1. Introduction
Anemarrhena asphodeloides, the dried rhizome of
A. asphodeloides Bunge (Fam. Liliaceae), is mainly distributed in China, Mongolia and other eastern Asian countries.
A. asphodeloides has been commonly used in traditional medicine in China, Japan, and Korea for thousands of years [
1]. In clinical applications of Traditional Chinese Medicine (TCM),
A. asphodeloides is able to teat febrile diseases, high fever and thirst, lung heat with dry cough, diabetes due to internal heat and constipation [
2]. Pharmacological studies revealed that
A. asphodeloides also possesses anti-microbial activity, decreases the blood glucose level, inhibits platelet aggregation, inhibits carcinomas, decreases radiation injuries and has anti-dementia activity [
3,
4,
5,
6].
As the decoction of the entire rhizome extract is taken orally, the pharmacological activity observed in humans is attributed to the entire rhizome extract, which includes steroidal saponins, flavonoids, pigments, polysaccharides,
etc. The main active components of
A. asphodeloides are steroidal saponins, with extremely diverse structures such as timosaponin BII (
1), anemarsaponin BIII (
2), timosaponin AIII (
3) and timosaponin E1 (
4) (
Figure 1), which have been shown to improve senile dementia and have anti-blood coagulation, anti-oxidant, anti-tumor, anti-osteoporosis, anti-inflammation, and blood sugar and blood pressure lowering effects [
7,
8,
9,
10,
11,
12,
13]. However, non-steroidal saponin small molecule ingredients such as flavonoids [
14,
15], organic acids [
16,
17], amino acids, nucleosides, oligosaccharides and a non-steroidal saponin macromolecule fraction comprising pigments and polysaccharides exist in
A. asphodeloides [
18]. Whether these non-steroidal saponins have pharmacological effects or can influence the absorption of the steroidal saponins ingredients and thus change their bioavailability in the gastrointestinal tract is still unclear.
Figure 1.
Chemical structures of timosaponinBII (1), anemarsaponin BIII (2), timosaponin AIII (3), timosaponin E1 (4) and ginsenoside Re (IS).
Figure 1.
Chemical structures of timosaponinBII (1), anemarsaponin BIII (2), timosaponin AIII (3), timosaponin E1 (4) and ginsenoside Re (IS).
Several analytical methods are reported for the determination of steroidal saponins in
A. asphodeloides, including thin-layer chromatography (TLC), gas chromatography (GC) and high-performance liquid chromatography (HPLC) with different detectors [
19,
20,
21]. However, these methods have some limitations including long analysis times and/or low sensitivity and thus are not suitable for the determination of steroidal saponins in biological fluids after administration of
A. asphodeloides extract. In this study, we hypothesized that the non-steroidal saponin ingredients in
A. asphodeloides might influence the pharmacokinetics of the active components in the steroidal saponin fraction of
A. asphodeloides. Therefore, a rapid, sensitive and selective LC-MS/MS method was developed to determine simultaneously four steroidal saponin components in rat plasma and applied to demonstrate the pharmacokinetic influences after comparing the pharmacokinetics of compounds
1–
4 in the steroidal saponins fraction from
A. asphodeloides when combined with different non-steroidal saponin fractions. The results are expected to be very helpful for evaluating the effect of non-steroidal saponin ingredients and guiding changes to the dosage form in clinical applications of this herb.
3. Experimental Section
3.1. Chemicals and Reagents
The reference standard of timosaponin BII (purity > 98%) was purchased from Chengdu Herb Purify Co., Ltd. (Chengdu, China). Anemarsaponin BIII (purity > 98%) and timosaponin AIII (purity > 98%) were obtained from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Timosaponin EI (purity > 98%) was purchased from Beijing Biohalfeast Technology Co., Ltd. (Beijing, China). Ginsenoside Re (purity > 98%) was purchased from the Chinese National Institute of Pharmaceutical and Biological Products (Beijing, China). HPLC grade acetonitrile and formic acid were purchased from Merck (Darmstadt, Germany). Deionized water was purified by an EPED water purification system (EPED, Nanjing, China). All other reagents used were of analytical grade. A. asphodeloides was collected from Anhui Province. A voucher specimen (Voucher No. HJ20141211) was deposited at the Shaanxi Collaborative Innovation Center of Chinese Medicinal Resource Industrialization (Xianyang, China).
3.2. Chromatographic Conditions
Chromatographic analysis was performed on an Acquity UPLC system (Waters Corp., Milford, MA, USA), consisting of a binary pump solvent management system, an online degasser, and an autosampler. An Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) was employed and the column temperature was maintained at 35 °C. The mobile phase was composed of A (0.1% formic acid) and B (acetonitrile) using a gradient elution of 20%–25% B at 0–1.0 min, 25%–30% B at 1.0–3.0 min, 30%–90% B at 3.0–3.1 min, 90%–95% B at 3.1–4.0 min, 95%–20% B at 4.0–4.2 min, at a flow rate set at 0.4 mL/min. The autosampler was conditioned at 4 °C and the injection volume was 2 μL.
3.3. Mass Spectrometric Conditions
Mass spectrometry analysis was performed using a Xevo TM triple quadrupole mass spectrometer (Waters Crop., Milford, CT, USA) equipped with an electrospray ionization source (ESI). The ESI source was set in negative ionization mode. The parameters in the source were set as follows: capillary voltage 3.0 kV; source temperature 150 °C; desolvation temperature 550 °C; cone gas flow 50 L/h; desolvation gas flow 1000 L/h. Analytes were performed by using multiple-reaction monitoring (MRM) mode. The cone voltage and collision energy were optimized for each analyte and selected values are given in
Table 6. All data collected in centroid mode were acquired using Masslynx4.1 software (Waters Crop, Version 4.1, 2010) and post-acquisition quantitative analysis was performed using the TargetLynx program (Waters Crop, 2010).
Table 6.
Precursor/product ion and parameters or MRM of compounds used in this study.
Table 6.
Precursor/product ion and parameters or MRM of compounds used in this study.
Analytes | Retention Time (min) | MRM Transitions (precursor → product) | Cone Voltage (V) | Collision Energy (eV) |
---|
Timosaponin BII | 2.48 | 919.5→757.4 | 52 | 28 |
Anemarsaponin BIII | 3.74 | 901.5→739.4 | 68 | 30 |
Timosaponin AIII | 4.21 | 739.4 →577.4 | 66 | 40 |
Timosaponin E1 | 1.95 | 935.3→773.2 | 64 | 34 |
Ginsenoside Re | 2.33 | 945.6→637.4 | 54 | 34 |
3.4. Preparation of Administered Samples
The dried A. asphodeloides were chopped into slices before using. The A. asphodeloides was immersed in water (1:10, w/v) and extracted twice by refluxing for 2 h. After filtration, the supernatant was condensed to a certain volume under reduced pressure, and 95% ethanol was added to the water extract filtrates until the concentration of ethanol was 80%. The precipitate was filtered as the macromolecular fraction (MF), the ethanol supernatant was concentrated under reduced pressure to a certain volume under vacuum and then purified by gradient elution with water, 40% ethanol and 70% ethanol from an AB-8 MARO porous resin column and then the water-eluted fraction was combined with the 40% ethanol-eluted fraction as the small molecule fraction (SF) and the 70% ethanol eluted fraction was the A. asphodeloides saponins extract (ASE). All of the residues were lyophilized and the resulting dry powder was stored at 4 °C before usage. The contents of four steroidal saponins in ASE dry powder were measured quantitatively by the external standard method using the same chromatography conditions as described above. The contents of timosaponin BII, anemarsaponin BIII, timosaponin AIII and timosaponin E1 in ASE were 429.12, 89.81, 59.13 and 41.25 mg/g, respectively.
3.5. Preparation of Standard Solution and Quality Control (QC) Samples
Stock solutions were separately prepared by dissolving the four accurately weighed standard reference compounds in a mixture of 50% acetonitrile. Then, the four stock solutions were mixed and diluted with 50% acetonitrile to prepare a final mixed standard solution containing 650.00 ng/mL of timosaponin BII, 620.00 ng/mL of anemarsaponin BIII, 485.00 ng/mL of timosaponin AIII, 530.00 ng/mL of timosaponin E1. A series of working solutions were freshly prepared by diluting mixed standard solution with 50% acetonitrile to the appropriate concentration. The internal standard solution of ginsenoside Re was prepared to the concentration of 920 ng/mL in 50% acetonitrile. For the validation of the method, three concentrations of standard solution containing timosaponin BII (5.20, 52.00 and 520 ng/mL), anemarsaponin BIII (4.96, 49.60 and 496.00 ng/mL), timosaponin AIII (3.88, 38.8 and 388 ng/mL), timosaponin E1 (4.24, 42.40 and 424.00 ng/mL) were used for preparing the QC plasma samples.
3.6. Preparation of Plasma Samples
Frozen plasma samples were unfrozen at room temperature and treated as follows: to each 200 µL plasma sample, 20 µL of IS (920 ng/mL ginsenoside Re) working solution and 600 µL of acetonitrile were added. After vortexing for 2 min and centrifugation at 13,000 rpm at 4 °C for 10 min the supernatant was transferred into a new tube and evaporated to dryness in a rotary evaporator at 39 °C and the residue was reconstituted in 100 µL of 0.1% formic acid–acetonitrile (50:50, v/v), vortexed for 2 min and centrifuged at 13,000 rpm at 4 °C for 10 min. The supernatant was transferred to an autosampler vial and an aliquot of 2 μL was injected onto the UPLC-MS/MS system for analysis.
3.7. Method Validation
3.7.1. Assay Specificity
The specificity of the method was evaluated by comparing the chromatograms of six different batches of blank plasma samples, plasma samples spiked with the timosaponin BII, anemarsaponin BIII, timosaponin AIII, timosaponin E1 and IS, and plasma samples obtained from rats administered ASE.
3.7.2. Linearity and lower limits of quantification (LLOQ)
The calibration curves were determined by plotting the peak area ratio (Y) of analytes to IS versus the nominal concentration (x) of analytes with weighted (1/x2) least square linear regression. The lower limit of quantitation (LLOQ) of the assay was defined as the lowest concentration on the standard curve that can be quantitated with accuracy within 20% bias of the nominal concentration and RSD.
3.7.3. Precision and accuracy
The intra-day and inter-day precision and accuracy were determined by quantifying three concentration levels of QC samples (six samples for each concentration level) on the same day and on three consecutive validation days, respectively. The precision was expressed as the relative standard deviation (RSD), and the accuracy by (mean measured concentration/spiked concentration) × 100%.
3.7.4. Recovery and matrix effect
The extraction recoveries of analytes at three QC levels were determined by comparing the peak area of each analyte extracted from plasma samples with that of post-extraction spiked plasma blank. The matrix effect was evaluated by comparing the peak areas of the analytes obtained from six plasma samples with the analytes spiked after extraction, at three concentration levels, to those from the neat standard solutions at the same concentrations. The extraction recovery and matrix effect of IS were also evaluated using the same procedure.
3.7.5. Stability
The stability experiments were measured by analyzing replicates (n = 6) of three QC samples during the sample storing and processing procedures. For all stability experiments, freshly prepared stability testing QC samples were evaluated by using freshly prepared standard curve for the measurement. The post-preparation stability was tested by determined of the extracted QC samples stored in the auto-sampler (4 °C) for 24 h. The freeze and thaw stability were determined after three freeze-thaw cycles (−80 °C to room temperature). Long-term stability in rat plasma stored at −80 °C was studied for a period of one month.
3.8. Pharmacokinetic Study
Male Sprague-Dawley rats (250–280 g) were obtained from the Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). All animals were kept in an environmentally controlled breeding room (temperature: 20–25 °C, humidity: 55%–65%) for 1 week before the experiments started. Animal welfare and experimental procedures were strictly in accordance with the ethical norms of the Nanjing University of Chinese Medicine. All rats were fasted for 12 h with free access to water prior to the experiments, twenty-four rats were divided into four groups and then were given a single dose of ASE, ASE-SF, AS-MF, AS-M-SF. A 40 mg/mL ASE (the amount of MF, SF added to group AS-MF, ASE-SF, AS-M-SF were based on the proportion extracted from A. asphodeloides) aqueous solution in each group was administered orally at a dose of 400 mg/kg, which contained 171.61, 35.92, 23.62, 16.53 mg/kg of timosaponin BII, anemarsaponin BIII, timosaponin AIII, timosaponin E1, respectively.
About 400 μL blood samples were collected from venipuncture before intragastric gavage and at 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24 h after a single oral administration. The blood samples were immediately transferred to heparinized tubes and centrifuged at 3000 rpm for 10 min, and the supernatant was transferred into 2.0 mL Eppendorf tubes and stored at −80 °C prior to analysis. Blank plasma was obtained from the rat without oral administration and was used to investigate the assay development and validation.
3.9. Statistical Analysis
To calculate the pharmacokinetic parameters of analytes in different group, concentrations–time dada were analyzed by DAS 3.2 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China, 2011). Data were measured as the mean ± standard deviation (S.D.) with triplicate measurements. The identification of significances between different groups was executed with Student’s t-test. A p value < 0.05 was considered statistically significant.