3.7.5 Study on plasma kinetics of trans- and cis-crocetin in rats

The trans-crocetin and cis-crocetin were fully ground into powder, and an appropriate amount of sample was added to 0.5% CMC-Na solution for vortex mixing. According to the dose of 300 mg/kg (administration volume: 1 mL/100 g), the trans-crocetin and cis-crocetin suspensions were prepared.

Twelve healthy male Wistar rats weighing 200 ± 20 g were divided into two groups, with six rats in each group. Fasting but not water was prohibited within 12 h before administration. Before administration, 300 μL of blood was collected from the fundus venous plexus as blank plasma. The rats in the two groups were given trans-crocetin and cis-crocetin by intragastric administration at a dose of 300 mg/kg. Approximately 300 μL of blood was collected from the fundus venous plexus at 10, 15, 30, 60, 120, 240, 360, 480, 720, and 1,440 min after administration and placed in a centrifuge tube containing heparin sodium. The collected blood samples were centrifuged at 4,000 r/min for 15 min to separate the upper plasma and were stored at −80°C.

In total, 80 μL of rat blank plasma was pipetted, 240 μL of methanol was added, and then 30 μL of internal standard solution was added, followed by vortex mixing for 3 min and centrifugation at 4°C for 15 min at a low temperature at a speed of 12000 r/min. The supernatant after centrifugation was aspirated in EP tubes, in 37°C, vacuum centrifugal concentrator to evaporate the solvent, and then 80 μL of methanol was solubilized, followed by sonication for 5 min and vortex mixing for 3 min. The supernatant was centrifuged at 12,000 r/min at 4°C for 10 min, and then the sample was injected into the sample for detection according to the chromatographic conditions under 2.7.2.

Pharmacokinetic software DAS 2.0 was used to calculate the relevant pharmacokinetic parameters; GraphPad 8.0.1 and Origin Graph 8.0 software were used to process and analyze the data and to plot the drug–time curves of trans- and cis-crocetin; SPSS 22 statistical software was used to calculate whether the results were statistically different.