L. is a mangrove shrub belonging to the Acanthus
genus in the Acanthaceae family which grows in tropical and subtropical intertidal habitats. Acanthus ilicifolius herb (AIH, “laoshule” in Chinese), the dry plant of A. ilicifolius
L., is a folk medicine to treat acute and chronic hepatitis, lymphatic intumescence, spleen enlargement, paralysis, and rheumatism [1
]. Modern pharmaceutical studies have demonstrated that the extracts of AIH possess hepatoprotective, antioxidant, anti-inflammatory, anticarcinogenic, and antibacterial activities [6
]. Numerous chemical constituents have been isolated from AIH, including phenylethanoid glycosides (PhGs), alkaloids, flavones, lignans, triterpenoid saponins, and sterols [11
]. Among them, PhGs were reported as one family of the main components from AIH, such as acteoside, isoacteoside, martynoside, and crenatoside with potential pharmacological effects [18
]. The pharmacologic actions of AIH extracts, as well as these four PhGs, have been reported, whereas no pharmacokinetics have been studied till now.
During our ongoing research of pharmacodynamic material basis of medical plants, we found that AIH and its efficient components, PhGs, exhibited potent hepatoprotective, antiviral, and antioxidant activities. Specifically, PhGs were found to be abundant in AIH, of which acteoside and isoacteoside were the main components, while martynoside and crenatoside were in relatively lower contents. In the present study, the pharmacokinetics of AIH were investigated with these four PhGs as representatives in rat plasma after intragastric administration of AIH extract. To achieve this purpose, a simple, sensitive and rapid ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method was established, first, for the simultaneous and quantitative determination of the four PhGs in rat plasma. Herein, we report the establishment and optimization of UPLC-MS/MS method, the validation of the established method, and the pharmacokinetic study of AIH.
3. Materials and Methods
3.1. Chemicals and Reagents
The authentic phenylethanoid glycoside compounds were purchased as follows: acteoside (purity 98.0%) from Dalian Meilun Biological Technology Co., Ltd. (Dalian, China); isoacteoside (purity 98.0%) from Chengdu Push Bio-Technology Co., Ltd. (Chengdu, China); and genistein (internal standard, IS) from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Martynoside and crenatoside (>98.0% purity) were isolated from AIH in our laboratory and identified by combination of NMR, HPLC, and MS. HPLC-grade acetonitrile (MeCN) and methanol were purchased from Fisher Scientific Co., Ltd. (St. Louis, MO, USA). HPLC-grade formic acid was obtained from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China), and experimental water was purified by a Milli-Q Reagent Water System (Millipore, Burlington, MA, USA).
3.2. Preparation of AIH Extracts
The plant A. ilicifolius L. was collected from Jiangmen, Guangdong Province, China and authenticated by Professor Feng-Qin Zhou, Shandong University of Traditional Chinese Medicine. Voucher specimen number for A. ilicifolius L. is 2018060805. Voucher specimen of the plant is deposited at the Key Laboratory of Marine Drugs, the Ministry of Education of China, Ocean University of China, Qingdao, China.
The whole plant was dried in the shade and ground into crude powder. The crude powder (200 g) was immersed in 95% ethanol (v/w, 10:1) for 1 h, and then heated to reflux at 80 °C for 2 h. The extraction solution was filtered, and the residue was refluxed again in 95% ethanol (v/w, 8:1) at 80 °C for 2 h. The filtrate was pooled together and concentrated by a rotary evaporator to dryness at 45 °C. Finally, the product was dissolved in distilled water to acquire the AIH extract for testing with the concentration of 5.0 g crude herb/mL. This extract sample was stored at 4 °C until use.
Ten male Sprague-Dawley (250–280 g) rats were purchased from Jinan Pengyue Experimental Animal Center (SCXK (Lu) 20140007). The animal experiments were approved by the Animal Ethics Committee of Marine Biomedical Research Institute of Qingdao (MBRI-2018-0606), and the guidelines of the institute were strictly followed. All rats had free access to water and food, and were maintained in an environmentally controlled breeding room under the following conditions: 20 ± 2 °C temperature, 60–70% relative humidity, and 12 h light/dark for 1 week before the experiment operated. After fasted for 12 h with free access to water, blank plasma was obtained from four rats after intragastric administration of 2.0 mL/kg water for the UPLC-MS/MS method validation, and the other six rats which were administrated with AIH extract were used for the pharmacokinetic study as in Section 2.4
3.4. Preparation of Stock Solutions, Calibration Samples, and Quality Control Samples
The stock solutions of four PhGs, acteoside, isoacteoside, martynoside, and crenatoside, were prepared with methanol as a solvent. A stock solution of the PhGs mixture was prepared by combining these four PhGs to attain the final concentrations of 10,000 ng/mL acteoside, 1000 ng/mL isoacteoside, 2000 ng/mL martynoside, and 2000 ng/mL crenatoside. The working solutions were obtained from the stock solution by sequential dilution with methanol at the concentrations of 20.0–10,000 ng/mL acteoside, 2.0–1000 ng/mL isoacteoside, 4.0–2000 ng/mL martynoside, and 4.0–2000 ng/mL crenatoside. Calibration samples were prepared by adding 5 μL working solutions to 45 μL blank plasmas in 1.5 mL Eppendorf tubes. Therefore, the final calibration samples contained 2.0–1000 ng/mL acteoside, 0.2–100 ng/mL isoacteoside, 0.4–200 ng/mL martynoside, and 0.4–200 ng/mL crenatoside. The quality control (QC) samples were prepared in the same way as calibration samples, with the final dilutions of 5.0, 50, and 800 ng/mL acteoside, 0.5, 5, and 80 ng/mL isoacteoside, 1.0, 10, and 160 ng/mL martynoside, and 1.0, 10, and 160 ng/mL crenatoside. The stock solution of the internal standard (IS), geistein, was prepared in methanol at the concentration of 1.0 mg/mL. The IS working solution of 1000 ng/mL was obtained by diluting the stock solution with methanol.
3.5. Pretreatment of Calibration Samples and QC Samples
The 10 μL IS working solution and 140 μL methanol were added to each calibration sample (50 μL) and QC sample (50 μL). The mixture was vortexed for 60 s and centrifuged at 15,000× g for 15 min to separate the precipitated protein. Then, 2 μL of supernatant of the mixture was used for the UPLC-MS/MS analysis.
3.6. Instrumentation and Chromatographic Conditions
A Waters ACQUITY™ UPLC system (Waters Corp., Milford, MA, USA) was interfaced with a Waters Xevo™ TQ/MS (Waters, USA) equipped with an electrospray ionization (ESI) source. Separation of the PhGs was performed on an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm), and the column temperature was maintained at 40 °C during the analysis. The mobile phase consisted of MeCN (solvent A) and 0.1% formic acid in water (solvent B) at a flow rate of 0.4 mL/min. The gradient elution conditions were as follows: 0–1 min, 2–2% A; 1–1.5 min, 2–10% A; 1.5–7 min, 10–43% A; 7–8 min, 43–95% A; 8–9 min, 95% A; 9–10 min, 95–2% A. The injection volume was 2 μL. The detection wavelength was at 330 nm.
The PhGs were detected and quantified by multiple reaction monitoring (MRM) in negative ionization mode (ESI−
). The MS parameters of the ionization source were as follows: source temperature, 150 °C; capillary voltage, 3.15 kV; desolvation gas temperature, 400 °C; desolvation gas flow rate, 800 L/h; and cone gas flow rate, 150 L/h. Other optimized parameters, collision energies, and cone voltages are shown in Table 1
. All raw data were processed using MassLynx V4.1 workstation (Waters Corp., Milford, MA, USA).
3.7. Method Validation
The selectivity of the method was assessed by chromatograms of blank plasma, blank plasma spiked with working solution of the four PhGs at the lowest limit of quantification (LLOQ) together with working solution of IS, and test plasma acquired at 30 min after intragastric administration of AIH extract. The samples were prepared and pretreated in the same approaches as in Section 3.4
and Section 3.5
Various concentrations of calibration standards (2.0, 5.0, 10, 25, 50, 100, 200, 400, 800, and 1000 ng/mL acteoside; 0.2, 0.5, 1.0, 2.5, 5.0, 10, 20, 40, 80, and 100 ng/mL isoacteoside; and 0.4, 1.0, 2.0, 5.0, 10, 20, 40, 80, 160, and 200 ng/mL martynoside and crenatoside) were processed according to the above procedures for sample preparation. The calibration curve was constructed by plotting analyte-to-IS peak area ratio (y) versus the concentration (x, ng/mL) of analyte and fitted to linear regression (y = ax + b) using 1/x as the weighting factor. The calibration curves were acceptable only if their correlation coefficients (r2) represented linearity of 0.99 or greater. The calibration curve was established daily throughout the method development and pharmacokinetic analysis. The LLOQ was determined by testing the lowest analytical concentration of the calibration curve.
The accuracy and precision of the method were evaluated by analyzing QC samples at three concentrations prepared as in Section 3.4
with six replicates. To determine the intra- and inter-day accuracy and precision, six replicates at each concentration level were analyzed for five consecutive days. Accuracy was expressed as relative error (RE, %) values within ±15%, and precision was described as relative standard deviation (RSD, %) values less than 15%.
The extraction recoveries at different QC levels were investigated by comparing the mean peak areas of the PhGs pipetted into blank plasma before and after protein precipitation, respectively. The absolute matrix effect expressed as matrix factor (MF) was evaluated by comparing the peak areas of the PhGs pipetted into rat plasma after protein precipitation with those dissolved in the initial mobile phase solution. The relative matrix effect was assessed based on the peak areas of the PhGs pipetted into six different individual sources of rat plasma. The IS normalized MF was determined by the absolute MF of analyte over that of the IS.
The stabilities of the four PhGs were determined by using QC samples in different conditions. Freeze-thaw stability was assessed after three freeze-thaw cycles (from −80 °C to room temperature). Short-term stability was determined after exposure of the QC samples at room temperature (25 °C) for 10 h. Long-term stability was assessed by exposing the samples at −20 °C for 20 days. The samples stored in the autosampler at 4 °C for 24 h were used to evaluate the post-preparative stability. Each QC concentration level was prepared in six replicate samples.
3.8. Pharmacokinetic Study
AIH extract was given to the six rats at a dose of 10.0 g crude herb/kg body weight by intragastric administration. Blood samples (200 μL) were collected from the fossa orbitalis vein before dosing and at the time points of 5, 15, 30, and 45 min and 1, 2, 4, 6, 8, and 12 h after administration and transformed into heparinized Eppendorf tubes. Then, the blood samples were centrifuged at 12,000 × g
, 4 °C, for 10 min. Each rat plasma sample (50 μL) was prepared in the same approaches as in Section 3.5
3.9. Data Analysis
Drug and Statistics (DAS) 3.2.8 software (Shanghai University of Traditional Chinese Medicine, Shanghai, China) was applied to calculate the pharmacokinetic parameters (t1/2, tmax, Cmax, AUC0–t, and AUC0–∞) of the four PhGs. All data were shown as mean ± standard deviation (SD).