|Year : 2021 | Volume
| Issue : 2 | Page : 246-253
Pharmacokinetic and relative bioavailability of three secoiridoid glycosides in beagle dog plasma after oral administration of conventional and enteric-coated capsules of Gentianella acuta extract
Zhi-Bin Wang1, Kai Li1, Meng Wang1, Gao-Song Wu1, Yu-Jin Bi2, Hai-Xue Kuang1
1 College of Pharmacy, Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of Chinese Medicine, Harbin 150040, China
2 SunGen Pharma, LLC, 303C Collage Road East, Princeton, NJ08540, USA
|Date of Submission||01-Sep-2020|
|Date of Acceptance||19-Oct-2020|
|Date of Web Publication||24-May-2021|
Prof. Hai-Xue Kuang
Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of Chinese Medicine, 24 Heping Road, Xiangfang District, Harbin 150040
Source of Support: None, Conflict of Interest: None
Objective: To simultaneously investigate the pharmacokinetics of gentiopicroside, sweroside, and swertiamarin, which are constituents of Gentianella acuta, by developing and validating a simple, sensitive, and fast ultra-high-performance liquid chromatography–tandem mass spectrometry method. Materials and Methods: Blood samples were collected from the forward limb veins of six beagle dogs following oral gavage with G. acuta, the whole plant extract (39.90 mg/kg). Plasma samples were processed using liquid–liquid extraction. The analytes and paeoniflorin (internal standard [IS]) were separated using an Acquity® UPLC ethylene bridged hybrid amide column (2.1 mm × 100 mm, 1.7 μm) with isocratic elution using a mobile phase consisting of acetonitrile and 0.1% formic acid in water (80:20, v/v) at a flow rate of 0.4 mL/min. Quantification was performed using multiple reaction monitoring of the fragmentation transitions at m/z 401.1s594;179.0, 403.1→195.0, 419.1→179.0, and 525.2→449.1 for gentiopicroside, sweroside, swertiamarin, and the IS, respectively. Results: The linearity of the analytical response was good and the calibration curves were linear over concentration ranges of 1.20–192.0, 0.40–159.0, and 0.20–209.3 ng/mL for gentiopicroside, sweroside, and swertiamarin, respectively. The extraction recovery was in the range of 84.72%–91.34%, 84.58%–93.43%, and 82.75%–91.37% for gentiopicroside, sweroside, and swertiamarin, respectively. Conclusions: The method was successfully used to evaluate the pharmacokinetic parameters of gentiopicroside, sweroside, and swertiamarin in beagle dogs.
Keywords: Beagle dog, Gentianella acuta, pharmacokinetics, secoiridoid glycosides, ultra-high-performance liquid chromatography–tandem mass spectrometry
|How to cite this article:|
Wang ZB, Li K, Wang M, Wu GS, Bi YJ, Kuang HX. Pharmacokinetic and relative bioavailability of three secoiridoid glycosides in beagle dog plasma after oral administration of conventional and enteric-coated capsules of Gentianella acuta extract. World J Tradit Chin Med 2021;7:246-53
|How to cite this URL:|
Wang ZB, Li K, Wang M, Wu GS, Bi YJ, Kuang HX. Pharmacokinetic and relative bioavailability of three secoiridoid glycosides in beagle dog plasma after oral administration of conventional and enteric-coated capsules of Gentianella acuta extract. World J Tradit Chin Med [serial online] 2021 [cited 2021 Jun 16];7:246-53. Available from: https://www.wjtcm.net/text.asp?2021/7/2/246/316609
| Introduction|| |
Herbal medicines have had a long history of clinical use for thousands of years, playing an important role in maintaining people's health and curing diseases, especially complicated and chronic diseases. In contrast to modern medicines, herbal medicines are frequently used to treat chronic diseases. Plants are still the richest and most cost-effective source of innovative drugs, which significantly affect chronic diseases. Therefore, herbal medicines are widely accepted by many countries., Pharmacokinetic studies of the active components in herbal medicines are important to illustrate their intracorporeal process. Gentianella acuta (Michx.) Hulten is a well-known herbal medicine used in Ewenki folk medicine and Mongolian medicine. In Mongolian medicine, it is widely used for the treatment of jaundice, hepatitis, and fever. In Ewenki Folk Medicine, this herb is mainly used for the treatment of arrhythmia and coronary heart disease.,
Secoiridoid glycosides and iridoids are very important active components that can be found in many plants., Secoiridoid glycosides have been shown to be major bioactive components in G. acuta,, and including the three most abundant components, gentiopicroside, sweroside, and swertiamarin. These compounds exhibit a wide range of bioactivities, such as cardiovascular, antihepatotoxic, anti-inflammatory, sedative, and immunomodulatory activities.,
To date, analytical methods have been developed and published in the literature for the determination of gentiopicroside, sweroside, and swertiamarin in rat plasma and other biological samples using high-performance liquid chromatography (HPLC),,, capillary electrophoresis,, and LC-tandem mass spectrometry (MS/MS).,,,, Although numerous studies have investigated the pharmacological activities of gentiopicroside, sweroside, and swertiamarin, their simultaneous determination and pharmacokinetic studies in beagle dog plasma have not been reported. Therefore, it would be expedient to develop an ultra-HPLC (UHPLC)-MS/MS method for the simultaneous determination of gentiopicroside, sweroside, and swertiamarin in beagle dog plasma to enhance the understanding of their pharmacokinetics and pharmacological activities for clinical practical applications.
In this study, we developed a sensitive and selective UHPLC-MS/MS method for the quantification of three secoiridoid glycosides in beagle dog plasma. The method was subsequently fully validated and applied to a pharmacokinetic study of these secoiridoid glycosides after oral administration of G. acuta extract. The method offered a short run time (1.5 min) and high sensitivity for the analytes.
| Materials and Methods|| |
Chemicals and reagents
Gentiopicroside, sweroside, swertiamarin, and paeoniflorin (the internal standard [IS]) were purchased from Nanjing Jingzhu Bio-technology Co., Ltd. (Nanjing, China). The purity of swertiamarin and paeoniflorin was >98%. The chemical structures of the analytes are shown in [Figure 1]. HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (NJ, USA), and HPLC-grade formic acid was purchased from Dikma Technologies Inc. (CA, USA). Isopropanol and ethyl acetate (analytical grade) were purchased from Tian in Fuyu Fine Chemical Co., Ltd. (Tianjin, China). Deionized water was purchased from A. S. Watson Group Ltd. (Hong Kong, China).
|Figure 1: The chemical structures of analytes (1. gentiopicroside; 2. sweroside; 3. swertiamarin)|
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The whole grass samples of G. acuta were collected from the Daxing' an Mountains, dried, and then identified by Professor Wang Zhenyue of the Chinese Medicine Resources, College of Pharmacy, Heilongjiang University of Chinese Medicine.
Six male beagle dogs were provided by the Shenyang Kangping Institute of Laboratory Animal (SCXK [Liao] 2014-0003) and housed under controlled laboratory environmental conditions of 25°C ± 2°C and a natural light-dark photoperiod. All animal experiments were approved by the Institutional Animal Ethics Committee of Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang.
Plasma sample preparation
A 100 μL sample of beagle dog plasma was mixed with 20 μL IS (241.0 ng/mL) and 50 μL 0.2% formic acid in tubes, which were vortexed for 1 min, and then 2 mL ethyl acetate-isopropanol (9:1, v/v) was added. Then, the tubes were vortexed for 3 min, centrifuged at 6000 rpm for 10 min, and the supernatant was collected and dried at 35°C under a gentle stream of nitrogen. The dried residue was reconstituted with 100 μL of the mobile phase, vortexed for 3 min, transferred to a 1.5 mL centrifuge tube, and centrifuged at 12,000 rpm for 10 min. Finally, a 2 μL aliquot was directly injected into the UHPLC-MS/MS for analysis.
Instrumentation for ultra-high-performance liquid chromatography–tandem mass spectrometry
The UHPLC was performed using a Waters Acquity® UPLC system (Waters, Milford, MA, USA) equipped with a quaternary pump, an auto-sampler, and a column oven. An AB Sciex 4000 QTRAP™ mass spectrometer (AB Sciex, Foster, CA, USA) was used for mass spectrometric detection. The sample solutions were separated using an Acquity® UPLC ethylene bridged hybrid (BEH) amide column (2.1 mm × 100 mm, 1.7 μm) maintained at 40°C. The following isocratic program was used for the elution: Mobile phase A (water containing 0.1% formic acid) and B (acetonitrile) (80:20, v/v) run at a flow rate of 0.4 mL/min. The sample injection volume was 2 μL.
For the MS/MS detection, electrospray ionization (ESI) source was operated in the negative ion mode and the optimal parameters were as follows: curtain gas, 10 psi; ion source gas 1, 55 psi; ion source gas 2, 55 psi; source temperature, 450°C; and ionspray voltage, −4500 V (all gases: Nitrogen). Quantification was performed using multiple reaction monitoring, and the quantitative parameters are listed in [Table 1].
|Table 1: List of selected multiple reaction monitoring parameters, declustering potential, collision energy, entrance potential, and collision cell exit potential for each analyte and internal standard|
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Preparation of standards and quality control samples
Stock solutions of swertiamarin (0.930 mg/mL), gentiopicroside (0.640 mg/mL), and sweroside (0.530 mg/mL) were prepared in methanol. The stock solutions were further diluted with methanol to prepare a series of working concentrations. The stock solution of the IS was prepared at an initial concentration of 0.964 mg/mL, from which the working solution (241.0 ng/mL) was prepared using methanol as the diluent. All solutions were immediately stored at 4°C before use.
Mixed calibration standards samples for gentiopicroside (1.200, 6.000, 12.00, 24.00, 48.00, 96.00, and 192.0 ng/mL), sweroside (0.994, 4.969, 9.938, 19.88, 39.75, 79.50, and 159.0 ng/mL), swertiamarin (1.308, 6.541, 13.08, 26.16, 52.33, 104.65, and 209.3 ng/mL), and the IS (241.0 ng/mL) were prepared by spiking 100 μL blank plasma with moderate amounts of the working solutions. The mixed quality control (QC) samples (low, medium, and high) were prepared separately using the same procedure (2.400, 24.00, and 153.6 ng/mL for gentiopicroside; 1.988, 19.88, and 127.2 ng/mL for sweroside; and 2.616, 26.16, and 167.4 ng/mL for swertiamarin).
Preparation of whole plant extracts of Gentianella acuta
Whole G. acuta plant samples (100.1 g) were extracted with 70% ethanol (1:10, w/v) by refluxing twice for 1.5 h each time. The filtrates were combined and the methanol was evaporated using a rotary evaporator. Then, 1 g of the whole plant extract of G. acuta was found to contain gentiopicroside, sweroside, and swertiamarin (1.73, 0.98, and 0.60 mg, respectively), determined using HPLC.
The method was fully validated for selectivity, specificity, linearity, accuracy, precision, recovery, lower limits of quantification (LLOQ), matrix effect, and stability according to the guidelines set by the US Food and Drug Administration and previously published literature.,,,
The specificity was evaluated by comparing chromatograms of blank plasma from six different beagle dogs, blank beagle dog plasma spiked with analytes and the IS, and beagle dog plasma samples collected after oral administration of the G. acuta whole plant extract to test for potential interference from the retention time of analytes and the IS.
The calibration curves were evaluated using linear regression with a weighting factor (1/x2) based on the peak area ratios of the analytes and IS. The LLOQ was defined as the lowest concentration that could be determined with both relative error (RE) and relative standard deviation (RSD) within 20%.
Intraday and interday precision and accuracy were estimated by repeated analysis of the low, medium, and high QC samples (2.400, 24.00, and 153.6 ng/mL for gentiopicroside; 1.988, 19.88, and 127.2 ng/mL for sweroside; and 2.616, 26.16, and 167.4 ng/mL for swertiamarin, respectively) for 3 consecutive days. The variability of determination and accuracy were expressed as the RSD and RE, respectively.
To evaluate the matrix effect, blank dog plasma was extracted and then spiked with the analytes at three QC levels (2.400, 24.00, and 153.6 ng/mL for gentiopicroside; 1.988, 19.88, and 127.2 ng/mL for sweroside; and 2.616, 26.16, and 167.4 ng/mL for swertiamarin) in six replicates. The corresponding peak areas of the deproteinated blank plasma samples were compared with those of the pure standard solution at equivalent concentrations, and this peak area ratio was defined as the matrix effect. The matrix effect of the IS was similarly evaluated.
The extraction recovery of the analytes at the three QC levels (2.400, 24.00, and 153.6 ng/mL for gentiopicroside; 1.988, 19.88, and 127.2 ng/mL for sweroside; and 2.616, 26.16, and 167.4 ng/mL for swertiamarin) were evaluated by comparing the peak area ratios of regular pretreated QC samples with those of unextracted spiked samples at corresponding concentrations in six replicates. The extraction recovery of the IS was similarly evaluated.
The stability of the analytes in dog plasma samples was assessed by analyzing QC samples at three QC levels (2.400, 24.00, and 153.6 ng/mL for gentiopicroside; 1.988, 19.88, and 127.2 ng/mL for sweroside; and 2.616, 26.16, and 167.4 ng/mL for swertiamarin) in five replicates under four different conditions: (1) short-term: 4 h at room temperature; (2) long-term: −20°C in the freezer for 14 days; (3) autosampler: in the UPLC autosampler for 24 h; and (4) freeze-thaw: after three consecutive freeze-thaw cycles (−20°C to room temperature). The samples were considered stable if assay values were within the acceptable limits of accuracy (±15%, RE) and precision (RSD within 15%).
The G. acuta whole plant extract (39.90 mg/kg) was filled in capsules and administered orally to six beagle dogs, who were all starved for 12 h before the experiment but allowed access to water. Approximately 0.6 mL blood samples were collected from the forward limb vein of each dog at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 h. The blood samples were immediately centrifuged at 3500 rpm for 5 min to obtain the plasma and then stored at −80°C for further analysis. The following pharmacokinetic parameters were analyzed using the drug and statistics (DA) software (version 2.0, Shanghai, China), the area under the plasma concentration curve (area under the curve [AUC]), terminal elimination rate constant (Ke), maximum plasma concentration (Cmax), time to reach Cmax (Tmax), and the terminal elimination half-life (T1/2). All parameters are described as means ± SD.
| Results and Discussion|| |
The composition of the mobile phase affects the separation and ionization of analytes and the IS and plays an important role in improving the peak shapes, background noise, detection sensitivity, and shortening the run time. In this experiment, we investigated the chromatograms using methanol–water and acetonitrile–water systems as the mobile phase. The results showed that acetonitrile caused lower background noise and better peak shapes than methanol did. Moreover, a higher sensitivity and shorter run time were observed with acetonitrile. Modifying the composition of the mobile phase could improve ionization of the ion source and increase the analyte response. To optimize the mobile phase system, different proportions of formic acid (0.05%, 0.1%, and 0.2%) were used and the best response was obtained from acetonitrile and 0.1% formic acid in water (80:20, v/v). This composition of the mobile phase increased responses, improved the peak shapes, and provided a shorter run time for the analytes and IS [Figure 2].
|Figure 2: Typical multiple reaction monitoring chromatograms of gentiopicroside (a), sweroside (b), swertiamarin (c), and IS (d). (1) Blank plasma, (2) blank plasma sample spiked with the three analytes at the LLOQ and IS, (3) blank plasma sample spiked with the three analytes at QC samples and IS (96.00 ng/mL for gentiopicroside, 79.50 ng/mL for sweroside and 104.63 ng/mL for swertiamarin), and (4) beagle dog plasma samples collected 2.0 h after oral administration of Gentianella acuta extract. IS: Internal standard, LLOQ: Lower limit of quantification|
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Similar retention times between analytes may cause the occurrence of cross-talk, which could significantly affect the results of the analysis. Therefore, it is vital to optimize MS parameters to acquire steady and sensitive responses for analytes. In the present study, the most difficult challenge was achieving the effective separation of gentiopicroside and sweroside. First, we tried several different reversed phase chromatography columns, including the Acquity® UPLC CSHTM fluoro-phenyl (2.1 mm × 150 mm, 1.7 μm), Acquity® UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm), and Acquity® UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm). These columns were not effective in isolating the peaks of gentiopicroside and sweroside. Then, we tried the Acquity® UPLC BEH amide column (2.1 mm × 100 mm, 1.7 μm), which also did not effectively separate gentiopicroside and sweroside, although its run time (1.5 min) was much shorter than that of the other columns. Moreover, the amide column provided better peak shapes and higher sensitivity that those of the other columns. In addition, there was no relevant cross-talk between gentiopicroside and sweroside in this study as previously reported.
Optimization of mass spectrometry conditions
For the MS conditions, positive and negative ion models were tested with various combinations of the mobile phase. The results showed that sweroside was highly responsive in the positive ion mode, whereas gentiopicroside and swertiamarin exhibited lower responses in the positive ion mode than in the negative ion mode. In the negative ion mode, no significant [M-H]-peak of sweroside was detected, but a strong and stable [M + HCOO]- signal was observed. Moreover, gentiopicroside, swertiamarin, and the IS also exhibited a strong and stable signal in the [M + HCOO]-mode. Based on these findings, ESI was used in the negative ion mode to quantify the analytes. The full-scan product ion spectra of the analytes and IS are shown in [Figure 3].
|Figure 3: Full-scan product ion spectra of (a) gentiopicroside, (b) sweroside, (c) swertiamarin, and (d) paeoniflorin (internal standard) in the negative ionization mode|
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It is very important to choose a suitable chemical compound with similar characteristics to the analytes as an IS. Among several compounds tested for used as possible IS, amygdalin, paeoniflorin, and butylparaben exhibited good responses in the negative ion mode. However, the polarity of the three analytes was too great for amygdalin and butylparaben. Paeoniflorin was more suitable as an IS because it exhibited a shorter retention time and peak shape than those of the other tested compounds, and no significant direct interference was observed.
Optimization of sample pretreatment
The sample treatment method is important for improving extraction recovery and reducing the matrix effect. Protein precipitation, liquid–liquid extraction, and solid–liquid extraction are the three major methods of plasma treatment. In this study, we first used methanol or acetonitrile to precipitate the protein, and the results showed that the samples exhibited a severe matrix effect and low extraction recovery. Consequently, a liquid–liquid extraction method was explored. Based on previous literature reports,, we used different proportions of ethyl acetate and isopropanol and the results showed that a 9:1 (v/v) ratio exhibited optimum recovery with no significant matrix effects for the three analytes and the IS. In addition, our analytical samples were weakly acidic, and therefore, we added different proportions of formic acid solution and found that 50 μL of 0.2% formic acid increases the recovery and further reduces the matrix effects. Therefore, we evaluated the plasma treatment method using 50 μL 0.2% formic acid, before adding 2 mL ethyl acetate–isopropanol (9:1, v/v) to 100 μL plasma for all the samples.
Specificity and selectivity
The typical chromatograms of gentiopicroside, sweroside, swertiamarin, and the IS in beagle dog plasma [Figure 2] showed retention times of 1.04, 1.06, 1.19, and 1.02 min, respectively. The analytes and the IS exhibited no significant interference and good selectivity and specificity at the retention times of the analytes.
Linearity and lower limit of quantification
Linear relationships were observed at ranges of 1.200–192.0 ng/mL for gentiopicroside, 0.994–159.0 ng/mL for sweroside, and 1.308–209.3 ng/mL for swertiamarin. The analyte regression equations are as follows: Y = 1.01 × 10-2X − 0.1214 (correlation coefficient [r] = 0.9950, gentiopicroside); Y = 0.19 × 10-2X − 0.0013 (r = 0.9960, sweroside); Y = 0.24 × 10-2X + 0.0016 (r = 0.9975, swertiamarin), where Y represents the peak area ratio of each analyte to the IS and X is the nominal concentration of the analyte. The LLOQs were 1.20, 0.40, and 0.12 ng/mL for gentiopicroside, sweroside, and swertiamarin, respectively.
Accuracy and precision
The accuracy and precision of gentiopicroside, sweroside, and swertiamarin were assessed by evaluating QC samples at three levels with six replicates [Table 2]. The intra- and interday precisions were 2.57 and 9.67%, respectively, and the accuracy was −4.26% to 7.14%. The results indicated that the method was reproducible and reliable for the analysis of beagle dog plasma samples.
|Table 2: Precision and accuracy of the analytes in beagle dog plasma (n=6)|
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Recovery and matrix effect
The extraction recovery and matrix effect were conducted with six replicates for the QC samples. As shown in [Table 3], the mean recovery of the analytes was >82.75% (RSD <9.76%), which indicated that the recovery was consistent and reproducible. The corresponding matrix effect ranged from 92.93% to 99.51% (RSD <7.81%), which indicated that there were no significant matrix effects. The overall average extraction recovery and matrix effect of the IS were 87.07% and 96.26%, respectively.
|Table 3: Extract recovery and matrix effect of the analytes in in beagle dog plasma (n=6)|
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The stability of gentiopicroside, sweroside, and swertiamarin during the sample storage and processing procedures was evaluated by analyzing the QC samples, and the results are presented in [Table 4]. The long-term (30 days), freeze-thaw, autosampler, and room temperature stability results indicated that gentiopicroside, sweroside, and swertiamarin were stable under all the investigated storage conditions because the bias in concentration was within ± 15% of nominal values. Consequently, it is a suitable method for pharmacokinetic studies.
|Table 4: Stability of the analytes in beagle dog plasma under various storage conditions (n=6)|
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The UHPLC-MS/MS method was successfully used for a pharmacokinetic study of gentiopicroside, sweroside, and swertiamarin in beagle dog plasma after oral administration of G. acuta extract. The mean plasma concentration–time profiles of gentiopicroside, sweroside, and swertiamarin are shown in [Figure 4], and the main pharmacokinetic parameters from the noncompartmental model are shown in [Table 5].
|Figure 4: Mean plasma concentration-time profiles of (a) gentiopicroside, (b) sweroside and (c) swertiamarin in beagle dogs after oral administration of the whole plant of G. acuta extract (n = 6)|
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|Table 5: Pharmacokinetic parameters of the analytes after oral administration conventional capsule and enteric-coated capsules to beagle dog plasma (mean±standard deviation, n=6)|
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First, we determined that the AUC from time 0 to t, AUC0-t) of enteric-coated gentiopicroside, sweroside, and swertiamarin capsules was 2.3, 2.2, and 2.1 times higher than that observed for the conventional capsule, respectively. This indicated that enteric-coated capsules could improve the hydrolysis of these compounds, thereby improving the absorption in vivo.
In addition, the plasma concentrations of gentiopicroside, sweroside, and swertiamarin increased to the mean Cmax concentrations of 96.46 ± 7.24, 78.50 ± 6.36, and 71.17 ± 2.39 ng/mL at 1.33 ± 0.24, 1.41 ± 0.19, and 1.08 ± 0.19 h, respectively, for the conventional capsules, whereas the corresponding values were 105.89 ± 15.20, 98.57 ± 8.25, and 67.23 ± 2.20 ng/mL at 1.42 ± 0.20, 1.75 ± 0.27, and 1.58 ± 0.20 h, respectively, for enteric-coated capsules. The T½ of gentiopicroside, sweroside, and swertiamarin was 9.23 ± 2.80, 14.25 ± 8.83, and 13.38 ± 4.43 for conventional capsule and 6.28 ± 1.72, 7.91 ± 2.15, and 9.78 ± 3.34 for enteric-coated capsules, respectively. The results showed that the absorption and elimination rate of enteric-coated capsules were slower than those of conventional capsules, probably because the enteric-coated capsules increased the intestinal hydrolysis capacity. These effects were attributable to the better absorptive permeability, transporter-mediated efflux, and hydrolytic metabolism of the enteric-coated capsules in the intestine.
The results showed that the concentration–time curve of swertiamarin had a double peak, which is consistent with a previous report. This phenomenon may be related to the intestinal absorption sites, enterohepatic circulation, and gastric emptying process., To ascertain the mechanism of the double-peak phenomenon in the pharmacokinetics, more detailed absorption studies are needed. In addition, the pharmacokinetic behavior of the three analytes from the G. acuta extract was different from that of other plant extracts.,,, The difference was likely due to other components of G. acuta affecting the metabolic processes of gentiopicroside, sweroside, and swertiamarin in vivo. Furthermore, it was also possible that the metabolic processes of the three analytes in beagle dog plasma differed from those in rat plasma.
| Conclusions|| |
In this study, a simple, sensitive, and fast UHPLC-MS/MS method was developed and validated for the simultaneous determination of gentiopicroside, sweroside, and swertiamarin in beagle dog plasma for the first time, to the best of our knowledge. This method had a shorter total run time of 1.5 min and higher sensitivity (LLOQs of gentiopicroside, sweroside, and swertiamarin were 1.20, 0.40, and 0.20 ng/mL, respectively). This method exhibited satisfactory linearity, accuracy, precision, sensitivity, matrix effect, and reproducibility. The results will provide helpful information for further clinical pharmacokinetic studies.
Financial support and sponsorship
This work was supported by the Research Project of Heilongjiang University of Chinese Medicine “Supporting Plan for Excellent Innovative Talents” (2018RCD03), Heilongjiang Provincial Science Fund Project (H2018056), Heilongjiang Post-doctoral Research Start Fund Project (LBH-Q16214), General Projects of NSFC (81973439, 81872979, and 81803686), and Research Fund of Heilongjiang University of Chinese Medicine (201504).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Sheng N, Zhi X, Yuan L, Zhang Z, Jia P, Zhang X, et al
. Pharmacokinetic and excretion study of three secoiridoid glycosides and three flavonoid glycosides in rat by LC-MS/MS after oral administration of the Swertia pseudochinensis
extract. J Chromatogr B Analyt Technol Biomed Life Sci 2014;967:75-83.
Garg V, Dhar VJ, Sharma A, Dutt R. Facts about standardization of herbal medicine: A review. J Chin Integr Med 2012;10:1077-83.
Calixto JB. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz J Med Biol Res 2000;33:179-89.
Chunliang W, Khasbagan BG. Ewenki folk medicinal plants and its comparison with mongolian medicine. Chin J Ethnomed Ethnopharm 2009;18:156-8.
Wang Z, Wu G, Liu H, Xing N, Sun Y, Zhai Y, et al
. Cardioprotective effect of the xanthones from Gentianella acuta
against myocardial ischemia/reperfusion injury in isolated rat heart. Biomed Pharmacother 2017;93:626-35.
Kumar S, Sati OP, Semwal VD, Nautiyal M, Sati S, Takeda Y. Iridoid glycosides from Lonicera quinquelocularis
. Phytochemistry 2000;53:499-501.
Tan RX, Kong LD, Wei HX. Secoiridoid glycosides and an antifungal anthranilate derivative from Gentiana tibetica
. Phytochemistry 1998;47:1223-6.
Liu Y, Shi W, Ni Y, Chao L, Wang T, Zhang Y. Isolation and identification of iridoidal glycosides from the whole plant of Gentianella acuta
. J Shenyang Pharm Univ 2016;33:702-5.
Ding Z, Liu Y, Ruan J, Yang S, Yu H, Chen M, et al
. Bioactive constituents from the whole plants of Gentianella acuta
(Michx.) Hulten. Molecules 2017;22:E1309.
Ni Y, Liu Y, Ruan J, Han L, Wang T, Zhang Y. Isolation and identification of iridoidal glycosides from the whole plant of Gentianella acuta
(II). Chin J Med Chem 2017;27:133-7.
Ghisalberti EL. Biological and pharmacological activity of naturally occurring iridoids and secoiridoids. Phytomedicine 1998;5:147-63.
Wu S, Ning Y, Zhao Y, Sun W, Thorimbert S, Dechoux L, et al
. Research progress of natural product gentiopicroside – A secoiridoid compound. Mini Rev Med Chem 2017;17:62-77.
Wang CH, Cheng XM, He YQ, White KN, Bligh SW, Branford-White CJ, et al
. Pharmacokinetic behavior of gentiopicroside from decoction of Radix Gentianae, Gentiana macrophylla
after oral administration in rats: A pharmacokinetic comaprison with gentiopicroside after oral and intravenous administration alone. Arch Pharm Res 2007;30:1149-54.
Wang CH, Cheng XM, Bligh SW, White KN, Branford-White CJ, Wang ZT. Pharmacokinetics and bioavailability of gentiopicroside from decoctions of Gentianae
and Longdan Xiegan Tang after oral administration in rats – Comparison with gentiopicroside alone. J Pharm Biomed Anal 2007;44:1113-7.
Jun C, Xue-Ming Z, Chang-Xiao L, Tie-Jun Z. Simultaneous determination of swertiamarin and its metabolites (5Z)-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3,4-c] pyridin-1-one and erythrocentaurin in broth of Aspergillus niger
by HPLC. Biomed Chromatogr 2008;22:191-5.
Takei H, Nakauchi K, Yoshizaki F. Analysis of swertiamarin in Swertia herb and preparations containing this crude drug by capillary electrophoresis. Anal Sci 2001;17:885-8.
Zhao S, Liu Q, Chen X, Hu Z. Separation and determination of gentiopicroside and swertiamarin in Tibetan medicines by micellar electrokinetic electrophoresis. Biomed Chromatogr 2004;18:10-5.
Li HL, Peng XJ, He JC, Feng EF, Xu GL, Rao GX. Development and validation of a LC-ESI–MS/MS method for the determination of swertiamarin in rat plasma and its application in pharmacokinetics. J Chromatogr B 2011;879:1653-8.
Feng B, Zhu H, Guan J, Zhao L, Gu J, Yin L, et al
. A rapid and sensitive UFLC-MS/MS method for the simultaneous determination of gentiopicroside and swertiamarin in rat plasma and its application in pharmacokinetics. J Pharm Pharmacol 2014;66:1369-76.
Sheng N, Yuan L, Zhi X, Cui C, Zhang Z, Jia P, et al
. Application of a liquid chromatography-tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion studies of sweroside in rats. J Chromatogr B Analyt Technol Biomed Life Sci 2014;969:1-1.
Wu Y, Ai Y, Wang F, Ma W, Bian Q, Lee DY, et al
. Simultaneous determination of four secoiridoid and iridoid glycosides in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry and its application to a comparative pharmacokinetic study. Biomed Chromatogr 2016;30:97-104.
Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 2003;75:3019-30.
Rocci ML Jr., Devanarayan V, Haughey DB, Jardieu P. Confirmatory reanalysisof incurred bioanalytical samples. AAPS J 2007;9:E336-43.
Fast DM, Kelley M, Viswanathan CT, O'Shaughnessy J, King SP, Chaudhary A, et al
. Workshop report and follow-up – AAPS Workshop on current topics in GLP bioanalysis: Assay reproducibility for incurred samples – Implications of Crystal City recommendations. AAPS J 2009;11:238-41.
Yadav M, Shrivastav PS. Incurred sample reanalysis (ISR): A decisive tool in bioanalytical research. Bioanalysis 2011;3:1007-24.
Li L, Liang S, Du F, Li C. Simultaneous quantification of multiple licorice flavonoids in rat plasma. J Am Soc Mass Spectrom 2007;18:778-82.
Zhao L, Sun X, Xiong Z, Wang S. Simultaneous determination of mosapride and its active des-p-fluorobenzyl and 4'-N-oxide metabolites in rat plasma using UPLC-MS/MS: An application for a pharmacokinetic study. Talanta 2015;137:130-5.
Mustafa AM, Caprioli G, Ricciutelli M, Maggi F, Marín R, Vittori S, et al
. Comparative HPLC/ESI-MS and HPLC/DAD study of different populations of cultivated, wild and commercial Gentiana lutea
L. Food Chem 2015;174:426-33.
Cai F, Xu W, Wei H, Sun L, Gao S, Yang Q, et al
. Simultaneous determination of active xanthone glycosides, timosaponins and alkaloids in rat plasma after oral administration of Zi-Shen Pill extract for the pharmacokinetic study by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2010;878:1845-54.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]