Licorice, also called Gancao (GC) in China, was derived from the dried roots and rhizomes of the Glycyrrhiza species. It was widely used for flavoring additives and confectionery in the food industry due to its sweet taste in most parts of the world [1
]. Additionally, Gancao was clinically used for treating wounds, diabetes, cough, and tuberculosis in China, Japan, Korea, and India [3
]. As the most frequently used herbal medicine worldwide, Gancao appears in about 60% of traditional Chinese medicine (TCM) prescriptions [7
] and confectionery products [10
]. To date, more than 400 compounds have been isolated from Gancao, of which the main constituents being saponins and flavonoids [11
], which are believed to be useful in pharmacology. Among these compounds, liquiritin (LQ), isoliquiritin (ILQ), liquiritigenin (LG), isoliquiritigenin (ILG) and glycyrrhizin (GLY) gained great attention for their validated anti-inflammatory and anti-virus effects [12
]. The chemical structure of each compound is shown in Figure 1
, commonly named Jiegeng (JG) in China, was the root of Platycodon grandiflorum A.DC
family). Traditionally, it has been consumed as food, as well as a folk remedy for ameliorating bronchitis, asthma, pulmonary tuberculosis, and inflammatory conditions [14
]. To date, more than 40 triterpenoid saponins, have been reported to existing in Jiegeng [14
]. Gancao was usually used together with Jiegeng to treat pharyngitis, cough, etc. [19
]. Gancao and Jiegeng, at a ratio of 2:1, was first described as Jiegeng Gancao Decoction or Jiegeng Tang (JG–GC) in the medical classic ‘Shang-han-lun’ to treat abscess of lung based on its demonstrated capacity to act as an expectorant, resolving phlegm and improving air flow to the lungs [20
]. It was reported that these two plants had a distinct synergistic effect working as an anti-inflammatory and eliminating phlegm [21
Several studies have investigated the pharmacokinetics of the flavonoids and saponins of Gancao [22
]. Based on these studies, the pharmacokinetic profiles of the main active components of Gancao, liquiritin, liquiritigenin and glycyrrhizic acid, were systematically determined. However, the effects of Jiegeng on the pharmacokinetics of the active components in Gancao were still unclear.
In this study, the influence of Jiegeng on the pharmacokinetics of five active constituents—liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin and glycyrrhizin and one metabolite of glycyrrhizin, glycyrrhetinic acid (GA)—in Gancao were evaluated. Furthermore, Caco-2 cell transport and fecal hydrolysis experiments were employed to investigate the absorptive and metabolic behavior of the six active components mentioned above in Gancao alone and in Jiegeng Gancao decoction.
3. Materials and Methods
3.1. Reagents and Materials
Reference compounds of liquiritin (LQ), isoliquiritin (ILQ), liquiritigenin (LG), isoliquiritigenin (ILG), glycyrrhizin (GLY), glycyrrhetinic acid (GA) and hesperidin (internal standard, IS) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of each compound was more than 98% (HPLC). The two plant materials of Gancao and Jiegeng were purchased from Nanjing Yifeng Pharmacy (Nanjing, China) and were authenticated by Dr. Shengjin Liu (Department of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China).
HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany), and formic acid with a purity of 99% was of HPLC grade (ROE, Newark, NJ, USA). Deionized water (18 MΩ) was prepared by a Milli-Q system (Millipore, Milford, MA, USA) for UHPLC analysis and all solutions or dilutions. Other reagents and chemicals were of analytical grade. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 0.1% EDTA-1.25% trypsin (EDTA/trypsin), penicillin-streptomycin, nonessential Amino Acids and Hank’s Balanced Salt Solution without Calcium & Magnesium (HBSS) were all obtained from Gibco Laboratory (Invitrogen Co, Grand Island, NY, USA).
3.2. Preparation of Sample Extracts
Jiegeng Gancao decoction (JG–GC) was composed of Jiegeng (100 g) and Gancao (200 g), and the plants were immersed in 13-fold deionized water for 0.5 h and then decocted to boil for 2 h. The extracted solution was filtered through two layers of gauze. Ethanol was added to precipitate the polysaccharide and protein. This sample was kept for 24 h at 4°C and then filtered through the filter paper. Finally, the filtrate was concentrated to obtain JG–GC with a final concentration of 3.0 g/mL (equivalent to dry weight of raw materials). Gancao decoction (GC, 200 g) was prepared as the above identical procedure, and the final concentration was 2.0 g/mL. All the extracts were kept in −70 °C until use.
Male Sprague-Dawley rats (weighing 200 ± 20 g) were supplied by the Animal Core Facility of Nanjing Medical University (Certificate No. SCXK-2008-0033) and kept in the laboratory condition (temperature, 22 ± 2 °C; relative humidity, 45~60%). The animals were acclimatized to the facilities for 3 days, and then fasted for 12 h but allowed free access to water prior to experiment. The animal experiments were conducted in accordance with protocols approved by the Animal Ethic Committee of Nanjing University of Chinese Medicine.
3.4. Pharmacokinetic Study
The rats were randomly divided into two experimental groups (n = 6 per group), including JG–GC group and GC group. Rats were administrated with JG–GC or GC at oral dose at 30 g/kg and 20 g/kg. The concentration of LQ, ILQ, LG, ILG and GLY in JG–GC and GC were determined using our validated UHPLC method, and the contents of these compounds in JG–GC and GC were shown in Table 3
. About 250 µL blood samples were collected into heparinized tubes via the postorbital venous plexus veins at 0.083, 0.167, 0.33, 0.67, 1, 2, 4, 8, 12, 24, 36 and 48 h after drug administration.
The samples were immediately centrifuged at 5000 rpm for 6 min, and 100 µL of plasma was spiked with 10 µL IS solution (92 ng/mL) by vortexing for 2 min. All analytes could be extracted from plasma with one-step precipitation by adding 300 µL methanol and vortexing for 5 min. The well-vortexed solutions were centrifuged at 17,000 rpm for 10 min. Finally, 5 µL of the supernatant was injected into the UHPLC-MS/MS system for analysis.
To obtain the pharmacokinetic parameters of the six analytes, the time-concentration data was processed by non-compartmental analysis (DAS 3.2.6 software, Chinese Pharmacological Association, Beijing, China). The pharmacokinetic parameters, including Tmax, Cmax, t1/2, AUC (0–t), and AUC(0–∞) of the four flavonoids (LG, ILG, LQ, ILQ) and two saponins (GLY, GA) in both groups were calculated and compared. The data was presented as mean ± SD for each group, if not specified otherwise. Significance is assessed by Student’s t-test, with p < 0.05 considered to be statistically significant.
3.5. Cell Culture
The human colon adenocarcinoma cell line, Caco-2, cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cell passages from the 30th to 60th generations were used in the experiment. Cells were maintained at 37 °C in an atmosphere of 5% CO2 and 90% relative humidity, growing routinely on 75 cm2 plastic culture flasks (Becton Dickinson, Franklin Lakes, NJ, USA) in DMEM, with 10% (v/v) FBS, 1% (v/v) non-essential amino acid solution, and 1% (v/v) penicillin–streptomycin. The medium was replaced every 2–3 days after incubation. The cells were seeded at a density of 5 × 104 cells/cm2 onto a permeable polycarbonate insert (0.6 cm2, 0.45 μm pore size, Millipore, Billerica, MA, USA) in 24-well tissue culture plates (NUNC, Roskilde, Denmark) and grown for 21 days before the transporter experiment. The resistances of monolayers were assessed using Millcell ERS-2 transmembrane resistance meter (Millipore, Billerica, MA, USA) on days 3, 5, 10, 14, 18 and 21. The transepithelial electrical resistance (TEER) values were calculated to examine its integrity. Only the Millicell membranes with monolayer cells that met the defined criteria with TEER values above 300 Ω·cm2 were used for transport studies.
3.6. Transport of LQ, LG and GLY across Caco-2 Cell Monolayer
The Caco-2 cell monolayers were washed three times with HBSS (pH 7.4), and placed in a shaking incubator for 30 min to remove impurities. The transport study was conducted by adding 0.4 mL of LQ solution (1.4 µg/mL), LG solution (0.4 µg/mL), GLY solution (5.2 μg/mL), GC solution and JG–GC solution. The concentration of each analyte in the GC and JG–GC solutions was measured to be equal to the targeted analyte. Furthermore, the difference was statistically negligible. These solutions were applied to the apical side of the cell monolayer, with 0.6 mL of blank HBSS applied to the basolateral side. Aliquots of 0.1 mL and 0.15 mL of samples were taken sequentially from the donor and receiver sides, respectively, after shaking at 50 rpm for 0, 30, 60, 90, 120 min. Drug solutions and HBSS medium were added to the apical or basolateral side immediately to compensate for volume lost due to sampling. A 10 μL internal standard (92 ng/mL of hesperidin in acetonitrile) was added to 100 µL of sample, and the mixture was vortexed for 5 min. All samples were nitrogen-dried and reconstituted with 20 μL methanol for UHPLC-MS/MS analysis.
The apparent unidirectional permeability, from apical to basolateral side (Papp(a-b)), was calculated according to the following equation: Papp= [(dQ/dt)]/[A × C0], where the dQ/dt (µg/min) was the drug permeation rate, A was the cross sectional area (0.6 cm2), and C0 (µg/mL) was the initial concentration of each drug solution in the donor compartment at t = 0 min.
3.7. Fecal Lysates Preparation
Fresh feces were collected from ten male normal SD rats, of which about 1 g was immediately mixed with 9 mL ice-cold PBS (pH 7.4), and vortexed for 5 s followed by filtering through four layers of gauze. For preparing fecal lysates, the filtrate was centrifuged at 1000 rpm for 15 min. The pellet was re-dissolved in 10 mL ice-cold PBS, and the mixture was sonicated in an ice water bath for 45 min, followed by centrifugation at 15,000 rpm at 4 °C for 30 min. All supernatant was stored at −70 °C.
3.8. Metabolism of LQ, LG and GLY in Fecal Lysates
Thawed fecal lysates (100 µL) were transferred to disposable glass vials, and then 10 µL of each drug solution with 90 µL PBS was added to obtain a 200 µL incubation volume. The concentrations of the analytes in the drug solutions are listed in Table 4
. The mixture was shaken at 37 °C, and samples of 100 µL were collected at 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h, after which 10 µL of 92 ng/mL hesperidin was added. The mixture was vortexed for 2 min and centrifuged at 17,000 rpm for 10 min. The supernatant was injected into the UHPLC/MS-MS to determine the remaining concentration of each analyte. The experiments were carried out in triplicate.
3.9. Quantification of Flavonoids and Saponins in Biological Matrices
The UHPLC-MS/MS was an ultimate 3000 series UHPLC system (Thermo Fisher, San Jose, CA, USA), consisting of a quaternary pump, an autosampler, a vacuum degasser, and a statically controlled column apartment equipped with a Thermo scientific quantum vantage™ mass spectrometer system (Thermo Fisher, San Jose, CA, USA), which coupled with an electrospray Ionization (ESI) source. All data was analyzed by Xcalibur Software from Thermo-Fisher Scientific. Flavonoids and saponins in Gancao and hesperidin were monitored in the negative mode (ESI−). Some significant mass spectrometry parameters, such as spray voltage, shealth gas pressure, aux gas pressure, vaporizer temperature and capillary temperature were set at 3.0 kV, 45 arb, 25 arb, 450 °C and 350 °C, respectively. The Q1 and Q3 quadrupoles were set at unit resolution with different collision energy (CE) and S-lens. The quantification was performed by the selected reaction monitoring (SRM) of the transitions of m/z 254.964–134.939 for LG/ILG (CE of 18 eV; S-lens at 64 V), 416.996–254.947 for LQ/ILQ (CE of 22 eV; S-lens at 93 V), 821.134–350.827 for GLY (CE of 39 eV; S-lens at 160 V), 469.120–425.289 for GA (CE of 37 eV; S-lens at 160 V) and m/z 609.200–301.100 for hesperidin (CE of 27 eV; S-lens at 129 V), respectively. The chromatographic analysis was achieved on a Hypersil Gold C18 column (100 mm × 2.1 mm, 1.9 µm particle size; Thermo, San Jose, CA, USA). The mobile phase consisted of 0.05% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.4 mL/min. The UHPLC gradient system began with 15% B at 0–1 min, 15–70% B at 1–6 min, and 70–100% B at 6–7 min. The column temperature was maintained at 40 °C, while the sample-tray temperature was kept at 4 °C.
The UHPLC-MS/MS analysis of LQ, ILQ, LG, ILG, GLY and GA in rat plasma, HBSS buffers and fecal lysates have been studied [24
] and the methods have been validated, respectively. Other substances did not interfere with the detection of these components in each biological sample. The intra- and inter-batch precision and accuracy were < 20% for all quality control samples. In addition, all the substances tested in each biological matrix were stable, and the linear range and LLOQ of each analyte in different samples are shown in Table 5