Chemicals and materials

Liquiritin apioside (purity: 96.0%) was purchased from Shanghai Standard Technology Co., Ltd. (Shanghai, China). Timosaponin AIII (purity: 98.7%), ononin (purity: 98.5%) and glycyrrhizic acid (purity: 99.6%) were obtained from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Isoliquiritin (purity: 99.0%) was acquired from Chengdu DeSiTe Biotech Co., Ltd. (Chengdu, China). Mangiferin (purity: 98.0%), arginine (purity: 99.9%), citric acid (purity: 97.0%), isoleucine (purity: 99.9%), formononetin (purity: 98.0%), phenylalanine (purity: 99.9%) and cinnamic acid (purity: 98.8%) were provided by the National Institutes for the Control of Pharmaceutical and Biological Products (Beijing, China). Stable isotope-labeled [2',3',5',6'-D4]-naringin (D4-naringin, purity: 98.0%) was synthesized by Artis-chem Co., Ltd (Shanghai, China) as internal standards (IS).

MS grade methanol and acetonitrile were provided by Fisher Scientific Inc. (Fair Lawn, USA). Formic acid of MS grade was supplied by Sigma-Aldrich Co. (St. Louis, USA). Purified water was prepared by the Milli-Q system (Billerica, USA; electrical resistivity: 18.2 MΩ·cm) and then filtered through 0.22 μm microfiltration membrane before use. BHGZ decoction was provided by Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (Beijing, China).

Preparation of Baihu-guizhi decoction (BHGZD) powder

Neolitsea cassia (L.) Kosterm. (Guizhi), Glycyrrhiza uralensis Fisch. (Gancao), Anemarrhena asphodeloides Bunge (Zhimu), Oryza sativa L. (Jingmi) and Gypsum Fibrosum (Shigao) were authenticated and purchased from Beijing Tongrentang Co., Ltd (Beijing, China).

To prepare Baihu-Guizhi Decoction, Gypsum Fibrosum (60 g) was firstly extracted in 1,000 ml boiling water. After 30 min extraction, Glycyrrhiza uralensis Fisch. (5 g), Neolitsea cassia (L.) Kosterm. (10 g), Anemarrhena asphodeloides Bunge (15 g), Oryza sativa L. (30 g) was added to the aqueous for continuous extracted in boiling water for 30 min. Collected the first aqueous extract. Conducted the second extraction by immersing the residue in 800 ml boiling water for another 30 min and collected the second aqueous extract. Combined the extracts and concentrated to 300 ml volume. The BHGZD powder was obtained by drying out the concentrate in 70 ℃ oven overnight. Collected and weighed the powder. The final yield was 12 g crude materials per 1 g of BHGZD powder.

Animals

Male Lewis rats, weighing 200–240 g, were purchased from Beijing Charles River Experimental Animals Co. Ltd. (Beijing, China). Animals were maintained under specific pathogen-free environment with constant temperature and humidity (22 ± 2 ℃ with 55 ± 15% relative humidity; 12 h light/dark cycle). Animal experiments were approved and supervised by the National Institutes of Health guide for the care and use of Laboratory animals. Every effort was made to minimize both the employed animal numbers as well as the potential discomfort of the animals throughout the study.

Collection of rat serum

After 12 h food restriction, rats were subjected to single or multiple gavages of BHGZD. For single administration, rats were orally administrated with BHGZD powder at dose of 21.4 g/kg, which was equivalent to two-fold of clinical dose in human [7]. 200 μL venous blood was collected at 0 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h post gavage. For multiple administration, rats were intragastrically administered BHGZD (21.4 g/kg/day) once a day, for continuous 12 days. On day 12, 200μL venous blood was collected at 0 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h post gavage. Serum was freshly prepared by centrifugation and stored at – 80 ℃ till analysis.

Preparation of stock solutions, calibration samples and quality control samples

To prepare stock solution, 10 mg liquiritin apioside, timosaponin AIII, glycyrrhizic acid, mangiferin, cinnamic acid and D4-naringin (IS) were accurately weighed, dissolved in methanol to a final concentration of 1.0 mg/mL. Working solutions of each reference standard were further prepared by a series of equimultiple dilutions of stock solutions, respectively.

To conduct calibration curve, a series of calibration solutions were prepared by mixing 10μL working solution at indicated concentrations with 10μL IS and 80μL blank rat serum to achieve desired concentration range: glycyrrhizic acid and cinnamic acid at 5, 25, 125, 250, 500 and 1000 ng/mL; mangiferin and liquiritin apioside at 1, 5, 25, 50, 100 and 200 ng/mL; timosaponin AIII at 0.5, 2.5, 12.5, 25, 50 and 100 ng/mL.

For method validation, QC samples were prepared at a high, medium, and low concentration with blank rat serum, with the desired concentrations of glycyrrhizic acid and cinnamic acid at 15, 150 and 750 ng/mL; mangiferin and liquiritin apioside at 3, 30 and 150 ng/mL; timosaponin AIII at 1.5, 15 and 75 ng/mL.

Metabolic profiling of serum samples

To reveal the metabolic profiling of BHGZD in rat serum, a connected system of UFLC XR-hybrid triple quadruple time-of-flight mass spectrometer equipped with electrospray ionization source (ESI) was employed. Briefly, 100 μL serum was mixed with pre-cooled acetonitrile (containing 50 ng/mL IS) and vortexed vigorously for 2 min for protein precipitation. After centrifugation at 15,000g for 20 min, the supernatants were transferred and subjected to UFLC-Q-TOF-MS/MS for analysis.

Chromatographic separation was carried on a Kinetex C18 column (150 × 3.0 mm, 2.6 μm, Phenomenex, USA) using a Shimadzu UFLC XR system (Shimadzu, Japan), and mass detection on a 5600 plus quadrupole time-of-flight mass system (Triple TOF™ 5600 plus; AB Sciex, USA). The mobile phase was consisted of 0.1% aqueous formic acid (v/v; A) and methanol (B). The flow rate was 0.3 mL/min with the optimized gradient elution condition: 5%–95% B (0 − 40 min), maintained at 95% B for 5 min, 95%-5% B (45 − 50 min), followed by 2 min system equilibration. The flow rate was kept in 0.3 ml/min. The feature parameters of the mass spectrometer were described in our previously study [8]. Data analysis was processed using PeakView, Natural Products HR-MS/MS Spectral Library and MetabolitePilot software (all from AB Sciex, Forster City, USA).

Quantification of absorbed compounds

Quantification for five absorbed compounds of BHGZD in rat serum were achieved by using a Shimadzu ultra-high-performance liquid chromatography tandem ion trap quadrupole Q-TRAP 6500 plus mass spectrometry (UHPLC-Q-TRAP-MS/MS). Sample preparations were described above.

Chromatographic separation was carried out on a Poroshell 120 EC-C18 column (50 × 3.0 mm, 2.7 μm, Agilent, USA) with a flow rate 0.3 mL/min. The mobile phases were 0.1% formic acid–water (v/v, A) and methanol (B) and corresponding gradient elution condition: 20%—40% B (0 – 0.5 min), maintained at 40% B for 0.5 min, 40%—90% B (1.0 – 3.5 min), maintained at 90% B for 1.5 min, 90%—20% (5.0 – 5.3 min) and 20% (5.3 – 8.0 min) for UHPLC system equilibration.

The optimized parameters for the mass spectrometer were set as follows: curtain gas, 35 psi; collision gas, medium; ionspray voltage, 4500 V; temperature, 550 °C; ion source gas 1 and 2, 55 psi. With the multiple reaction monitoring (MRM) detection, the information of five analytes and IS were optimized as shown in Table 1, including quantitative ion pairs, declustering potential and collision energy. Data acquisition and analysis were achieved by OS-Q software (version 1.4.1.20719, AB Sciex, USA). Pharmacokinetic analysis was conducted by Drug and Statistic software (version 3.0, Shanghai, China).

Table 1 The optimal MRM quantification parameters of five analytes and ISQuantification of calcium in Gypsum Fibrosum and BHGZD

Quantification for calcium were carried out by inductively coupled plasma atomic emission spectrometry (Optima 8300, PerkinElmer, USA). The parameters of ICP-AES were set as follows: incident power, 1300 W; plasma gas flow rate, 12L/min; nebulization gas flow rate, 0.55 L/min. Briefly, 1 mL Gypsum Fibrosum or BHGZD extraction was mixed with 5 mL concentrated nitric acid and nitrified 12 h. Then the solution was adjusted the volume to a 100 mL volumetric flask and subjected to ICP-AES for analysis.