, respectively. 0.5 ml of blood samples was withdrawn from the ocular veniplex and placed in heparinized tubes at 5, 15, 30 min, 1, 2, 4, 6, 8, 12, and 24 hr. The blood samples were subsequently centrifuged at 4,000 rpm for 10 min (Sorvall ST16, Thermo Fisher Scientific) to prepare the plasma.

To quantify the plasma ENR, a liquid–liquid extraction procedure was applied to retrieve ENR from the plasma. In brief, 200 μl of plasma was mingled with 1.2 ml of dichloromethane, 100 μl of ultrapure water, and 100 μl of 200 μg/ml ofloxacin solution as an internal standard. After vortex for 2 min, the mixtures were centrifuged at 8,000 g for 10 min. The bottom liquid was transferred to new tubes and the extraction was repeated once. All collected organic extracts were pooled and dried under a gentle stream of nitrogen at 40°C, and then, the residues were reconstituted in 100 μl of methanol. After vortex mixing and centrifugation at 15777 ×g for 10 min, the supernatant was collected and then injected into HPLC system as described above, and the separation was performed on the C₁₈ analytical column (4.6 × 250 mm, 5 μm) maintained at 30°C. Mobile phase consisted of 0.025 M phosphate acid (adjusted pH to 3.0 with triethylamine)/acetonitrile (17:83) with the flow rate of 1.0 ml/min (Kawas et al., ²⁰¹⁸).