Reagents and animals
SGFD is composed of Paeoniae Radix Alba (Paeonia lactiflora Pall.), Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle (Glycyrrhiza uralensis Fisch.), and Aconiti Lateralis Radix Praeparata (Aconitum carmichaelii Debx.). All herbs were purchased from Nanjing Pharmaceutical Baixin Pharmacy (Nanjing, China), which were authenticated by Prof. Yufeng Xia, Department of Pharmacognosy, School of Traditional Chinese Pharmacy, China Pharmaceutical University. Voucher specimens of these herbs were stored in the Department of Pharmacognosy Laboratory of China Pharmaceutical University, labeled as 20170801 (Paeoniae Radix Alba, Baishao), 20170901 (Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle, Zhigancao), and 20170816 (Aconiti Lateralis Radix Praeparata, Fuzi).
Caffeine (purity > 98%) was obtained from Push Bio-technology Co., Ltd. (Chengdu, China). Antipyrine (IS, purity > 98%) was purchased from National Institutes for Food and Drug Control (Beijing, China). Dapsone and chlorzoxazone (purity > 98%) were obtained from Shanghai Guangrui Biological Technology Co., Ltd. (Shanghai, China). Coumarin (purity ≥ 98%) was purchased from Nanjing Jingzhu Bio-technology Co., Ltd. (Nanjing, China). Heparin sodium salt (194 USP units/mg) was purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany), and HPLC-grade formic acid was purchased from Shanghai Linen Science and Technology Development Co., Ltd. (Shanghai, China). Ultrapure water was prepared from a Milli-Q water purification system (Millipore, Milford, MA, USA). All other reagents were of analytical grade.
Male Sprague-Dawley rats (180–220 g) were supplied by Nanjing Qinglongshan Laboratory Animal Breeding Center (Nanjing, China). The animal study was performed according to the guideline of current ethical regulations for institutional animal care and use at China Pharmaceutical University. All animals were housed at controlled temperature 22 ± 2 °C, relative humidity 55 ± 10% in an SPF environment. The animals were provided tap water ad libitum and fed with standard rodent chow for five days. Before being orally administrated with SGFD extract, the rats were fastened with access to water for 12 hours.
Preparation of SGFD extracts
To prepare of the SGFD extract, 225 g Baishao, 225 g Zhigancao and 75 g Fuzi were mixed together and then macerated in 10-fold volume of double-distilled water for 30 min, and after boiling with high heat, keep slight boiling with gentle fire for 1 h followed by filtering with absorbent cotton. The decoction step was repeated two times. The filtrates were combined and concentrated under reduced pressure. The powder of SGFD (125 g) was obtained by freeze-drying machine and stored at -20 ℃ for further use. The assay of contents was calculated by the external standard method according to the LC-MS/MS method established by our laboratory. The average contents of the main components in SGFD were 29.06 mg/g for peoniflorin, 9.16 mg/g for liquiritin, 0.31 mg/g for benzoylmesaconine, 0.0029 mg/g for mesaconitine, and 16.99 mg/g for glycyrrhizic acid, respectively.
Effects of SGFD on activities of CYP 450 s
Ten rats were randomly divided into two groups (SGFD-treated and control), and administered SGFD (10 g/kg) or physiological saline for consecutive fourteen days. On day fifteen, all rats were orally administrated cocktail drugs containing caffeine (10 mg/kg), dapsone (20 mg/kg), coumarin (30 mg/kg), chlorzoxazone (20 mg/kg) and tolbutamide (5 mg/kg) and dissolved in an aqueous solution of 0.5% sodium carboxymethyl cellulose as probe substrates for CYP1A2, 3A4, 2A6, 2E1, and 2C9, respectively. Food intake was restricted 12 h before cocktail drugs administration. The blood samples were collected into heparinized polythene tubes at 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after administration of probe drugs. The plasma samples for chromatographic analysis were obtained by centrifugation (8000 r/min for 10 min at 4 ℃) of the blood samples and stored at -20 ℃ until analysis.
Plasma samples were thawed at room temperature, 100 µL of the plasma was mixed with 10 µL internal standard (antipyrine, 50 µg/mL) by vortexing for 30 s, extracted with 1.5 mL trichloromethane, the mixture was vortexed for 5 min and centrifuged at 12000 r/min for 10 min, the organic phase (1.4 mL) was transferred into another tube and evaporated to dryness in vacuum concentrator at 37 ℃. The residue was reconstituted with 50 µL methanol-water (50:50, v/v), vortex for 5 min and centrifugation at 12000 r/min for 10 min, and then the supernatant was used for chromatographic analysis.
The chromatographic separation was performed on a C18 column (4.6 mm × 150 mm, 5 µm, Dubhe). The flow rate was 1 mL/min and the injection volume was 20 µL. The mobile phase consisted of water (containing 0.1% phosphate acid, A) and acetonitrile (B). The gradient was set as follows: 0–12 min, a linear gradient of 5–22% B; 12–15 min, a linear gradient to 33% B; 15–30 min, a linear gradient to 45% B. The detect wavelength was set as follows: 0–22 min, 280 nm; 22–30 min, 220 nm. The column compartment was set at 40 ℃ and the automatic sample was held at room temperature.
Stock standards of caffeine, dapsone, coumarin, chlorzoxazone, tolbutamide and antipyrine (IS) were precisely weight and separately dissolved in methanol to achieve a concentration of 2.100 mg/mL, 2.010 mg/mL, 2.040 mg/mL, 5.100 mg/mL, 10.100 mg/mL and 2.000 mg/mL, respectively. Standard solutions were precisely in blank rat plasma to obtain final concentrations of 21, 105, 210, 1050, 2100, 10500 and 21000 ng/mL for caffeine, 20.1, 100.5, 201, 1005, 2010, 10050 and 20100 ng/mL for dapsone, 51, 102, 201, 1020, 2040, 10200 and 20400 ng/mL for coumarin, 102, 510, 1020, 5100, 10200, 20400 and 51000 ng/mL for chlorzoxazone, 101, 505, 1010, 5050, 10100, 50500, 101000 ng/mL for tolbutamide. The QC samples were prepared in blank plasma at final concentrations of 21, 1050, 21000 ng/mL for caffeine, 20.1, 1005, 20100 ng/mL for dapsone, 51, 1020, 20400 ng/mL for coumarin, 102, 5100, 51000 ng/mL for chlorzoxazone, 101, 5050, 101000 ng/mL for tolbutamide.
Methods were validated for specificity, linearity, accuracy, precision, extract recovery and stability, according to USA FDA guidelines [16].
Effects of SGFD on the pharmacokinetics of tofacitinib
For orally administration solution, tofacitinib was dissolved in an aqueous solution of 0.5% sodium carboxymethyl cellulose. Twelve rats were randomly divided into two groups, and were orally administrated SGFD (10 g/kg) or physiological saline fourteen days before given tofacitinib. On day fifteen, both groups were orally administrated tofacitinib (50 mg/kg). Food intake was restricted 12 h before the tofacitinib administration. The plasma concentrations were collected at prior to dosing and 0.083, 0.167, 0.25, 0.75, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after dosing. The plasma was separated from the blood by centrifugation (8000 r/min for 6 min at 4 ℃) and stored at -20 ℃ until analysis.
Plasma (100 µL) was mixed with 10 µL strychnine (200 µg/mL, IS). The mixture was vortexed for 30 s and added 1 mL ethyl acetate, and then was vortexed 5 min and centrifuged at 12000 r/min for 10 min. The supernatant (900 µL) was transferred into another tube and evaporated to dryness in vacuum concentrator at 37 ℃. The residue was reconstituted with 100 µL acetonitrile-water (25:75, v/v), vortex for 2 min and centrifugation at 12000 r/min for 10 min, and then the supernatant was used for chromatographic analysis.
The chromatographic separation was performed on a C18 column (4.6 mm × 150 mm, 5 µm, Dubhe). The flow rate was 1 mL/min and the injection volume was 20 µL. The mobile phase consisted of water (containing 0.1% phosphate acid, A) and acetonitrile (B). The gradient was set as follows: 0–1 min, a linear gradient of 10–13% B; 1–4 min, a linear gradient to 25% B; 4–10 min, an isocratic gradient at 25% B. UV detection was at wavelength of 287 nm, the column compartment was set at 40 ℃ and the automatic sample was held at room temperature.
To prepare the samples of the calibration curve, the blank plasma (90 µL) added various concentrations of tofacitinib to obtain final concentrations of 54.5, 109, 218, 545, 1090, 2180, 5450, 10900 and 21800 ng/mL. The QC samples were prepared in blank plasma at final concentrations of 54.5, 1090 and 21800 ng/mL for tofacitinib.
Methods were validated for specificity, linearity, accuracy, precision, extract recovery and stability, according to USA FDA guidelines [16].
Data analysis
Pharmacokinetic parameters were determined by non-compartmental modeling with the PK Slover software. The PK parameters including maximum plasma concentration (Cmax), the time to reach Cmax (Tmax), area under the curve (AUC), half-time t1/2), oral clearance per unit time (CL/F) and mean residence time (MRT). The differences were considered to be significant at p < 0.05 or extremely significant at p < 0.01 as determined by independent-sample t-tests.