1. Introduction
Gelsemium elegans Benth. (
G. elegans) is distributed in China and Southeastern Asia. It was widely used as a classic Chinese herbal medicine to treat malignant skin problems [
1]. Recent years, its anti-tumor [
2], anti-inflammatory [
3], and anxiolytic [
4] activities have also been well studied. On the other side,
G. elegans was well-known for its toxicity. Typical symptoms of intoxication include vomiting, blurred vision, muscular weakness, limb paralysis, dilated pupils, breathing difficulty, and convulsion. In instances of severe poisoning, the nervous system is depressed and death is caused by respiratory depression [
5,
6]. Its toxicities set a limit to the application of
G. elegans in clinical settings. It is critical finding effective methods to reduce the toxicity of
G. elegans based on the mechanism of action.
Glycyrrhiza uralensis Fisch (GU) is one of the most widely used herbal medicines. According to traditional Chinese medicine (TCM) theory, GU is primarily effective for fatigue and debilitation, asthma with coughing, and excessive phlegm. Moreover, it has a unique effect on moderating the characteristics of toxic herbs, which could be partly interpreted as detoxification [
7]. In the folk, intragastric administration of GU was used to detoxify
G. elegans, much of the work carried out on the role of GU on CYPs regulation and function, though further investigation was required for the mechanism of this detoxification method.
As the most abundant molecule among the alkaloids of
G. elegans, koumine (KM) has been demonstrated to exert numerous potent biological properties, such as anxiolytic and analgesic effects [
8]. However, it also possesses inhibitory effects on splenocyte proliferation and the humoral immune response [
9]. In the previous study, our group reported the pharmacokinetics study of gelsemine and koumine after oral administration of the extract of
G. elegans [
10], but the tissue distribution data in the literature is limited. Furthermore, there is no report about the influence of GU on the pharmacokinetics and tissue distribution of KM and we speculate that GU may have an impact on the pharmacokinetics of KM metabolized by inducing the CYP450 system. The aim of this work is to evaluate the pharmacokinetics and tissue distribution properties of KM in rats and to explore how these behaviors are altered by the pre-treated GU extract. It desrved further investigation so as to understand the possibility regarding the combination use of KM and GU. To achieve this, an ultra-liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of koumine in rat plasma, tissues, and rat liver microsome (RLM). In vivo pharmacokinetic and tissue distribution study could give a straightforward result for the influence of GU on KM. Moreover, GU is discussed for testing its potential on hepatic enzyme inductions by both in vitro metabolism and RT-qPCR study. The results of our study would provide a meaningful basis for evaluating the rationality for the detoxification effect of GU on
G. elegans.
3. Experimental
3.1. Materials and Reagents
Standard of KM (98% purity) were purchased from Qingdao JieShiKang Biotechnology (Qingdao, Shandong, China). The berberine (internal standards, I.S., 98% purity) was provided from National Institute for the Control of Pharmaceutical and Biological Products (0713-9906, Beijing, China). The NADPH-regenerating system containing glucose 6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PDH), NADPH+, and MgCl2 was purchased premixed from BD Biosciences (San Jose, CA, USA). HPLC-grade acetonitrile was purchased from Merck (Merck, Darmstadt, Germany). Deionized water was produced from a Milli-Q water purification system (Millipore, Billerica, MA, USA). Other reagents were of analytical grade. T10 basic ULTRA-TURRAX homogenizer (IKA company, Staufen, Germany).
Pharmacokinetic parameters were estimated using the drug and statistic (DAS) software. Statistical analysis was used the IBM spss statistics software and an independent-sample t test.
Glycyrrhiza uralensis Fisch was collected from the Bozhou Traditional Chinese Medicine Market of Anhui and identified by Professor Dongfang Zhang of China Medical University.
3.2. Animals
Male adult Sprague-Dawley rats (180–220 g) were supplied by the Experiment Animal Center, China Medical University. The experimental protocol was approved by the Animal Ethics Committee of China Medical University (Permit number: SYK2017–0019), and all animal studies were carried out according to the Guide for Care and Use of Laboratory Animals.
3.3. Preparation of Herbal Decoctions
The powdered GU was added to fourfold of distilled water and immersed for 30 min at room temperature. The mixture was heated at reflux for 1 h. The filtrates were extracted again in the same way. The combined extract solution was evaporated under reduced pressure to yield a dark brown residue. The extraction yield was 32.6%. For administration to the animals, the dose was calculated on the base of original herbs. Dried residues were reconstituted with distilled water and adjusted to such a volume that 1 mL of the final decoction contained 1 g of equivalent original herbal material.
3.4. Preparation of Calibration Standards and Quality Control (QC) Samples
The stock solution of KM (0.5 mg/mL) and IS (1 μg/mL) were separately prepared in methanol. The stock solution of KM was further diluted into a series of working standard solutions with methanol. The calibration standard solution were prepared by spiking 10 μL standard working solutions, which was evaporated to dryness by a gentle stream of nitrogen and then mixed with 100 μL blank biological matrix to yield calibration concentrations of 10–5000 ng/mL for plasma, 25–5000 ng/mL for tissues, and 50–50000 ng/mL for RLM. QC samples at low, middle, and high concentrations were prepared at 25 ng/mL, 300 ng/mL, and 4000 ng/mL for plasma, 100 ng/mL, 600 ng/mL, and 4000 ng/mL for tissues and 250 ng/mL, 2000 ng/mL, and 20,000 ng/mL for RLM.
3.5. Sample Preparation
An aliquot of 10 μL IS solution was added to 100 μL biological matrix. The mixture was then precipitated with 300 μL methanol. The mixture was vortexed for 1 min and centrifuged at 12,000 rpm for 15 min. The supernatant was transferred to a clean tube and dried under nitrogen gas. The residue was reconstituted with 50 μL methanol, after being centrifuged at 15,000 rpm for another 15 min, 5 μL of the sample solution was injected into the UPLC-MS/MS system for analysis.
Each weighed tissue sample was thawed and then homogenized in ice-cold physiological saline (1:2, w/v). Subsequent steps were identical to those that are described above.
3.6. Instruments and Analytical Conditions
An ACQUITY UPLC system (Waters Corp. Milford, MA, USA), equipped with cooling auto-sampler and column oven enabling temperature control was used for all analysis. Chromatographic separation was performed on an ACQUITY UPLC® BEH C18 column (3.0 × 50 mm, 1.7 µm) and the column temperature was maintained at 25 °C. The mobile phase consisted of acetonitrile (A) and water containing 0.05% formic acid (B). The gradient elution program was as follows: 0–0.5 min, 5% A, 0.5–2.1 min, 70% A, 2.1–3.0 min, 5% A. The flow rate was 0.4 mL/min and the injection volume was 5 µL.
Mass spectrometry was performed on a Waters Xevo TQD tandem quadrupole mass spectrometers (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) interface. The instrument was operated in positive ion MRM mode under the following setting parameters: capillary voltage 2.0 kV, cone voltage 44 V, source temperature 120 °C, desolvation temperature 500 °C, and desolvation gas 1000 L/h.
3.7. Method Validation
The method validation was fully conducted according to the guidelines of the Food and Drug Administration (FDA) for evaluating the specificity, linearity, precisions, accuracy, dilution integrity, extraction recovery, matrix effects, and stability. The method validation was seen
supporting information “method validation” section.
3.8. In Vivo Pharmacokinetics and Tissue Distribution Study
12 male SD rats were randomly divided into two groups (n = 6 per group), one as the single-dose of KM group, and the other as the repeated oral pre-treatment GU (GU + KM) group. Rats in the GU + KM group were administered GU decoction 3 g/kg by gavage once daily for 14 consecutive days. After 1 h of the last dosing, rats of the two groups were intravenously administered with 10 mg/kg KM. Blood sample (each approximately 0.3 mL) were collected into heprin sodium containing Eppendorf tubes from the suborbital vein at 0.017, 0.083, 0.17, 0.33, 0.50, 0.75, 1, 2, 4, 6 h post dose. The blood samples were immediately centrifuged at 4000 rpm for 10 min to collect plasma, which were then stored at −20 °C until further analysis by UPLC-MS/MS. Pharmacokinetics parameters were estimated by drug and statistic (DAS) software (version 3.0, Mathematical Pharmacology Professional Committee of China, Shanghai, China).
48 male SD rats were randomly divided into two groups (n = 24 per group), as above. After intravenous administration of KM, each group were further divided into four groups (n = 6 per group). To each group, heart, liver, spleen, lung, kidney, stomach and intestine were collected at 0.25, 0.5, 1 and 2 h. Tissue samples were rinsed with normal saline solution to remove the blood and were then weighed the wet weight and stored at −20 °C until further analysis by UPLC-MS/MS.
3.9. Preparation of RLM
Male SD rats were randomly divided into two groups of five animals each. The rats of GU-pretreated group received the decoction orally at a dose of 3 g/kg once daily for 14 days as well as the control group rats were given water only. On the day 15, rats were killed and the liver was rinsed in situ with 1.17% KCl via the hepatic portal vein and the dorsal aorta. The livers were removed immediately and then homogenized with buffer A (50 mM Tris-HCl buffer at pH 7.4 containing 0.2 M sucrose, 1:3,
w/
v). After the first centrifugation at 12,500×
g for 30 min, the supernatant was further centrifuged at 100,000×
g for 60 min at 4 °C. The microsomal pellets were suspended in buffer A with 20% glycerol. The microsomal protein content was quantified according to the method of Lowry et al. [
13].
3.10. In Vitro Metabolism of Koumine in RLM
Microsomes prepared from both the GU-pretreated and control groups were used to assess in vitro the metabolism of KM. The NADPH-regenerating system contained 1 mg/mL microsomal proteins, 1.1 mM NADPH+, 0.035 mM MgCl2, 2.34 mM G-6-P, 0.28 U/mL G-6-P dehydrogenase, serial concentration of koumine (final concentration 5–50 μM) in phosphate buffer (pH 7.4, 0.1 M) in a total volume of 200 μL. The mixture was incubated at 37 °C for 30 min based on our preliminary studies to ensure the linear metabolic clearance rate of KM. The reactions were terminated with an equal volume of ice-cold acetonitrile, and then the samples were analyzed by UPLC-MS/MS. The equation of KM reaction velocity (V) in liver microsomes was expressed as V = (C0 − Ct)/T/Cp, where C0 and Ct represent the initial concentration and the final concentration of KM in incubation solution, respectively, T is the reaction time (min) and Cp is the protein concentration (mg/mL). All of the values were expressed as mean ± SD (n = 5). The mean intrinsic clearance rate (CLint) for the in vitro incubation was estimated by Vmax/Km.
3.11. RNA Isolation, cDNA Synthesis and Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis of CYP3A1 mRNA
Approximately 100–200 mg of liver tissue obtained from the rats of both the GU-pretreated and control groups. The liver tissue was homogenized and total RNA was extracted using the Trizol reagent, following the protocol. The RNA concentration was determined by a NANOROP 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). 1 mg of total RNA of each sample was reverse transcribed by the ReverTra Ace qPCR RT kit (TOYOBO CO., LTD., Osaka, Japan), according to the manufacture’s manual. All of the templates were diluted 10-fold prior to use in RT-qPCR. The expression level of CYP3A1 was determined in 96-well plates by an Mx3000P PCR system (Agilent Technologies Inc., Santa Clara, CA, USA). All of the PCR reactions was carried out using SYBR® Green Realtime PCR Master Mix kit in a 13.5 μL reaction system containing 0.25 μL of each primer and 2 μL cDNA, 4.5 μL dd H2O, and 6.25 μL Ultra SYBR Mixture with ROX (TOYOBO CO., LTD.), following the manufacturer instructions. Primer sequences were as follows: for GAPDH, Forward primer: 5′-AGCCTCGTCCCGTAGACAAAA-3′, Reverse primer: 5′-TGGCAACAATCTCCACTTTGC-3′, for CYP3A4, Forward primer: 5′-TTCCCTCAACAACCCGAAGG-3′, Reverse primer: 5′-CTGCCCTTGTTCTCCTTGCT-3′. The reaction mixture was initially incubated at 95 °C for 2 min to denature DNA. Amplification was performed for 40 cycles of 94 °C for 30 s and 55 °C for 30 s and 72 °C for 30 s, and 72 °C for 5 min.