2.6. Pharmacokinetic study in rats

The pharmacokinetic study was investigated in Sprague Dawley rats (300–320 g) purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China) and the approval document is SCXK/2018‐0002. The rates were kept for 7 days in the environmentally controlled room (12/12 hr dark/light cycle, 25 ± 2°C, 50% humidity) before the experiments study for acclimatization to animal house conditions. All rats were given free access to diet and water. All animal experimental protocol used in this study was approved by the Guangdong Medical Laboratory Animal Center and performed in compliance with the relevant laws and institutional guidelines.

The rats were randomly divided into six groups (n = 5) and fasted for 12 hr before drug administration. A single dose of 400 mg/kg Ph solution, SCT‐Ph‐SNE, MCT‐Ph‐SNE, and LCT‐Ph‐SNE dissolved in 5% PEG400 solution was oral administration, respectively. The 0.4 ml of blood was taken from the retro‐orbital plexus under isoflurane anesthesia and then collected at predetermined intervals (5 min predose, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 12 hr postdose) after oral administration. The blood samples were centrifuged at 5,000 rpm for 10 min to separate plasma. The plasma samples were stored at −20°C until analyzed. After treatment, the Ph concentration of the plasma sample was measured by HPLC.

Before analyzing, plasma was thawed at 37°C. 0.2 ml sample was added to 0.2 ml acetonitrile and vortexed for 1 min. The mixture was centrifuged at 12,000 rpm for 5 min, and the upper phase was collected. Then, 20 μl sample was injected into HPLC for analysis on Ph concentration of plasma.

All HPLC analyses were performed on a Shimadzu LC‐20A HPLC with DIKMA C18 Reversed‐phase column (4.6 mm × 200 mm, 5 μm). The column temperature was maintained at 30°C, and the mobile phase was 30% acetonitrile in water with a delivery flow rate of 1 ml/min. PDA detector (detection wavelength 286 nm) was used. The pharmacokinetic parameters were calculated by the pharmacokinetic software WinNonlin Standard Edition v1.1 (Pharsight Corp., Mountain View, CA, USA). The Ph, Blank plasma + Ph, and plasma sample at 0.083h in rats were analyzed with HPLC based on the above chromatographic conditions. As shown in the Figure S1, the retention time of Ph in HPLC is approximately 14.2 min with a good separation to plasma constituents. The regression equation for Ph was y = 14885x + 2,780.3 (r = 0.9994), which was a good linear relationship between drug concentration and peak area. The precision was measured at six different concentrations (0.5, 1.0, 2.0, 5.0, 10.0, 20.0 μg·ml‐1). The percent relative standard deviation(RSD%) value is less than 2%.The mean extraction recovery was 82.02 ± 1.2%, 84.67 ± 1.5% and 81.51 ± 0.85% at 0.5, 10.0, 20.0 μg·ml‐1, respectively. The limit of quantization (S/N = 10) was estimated to be 0.3 μg/ml, and the limit of detection (S/N = 3) of phloretin was estimated to be 0.1 μg/ml in plasma. It indicated good accuracy and precision of the developed method.