Winnonlin software version 5

All data were presented as mean ± SEM. Statistical analysis between different groups was performed using two-tailed t-test and p-values less than 0.05 were considered to indicate statistical significance. The enzyme kinetic data of nifedipine and midazolam metabolism in RLM was fitted according to the typical Michaelis-Menten equation with GraphPad Prism 5.0 (GraphPad Software, CA, USA). Pharmacokinetic parameters were calculated by WinNonlin software version 5.2.1 (Pharsight Corporation, Mountain View, USA) based on non-compartmental analysis.

Eighteen rats with liver fibrosis (in the disease group) were selected for pharmacokinetics studies and randomly divided into three groups (n = 6): (1) Free-Cur (Cur dissolved in 20% Tween 80 at a concentration of 2 mg/mL); (2) Cur-NLCs; (3) Cur-mNLCs, respectively. All the solutions were administered by intraperitoneal injection at Cur dose of 15 mg/kg. The blood samples (1 mL) were collected from orbit at different time points, followed by centrifugation at 3000 rpm for 10 min. The supernatants or plasma samples were stored at −20 °C until they were analyzed.
Rat plasma (200 µL) were mixed with 2 µL of emodin (an internal standard), followed by extraction with 400 µL of methanol, vortexed for 1 min and centrifuged at 13,000 rpm for 10 min. The upper organic layer was transferred to a 1.5 mL tube and dried under nitrogen. The residuals were re-dissolved in 200 µL of methanol, vortexed for 3 min, and centrifuged at 13,000 rpm for 10 min again. The final supernatants (5 µL) were used for Cur content analysis by using LC-MS/MS as described below. The pharmacokinetics parameters were calculated by using a non-compartmental model (WinNonlin software version 5.2.1, Pharsight Corporation, Mountain View, CA).