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Abstract

Gastric ulcer (GU) is one of the major gastrointestinal disorder diseases, with increasing incidence and prevalence globally. Modified Xiaochaihu granules (MXCHG) have been used effectively for treating chronic gastritis and GU clinically. To investigate the pharmacokinetics and tissue distribution of MXCHG, an ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was established for the simultaneous determination of 8 bioactive ingredients (baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, berberine hydrochloride, saikosaponin a, and saikosaponin d) in rat plasma and various tissues using puerarin as an internal standard (IS). The biological samples were pretreated by protein precipitation with acetonitrile. The chromatographic separation was carried out on a C₁₈ column with a gradient mobile phase consisting of acetonitrile and 0.1% formic acid in water. All analytes and IS were quantitated through ESI in the positive/negative ion multiple reaction monitoring mode. The mass transitions were as follows: m/z 445.0 → 268.5 for baicalin, m/z 458.7 → 282.8 for wogonoside, m/z 269.2 → 222.6 for baicalein, m/z 417.0 → 254.8 for liquiritin, m/z 822.1 → 350.8 for glycyrrhizic acid, m/z 336.0 → 319.9 for berberine hydrochloride, m/z 780.3 → 618.5 for saikosaponin, and m/z 415.0 → 294.6 for the IS. The validated method was successfully applied to the pharmacokinetics and tissue distribution study of 8 compounds in rat plasma and tissues after the intragastric administration of MXCHG. The results demonstrated that 8 components were distributed widely and rapidly in various rat tissues after intravenous administration. Tissue deposition of the compounds in the rats was mainly in the small intestine and stomach. The present study can provide more useful information to guide the clinical use of MXCHG and the developed analytical method can also be applied for further clinical pharmacokinetic studies.

Keywords

bioactivity modified Xiaochaihu granules gastric ulcer UPLC-ESI-MS/MS pharmacokinetics tissue distribution

Gastric ulcer (GU) is a common alimentary system disease and its incidence is increasing worldwide. According to WTO’s statistics, the incidence of GU was up by 80%. The prevalence of GUs in Western countries is 2.4%, with annual incidence rates ranging from 0.10% to 0.19%.^1
-3 In China, 12 million people have GUs, the highest incidence of GUs in the world.^4,5 A variety of factors contribute to the progression of GU, including poor diets, Helicobacter pylori (H. pylori) infection, gastric mucosa ischemia, smoking, psychological stress, the excessive use of nonsteroidal anti-inflammatory drugs, and alcohol.^6,7 Ethanol is a well-known cause of gastric damage by altering protective factors, including decreasing mucus production and blood circulation within the mucosa.⁸ Many different experimental models of GU induction have been developed for pharmacological research.⁹

Until now, conventional medical therapy has been used in the prevention and treatment of GUs.^10,11 However, its clinical utility is limited by a lack of efficacy and severe side effects.¹² Traditional Chinese medicine (TCM) has received more attention and acceptance worldwide in recent years because both clinical and experimental studies have demonstrated that TCM exhibited therapeutic benefit for GUs with fewer side effects.^13,14 Moreover, the cost of TCM for GUs is only about one-sixth that of Western medicine.^1
-3 Therapies collectively called TCM, which includes Chinese herbal medicine, the extracts of single ingredients or mixtures, and prescription medication, are commonly used to treat GUs.^15
-17

Modified Xiaochaihu granules (MXCHG) have been used clinically for treating chronic gastritis and GU for more than 10 years in southern China. Modified Xiaochaihu granules contains Chaihu (Radix bupleuri), Huangqin (Radix Scutellariae), Renshen (Ginseng), Banxia (Pinellia Tuber), Gancao (Radix Glycyrrhizae), Shengjiang (Rhizoma Zingiberisrecens), Dazao (Fructus Jujubae), Huanglian (Rhizoma Coptidis), Ganjiang (Rhizoma Zingiberis), Fuling (Poria), and Baizhu (Rhizoma Atractylodis Macrocephalae). The secondary metabolites of Chinese herbal medicines include many different classes of compounds, which are difficult to isolate and concentrate from Chinese herbal preparations. Our previous study demonstrated the efficiency of MXCHG for treating GUs.¹⁸ Several active components have been identified in vitro and in vivo, including flavonoids, such as baicalin, wogonoside, baicalein, and liquiritin; alkaloids, such as berberine; and triterpenoid saponins, such as saikosaponin a, saikosaponin d, and glycyrrhizic acid.¹⁹ For bioactivity, baicalin was shown to protect against ethanol-induced chronic gastritis in rats by inhibiting the Akt/NF-κB pathway.²⁰ Wogonoside not only dose-dependently decreased the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 but also inhibited the release of pro-inflammatory cytokines such as tumor necrosis factor-alpha and interleukin-6 in lipopolysaccharide-induced RAW264.7 cells.²¹ Baicalein displayed gastroprotective effects. It significantly inhibited the formation of acute ulcers induced by acidified ethanol, reduced the inflammatory process, stimulated the cellular antioxidant mechanism, and increased the amount of mucus.²² Berberine exerted therapeutic effects on H. pylori-induced chronic atrophic gastritis, and the anti-inflammatory property of berberine was related to the suppression of the IRF8-IFN-γ signaling axis.²³ Saikosaponin a and saikosaponin d showed potent anti-inflammatory activity through inhibitory effects on NF-κB activation, iNOS and COX-2, and pro-inflammatory cytokines.²⁴ However, the saikosaponins were poorly absorbed in vivo and difficult to detect in plasma. To observe the pharmacokinetics and tissue distribution of saikosaponins and provide a reference for their clinical usage, 4 times the human equivalent dose, 22.4 g/kg MXCHG, was administered in the current study based on the previous experimental results.²⁵

The composition of TCM is complex, including multiple components that display synergistic effects on multiple systems or multiple targets. Many pharmacokinetic studies on the active components in TCM prescriptions in animal models have focused on single herbs.^20
-22 However, to the best of our knowledge, reports on both the pharmacokinetics and the tissue distribution of several bioactive compounds in TCM prescriptions are rare.^23,24In the present study, a sensitive and selective ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed for the simultaneous determination of 8 bioactive compounds in rat plasma and 6 tissue samples in ethanol-induced rats after a single oral administration of 22.4 g/kg MXCHG. The aim of the current study was to provide a systematic analysis of MXCHG to establish more useful information to guide the clinical use of MXCHG and develop an analytical method for further clinical pharmacokinetic studies.

Methodology

Chemicals and Materials

Authentic standards of baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, berberine hydrochloride, saikosaponin a, saikosaponin d, and puerarin (internal standard, IS) were obtained from the National Institutes for Food and Drug Control (Beijing, China). The purity of all the references and internal standards was over 98%. Mass grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany) and experimental water was purified by a Millipore Alpha-Q system (Bedford, MA, United States). Formic acid and other chemicals and reagents were of analytical grade.

Modified Xiaochaihu granule was prepared by the Dongguan Hospital of Traditional Chinese Medicine (Dongguan, China). A voucher specimen was stored in the Key Laboratory of Traditional Chinese Medicine Resources and Traditional Chinese Medicine Chemistry at Hubei University of Chinese Medicine. The batch numbers for 3 parallel samples were 150504, 150505, and 150506, and each individual package weighed 10 g.²⁵

Experimental Animals

Animal care and all the experimental procedures abided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley (SD) rats, weighing 200 ± 20 g were obtained from the Experimental Animal Center of Hubei Province (No. SCXK2012-0068, Wuhan, China). The animals were housed in cages for at least 1 week before experimentation, with ad libitum access to food and water. Prior to the experiments, all animals were fasted overnight, but had free access to water. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee and the local experimental Ethics Committee (Laboratory Animal Certificate no. SYXK2017-0067).

Ultraperformance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry Conditions

The analyses were performed on a Shimadzu 30AD liquid chromatography system (Shimadzu, Japan), equipped with an AB 4500 Triple Quad MS system (ABSCIEX, Framingham, MA, United States).

An Agilent Zorbax SB-C₁₈ column (2.1 mm × 100 mm, 1.8 µm) was used for the chromatographic separation, the mobile phase was 0.1% (v/v) formic acid in water (A) and acetonitrile (B) with a gradient elution (0-1 minute, 5% B; 1-4 minutes, 5%-98% B; 4-6 minutes, 98% B; 6-6.5 minutes, 98% B; 6.5-7 minutes, 98%-5% B). The flow rate was 0.4 mL/min, the column temperature was 40 °C, the autosampler temperature was 4 °C, and the injection volume was 2 µL.

Mass spectrometric detection was carried out on a triple quadrupole mass spectrometer with an ESI source in positive and negative ionization switching modes, and quantification was performed in the multiple reaction monitoring (MRM) mode. To separate and determine baicalin and berberine efficiently, the MS parameters were optimized as follows: ion spray voltage 5500 (−4500) V, ion source temperature: 500 °C, nebulizer gas pressure 35 psi, and dwell time 100 ms. Nitrogen served as the nebulizing agent and drying gas and was of high purity. The MS data were analyzed using Analyst 1.6 data software. The optimized precursor ion, daughter ion, declustering potential (DP), and collision energy (CE) of each analyte are listed in Table 1. The product ion mass spectra of 8 analytes and the IS are shown in Supplemental Figure S1.

Table 1

Retention Time, Multiple Reaction Monitoring Transitions, and Mass Spectrometric Parameters of the Analytes and Internal Standard.

Analyte	Retention time (min)	Precursorion (m/z)	Daughterion (m/z)	ESI	DP (V)	CE (V)
Berberine hydrochloride	3.1	336.0	319.9	+	60	43
Baicalin	3.1	445.0	268.5	−	−101	−34
Wogonoside	3.3	458.7	282.8	−	−78	−22
Baicalein	3.6	269.2	222.6	−	−113	−34
Liquiritin	2.9	417.0	254.8	−	−100	−25.6
Glycyrrhizic acid	3.5	822.1	350.8	−	−270	−56
Saikosaponin a	3.6	780.3	618.5	−	−220	−54
Saikosaponin d	3.9	780.3	618.5	−	−220	−54
Puerarin (IS)	2.7	415.0	294.6	−	−34	−30

CE, collision energy; DP, declustering potential; ESI, electrospray ionization; IS, internal standard.

Preparation of Calibration Standard Solutions, Internal Standard Solution, Quality Control Solutions, and Modified Xiaochaihu Granule Solution

A mixed stock standard solution including baicalin (0.408 µg/mL), wogonoside (0.199 µg/mL), baicalein (2.01 µg/mL), liquiritin (0.106 µg/mL), glycyrrhizic acid (10.45 µg/mL), berberine hydrochloride (0.04 µg/mL), saikosaponin a (10.1 µg/mL), and saikosaponin d (10.15 µg/mL) was prepared in methanol and diluted to produce a series of working standard solutions. The IS stock solution of puerarin (105 µg/mL) in methanol was diluted to obtain an IS working solution (0.105 µg/mL). Working solutions used for spiking the plasma/tissue homogenates were freshly prepared by diluting the stock solutions with methanol to the appropriate concentrations. Modified Xiaochaihu granules were dissolved in warm water to obtain a 1 g/mL solution.

Calibration standards were prepared by freshly spiking the appropriate working and IS working solutions (20.0 µL) into blank pooled plasma and tissue homogenates (200.0 µL) to prepare different concentrations (Supplemental Table S1) of 8 active compounds in plasma and tissue homogenates, then processed as described in the preparation of sample solutions (see the Tissue Distribution Study and Method Validation sections). Quality control (QC) samples (at different concentrations, Supplemental Tables S2-S4) used for the recovery, matrix effect, intraday and interday accuracy, precision, and stability studies were prepared in the same way as the calibration standards.

Pharmacokinetic Study

Acute gastric lesions were induced by the intragastric administration of absolute ethanol according to a published method.²⁶ Eighteen rats were randomly divided into 3 groups, each consisting of 6 animals. The blank group received vehicle (0.9% NaCl) throughout the course of the experiments. The ulcer group was intragastrically administered absolute ethanol (0.5 mL/kg body weight) to induce GUs (GU group). The normal group received 0.9% NaCl. Modified Xiaochaihu granule (22.4 g/kg) was administered to the normal group and GU group after 1 hour.

After intravenous administration, blood samples (500 µL) were collected from the suborbital venous plexus into heparinized tubes at 0, 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 hours. The blood samples were centrifuged at 3500 rpm for 10 minutes at 4 °C, and plasma was separated and stored at −80 °C until the assays. The plasma samples were prepared as follows. The plasma sample (200 µL), 20 µL of IS working solution, and 20 µL of 1 mol/mL HCl were added to a 1.5 mL centrifuge tube, followed by the addition of 600 µL of acetonitrile. The tube was vortex mixed for 2 minutes and centrifuged at 13 000 rpm for 10 minutes. The upper layer was collected and dried under gentle N₂ streaming (37 °C). The dried residue was redissolved in 200 µL of the mobile phase and then centrifuged at 13 000 rpm for 10 minutes. The supernatant was injected into the UPLC-MS/MS system for analysis.

Tissue Distribution Study

The animal models for tissue distribution were the same as for the pharmacokinetic study. Seventy-eight SD rats were randomly divided into 3 groups (6 in the blank group, 36 in the normal group, and 36 in the ethanol-induced ulcer group) for tissue sample collection at 0.5, 1, 3, 6, 12, and 24 hours following the administration of 22.4 g/kg MXCHG. Six rats in each group were euthanized by decapitation. Tissues (including heart, liver, spleen, stomach, kidneys, and small intestine) were harvested and rinsed in ice-cold 0.9% NaCl to remove superficial blood. After blotting dry with filter paper, 0.5 g tissues were weighed accurately and homogenized in 4 volumes of 0.9% NaCl to prepare homogenates. These were stored at −80 °C until the assay. The tissue sample preparation procedure was the same as that for the plasma samples in the Pharmacokinetic Study section.

Method Validation

Specificity

Blank plasma/tissues, blank plasma/tissues spiked with serial concentrations of baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, berberine hydrochloride, saikosaponin a, and saikosaponin d, and the rat plasma collected 1.0 hour after the administration of 22.4 g/kg concentrate of MXCHG (n = 6) were analyzed. All samples were treated as in the Tissue Distribution Study and Method Validation sections, and analyzed by UPLC-MS/MS.

Linearity, lower limit of detection, and lower limit of quantitation

Calibration curves were established by plotting the ratios of the peak area of each analyte to the IS vs the plasma/tissues concentrations based on a weighted linear least-squares regression model (1/x ²). The lower limit of detection (LLOD) and the lower limit of quantitation (LLOQ) were used to determine the sensitivity of the assay based on the responses at signal-to-noise ratio (S/N) of 3 and 10, respectively. According to the international guidelines, the LLOQ was repeated 5 times to estimate the lowest concentration of the calibration curve with precision lower than 20% and accuracy of 80% to 120%.

Precision and accuracy

The intraday precision was analyzed in 6 replicate determinations of the control solutions (low, middle, and high concentration in the linear range), which were determined in 24 hours. Low, middle, and high concentrations of the control solutions in the linear range were prepared and analyzed on 3 consecutive days to determine the interday precision. Relative standard deviation (RSD%) was used to evaluate the intra- and interday precision. The relative error (RE%) was calculated according to the following formula: RE% = [(assayed value − nominal value)/nominal value] × 100%.

Extraction recovery and matrix effects

Three QC samples with low, medium, and high concentrations were prepared and analyzed. Meanwhile, the peak areas of the blank plasma/tissues extracted with acetonitrile and then spiked with baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, berberine hydrochloride, saikosaponin a, and saikosaponin d standards at different concentrations were analyzed. Then, the extraction recoveries of 8 effective substances were determined by comparing the spiked and the unspiked samples’ peak areas (n = 6). The matrix effect was determined by comparing the peak area of the pure standard solutions with the extracted blank plasma samples/tissues spiked with analytes at 3 different concentrations (low, medium, and high) in 6 replicates.

Stability

The stability of each analyte was measured by analyzing different QC samples under 3 different conditions. The short- and long-term stability was tested with different QC samples at room temperature for 8 hours and at −80 °C for 20 days, respectively. Freeze-thaw stability was determined with different QC samples after 3 freeze-thaw cycles.

Data Analysis

The rat plasma and tissue concentrations at different time points were analyzed by DAS 3.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). All pharmacokinetic parameters were processed by a noncompartmental method from the plasma concentration-time data, including the biological half-life (t _1/2), time to peak (t _max), the measured maximal concentration (C _max), area under curve (AUC_0-t), AUC from 0 to infinity (AUC_0-∞), the mean residual time (MRT), and clearance rate (CL). A t-test was employed to analyze the pharmacokinetic parameter results. The values are shown as the mean ± standard deviation from 6 independent measurements of 6 SD rats. A P-value of <.05 was considered statistically significant.

Results

Method Development

Optimization of ultraperformance liquid chromatography and tandem mass spectrometry conditions

According to the longer peak retention time of glycyrrhetinic acid compared with the other components, acetonitrile-water was selected as the mobile phase to satisfy the needs of large batch determinations of biological samples. In addition, experiments on the effects of aqueous solvents on each component certified that formic acid improved the chromatographic peak shape and ionization efficiency. Therefore, acetonitrile and 0.1% formic acid were selected as the mobile phase, and the analysis of all 8 compounds was performed successfully within only 7 minutes under this condition.

Optimization of the sample preparation procedure

Sample preparation should be optimized to achieve reproducibility, efficiency, and low cost. We compared the protein precipitation method and the liquid-liquid extraction method. The extraction recovery of 8 compounds was not significantly different between 2 methods. Thus, the protein precipitation method was selected as a simple and rapid procedure in this study. In addition, acetonitrile was shown to be the optimal precipitation solution because of its high extraction efficiency and repeatability.

Method Validation

Specificity

The specificity was tested by analyzing blank plasma; blank biological matrix samples spiked with baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, berberine hydrochloride, saikosaponin a, and saikosaponin d; and the plasma of rats intragastrically administered MXCHG. The 8 active ingredients were detected in 7 minutes without any endogenous interference. This method was fast and specific due to the high selectivity of the MRM mode. Typical chromatograms of blank rat plasma, blank rat plasma spiked with pure standard solutions, and plasma samples obtained 1.0 hour after the intragastric administration of 22.4 g/kg MXCHG are shown in Figure 1.

Figure 1

Typical multiple reaction monitoring chromatograms of 8 analytes and internal standard in rat plasma: (A) blank plasma, (B) blank plasma spiked with 8 analytes at lower limit of quantitation and internal standard, and (C) plasma sample collected at 1.0 hour after intragastric administration of modified Xiaochaihu granule (22.4 g/kg).

Linearity, lower limit of detection, and lower limit of quantitation

The correlation coefficients of the calibration curves were all over 0.9969, suggesting excellent linearity within the selected concentration range in the tested rat matrix. Lower limit of detection and LLOQ indicate the sensitivity of the equipment and method. The detailed results are shown in Supplemental Table S1. Hence, this method was considered sensitive for the quantification of 8 compounds.

Precision and accuracy

The intra- and interday precision (RSD) of all analytes was less than 14.5%, and the accuracy (RE) for all analytes ranged from −12.6% to 14.5%. The detailed results are shown in Supplemental Table S2, indicating that this method was precise and accurate enough to analyze the plasma and tissue samples.

Extraction recovery and matrix effects

Extraction recovery could reveal if the study methods were acceptable. In Supplemental Table S3, the extraction recoveries of 8 active compounds in rat plasma ranged from 78.1% to 104.8% with an RSD% of less than 14.6%, and from 80.1% to 106.8% in rat tissue with an RSD% of less than 13.4%. The matrix effect values of 8 active compounds in rat plasma ranged from 78.5% to 109.7% with an RSD% of less than 12.1%, and from 80.5% to 110.0% in rat tissue with an RSD% of less than 14.9%. The results showed that the matrix effect was negligible.

Stability

The results shown in Supplemental Table S4 demonstrated that all analytes were stable under the tested conditions. For plasma samples stored at room temperature for 8 hours, the RSD values were in the range of 1.21% to 14.3%, and those for samples stored in a freezer at −80 °C for 20 days were in the range of 1.2% to 13.2%. After 3 freeze-thaw cycles, the RSD values ranged from 2.1% to 11.3%. For the tissue samples stored short term at room temperature for 8 hours, the RSD values ranged from 0.18% to 14.8%, and from 0.15% to 14.4% in samples stored long term in a freezer at −80 °C for 20 days. After 3 freeze-thaw cycles, the RSD values ranged from 0.56% to 12.2%.

Pharmacokinetic Study

The validated analytical method was successfully applied to the intragastric administration pharmacokinetic study. The mean plasma concentration-time curves are shown in Figure 2, and the main pharmacokinetic parameters are listed in Table 2. Baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, and berberine hydrochloride could be detected in the GU rat plasma. In general, the C _max, AUC_0-t, AUC_0-∞, and CL values of baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, and berberine hydrochloride in the GU group were significantly higher than those in the normal group. The t _max and t _1/2 values were similar between the GU group and the normal group. Only t _max and t _1/2 of wogonoside exhibited significant differences between the GU group and the normal group. In addition, the MRT_0-∞ of baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, and berberine hydrochloride was significantly prolonged in the GU group. These data indicate a higher systemic exposure of baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, and berberine hydrochloride in the GU rat than in the normal rat.

Figure 2

Mean plasma concentration-time curves for 8 analytes after the intragastric administration of modified Xiaochaihu granule (22.4 g/kg) for the normal and gastric ulcer groups: baicalin (A), wogonoside (B), baicalein (C), liquiritin (D), glycyrrhizic acid (E), berberine hydrochloride (F), saikosaponin a (G), and saikosaponin d (H).

Table 2

Pharmacokinetic Parameters of Analytes in Rats After Oral Administration of Modified Xiaochaihu Granules (Mean ± SD, n = 6).

Analyte	Group	t _max (h)	t _1/2 (h)	C _max (μg/L)	AUC_0-t (μg/h/L)	AUC_0-∞ (μg/h/L)	MRT_0-∞ (h)	CL (L/h/kg)
Baicalin	Normal	1.58 ± 0.67	7.51 ± 0.18	35.44 ± 1.44	442.85 ± 7.72	448.78 ± 7.93	15.86 ± 0.81	29 202.27 ± 485.83
Baicalin	GU	2.69 ± 2.02	7.85 ± 0.34	55.02 ± 1.67**	756.72 ± 11.22**	767.24 ± 12.80**	17.96 ± 0.28**	49 926.61 ± 885.85**
Wogonoside	Normal	0.18 ± 0.17	6.03 ± 0.19	180.04 ± 4.78	2172.22 ± 26.93	2184.04 ± 28.42	12.11 ± 0.20	6546.01 ± 211.45
Wogonoside	GU	5.17 ± 2.04**	10.16 ± 2.21*	213.66 ± 4.62**	3282.31 ± 44.82**	3424.91 ± 110.55**	15.13 ± 0.78**	10 257.69 ± 133.07**
Baicalein	Normal	0.14 ± 0.09	5.82 ± 0.09	41.87 ± 1.11	457.52 ± 7.25	460.12 ± 7.46	16.57 ± 0.36	25 728.57 ± 258.67
Baicalein	GU	0.25 ± 0.00*	6.79 ± 0.21*	62.61 ± 0.69**	862.04 ± 7.56**	870.70 ± 8.72**	17.76 ± 0.34**	48 693.33 ± 785.67**
Liquiritin	Normal	0.14 ± 0.09	7.56 ± 0.28	32.34 ± 2.08	327.32 ± 8.46	331.17 ± 8.29	17.06 ± 0.76	26 710.33 ± 641.77
Liquiritin	GU	0.83 ± 0.26**	8.32 ± 0.48	68.43 ± 1.42**	815.12 ± 46.76**	831.34 ± 50.58**	17.92 ± 0.91**	67 672.55 ± 1646.94**
Glycyrrhizic acid	Normal	0.58 ± 0.20	7.36 ± 1.47	49.45 ± 0.12	442.55 ± 5.90	449.00 ± 13.19	13.02 ± 0.09	25 855.46 ± 1423.62
Glycyrrhizic acid	GU	0.92 ± 0.20*	11.05 ± 1.78*	62.22 ± 0.39**	769.86 ± 12.11**	868.53 ± 47.42**	18.47 ± 0.49**	49 922.15 ± 1400.37**
Berberine hydrochloride	Normal	0.58 ± 0.20	9.06 ± 1.07	14.77 ± 1.33	216.59 ± 11.92	223.39 ± 13.11	13.94 ± 0.99	44 551.00 ± 2913.58
Berberine hydrochloride	GU	0.92 ± 0.20*	11.56 ± 3.46	30.17 ± 1.89**	470.18 ± 23.19**	504.61 ± 33.31**	18.11 ± 1.65**	100 551.87 ± 5742.42**
Saikosaponin a	Normal	-	-	-	-	-	-	-
Saikosaponin a	GU	0.58 ± 0.20	15.30 ± 1.84	61.79 ± 4.81	1015.64 ± 62.34	1149.22 ± 85.09	21.52 ± 2.14	19 579.48 ± 1428.31
Saikosaponin d	Normal	-	-	-	-	-	-	-
Saikosaponin d	GU	0.083 ± 0	5.54 ± 1.91	38.02 ± 1.75	249.32 ± 28.99	250.60 ± 30.59	10.14 ± 3.39	90 495.98 ± 10 946.34

AUC, area under curve; CL, clearance rate; GU, gastric ulcer; MRT, mean residual time.

*.01 < P < .05 vs normal group.

**P < .01 vs normal group.

Tissue Distribution Study

The distribution of 8 compounds in the heart, liver, spleen, stomach, kidney, and small intestine of the normal and GU groups is shown in Figure 3. Baicalin, baicalein, and berberine hydrochloride were widely distributed into the extravascular system of the GU and normal rats’ bodies after the oral administration of MXCHG. In the GU and normal rats, the amount of liquiritin in the liver, stomach, and small intestine was higher than in other tissues. The amount of glycyrrhizic acid, saikosaponin a, and saikosaponin d in the stomach and small intestine was higher than in the other tissues. In the normal group, the amount of wogonoside in the spleen, kidney, and small intestine was greater than in the other tissues, but the concentration in the stomach, kidneys, and small intestine was higher in the GU group. The concentrations of 8 compounds in different tissues were greater in the GU group than in the normal group and more than in the GU group. These data indicate a higher stomach and small intestine exposure to 8 compounds in the GU rats compared with the normal rats.

Figure 3

Tissue distribution for 8 analytes after intragastric administration of modified Xiaochaihu granule (22.4 g/kg) in the normal and gastric ulcer groups: baicalin (A), wogonoside (B), baicalein (C), liquiritin (D), glycyrrhizic acid (E), berberine hydrochloride (F), saikosaponin a (G), and saikosaponin d (H). The data are presented as means ± SD (n = 6).

Discussion

The pharmacokinetic studies showed that 3 compounds (liquiritin, saikosaponin a, and saikosaponin d) were rapidly absorbed (t _max, 0.083-0.83 hours) and slowly eliminated because their plasma concentrations were higher than the LLOQ at 48 hours postdose. Previous reports have suggested that the concentration of liquiritin in rat plasma was extremely low following intragastric administration, whereas the content of liquiritigenin after hydrolysis by β-glycuronidase was far more than that of liquiritin, suggesting that liquiritin may undergo hydrolysis in the intestinal tract and phase II metabolism.^27,28 Although saikosaponin d is an enantiomer of saikosaponin a, in which the configuration of the β-hydroxy group is substituted with an α-hydroxy group, their pharmacokinetic behaviors are remarkably different.²⁹ For the other 5 compounds (baicalin, wogonoside, baicalein, glycyrrhizic acid, and berberine hydrochloride), double-peak phenomena were observed in the normal and GU groups, except for glycyrrhizic acid in the normal group, which may explain the moderate effect of licorice seen in other studies.^27,30,31 In the GU group, the first peak (at 1 hour postdose) rose and fell very quickly, and the second peak (at 10 hours postdose) remained at high levels for several hours. This may be ascribed to the enterohepatic circulation, a delay in gastric emptying, and multiple sites of absorption in the gastrointestinal tract.³²A high t _max allows baicalin and wogonoside to remain in the body for a longer time, improving therapeutic activities such as anti-inflammatory effects.²⁶ These effects may be due to the influence of the other constituents of MXCHG (baicalin and wogonoside) on the pharmacokinetic parameters in vivo. Overall, the pharmacokinetic behavior of 8 active compounds partly revealed the clinical effect of MXCHG as an anti-GU agent.

For tissue distribution, the small intestine is the most important functional gastric tissue in both the normal and experimental rats. The stomach is the second functional tissue in the GU group, except for baicalin. Moreover, the compound concentrations in the small intestine and stomach in the GU rat were higher than those in normal rats in the order of baicalin > wogonoside > saikosaponin a > glycyrrhizic acid > baicalein > berberine hydrochloride > liquiritin > saikosaponin d. Baicalin was also widely and rapidly distributed in the liver.³³ For both groups, the peak concentration appeared at 0.5 hours in the stomach and small intestine, followed by a gradual decline until the last time point measured. In the liver, the peak concentration of baicalin appeared at 0.5 hours in the normal group and 1 hour for the GU group. These results indicate that GUs could delay the distribution of baicalin in the liver. Wogonoside was mainly distributed in the spleen and kidney in the normal group, but in the stomach and small intestine in the GU group, showing that the target site could be changed for some diseases.^29,34 Baicalein was widely and rapidly distributed in all 6 tissues.^29,34 In the time study, the tendency was almost similar in all tissues except the small intestine, in which the peak concentration appeared at 3 hours in the normal group and 1 hour in the GU group.^35,36 These results indicate that a GU could accelerate the distribution of baicalin in the small intestine. ^37,38 Liquiritin and glycyrrhizic acid are derived from licorice.³⁹ Liquiritin was distributed to the liver, and the peak concentration appeared at 1 hour, indicating that a GU could prolong the absorption of liquiritin in GU rats.^40,41 Glycyrrhizic acid exhibited bimodal phenomena in the stomach and small intestine concentration-time profiles, probably due to reabsorption and distribution into the hepatoenteral circulation in the GU group.⁴² Berberine hydrochloride was extensively distributed to all 6 tissues in the order of small intestine > stomach > liver > heart > spleen > kidneys in the GU rats, indicating the slow and incomplete absorption of berberine in the stomach and intestine to be suitable as a gastric medicine.⁴³ Because of the similar structure of saikosaponins a and d, the tissue distributions were similar with small concentrations in the tissues in the order of intestine > stomach > liver > heart > kidneys > spleen in the normal and GU rats. The concentration of saikosaponins in the GU group was higher than in the normal group, indicating that the acidic in vivo environment was beneficial for the absorption of saikosaponins in the small intestine and stomach.^24,44 Little research has been conducted on the distribution of saikosaponins a and d in both the small intestine and stomach.

This was the first study to examine the tissue distribution profiles of multiple bioactive components after the intragastric administration of MXCHG. The results of the tissue distribution study showed that 8 compounds in MXCHG were mainly distributed in the gastrointestinal tract, which provided the material basis for its pharmacological actions in the treatment of GUs. Moreover, gut microbiota, known as a complex ecological community colonized in the gastrointestinal tract, can interact with host factors to affect normal physiology and diseases. The main functional tissue for the MXCHG was the stomach and small intestine, which contain abundant microbiota. Therefore, the relationship between altered gut microbiota and changed pharmacokinetic behavior should be further investigated.

Conclusion

Traditional Chinese medicine prescriptions have been widely used for the prevention and treatment of various diseases for thousands of years in China. With changes in human disease, many classic prescriptions, such as MXCHG, have been modified to adapt to new symptoms. Traditional Chinese medicine prescriptions show synergistic therapeutic efficacy through the interaction of multiple components. The pharmacokinetic parameters of a single component cannot represent the pharmacokinetics characteristics of TCM prescriptions. After oral administration, the components enter the blood, but metabolites may also be present at lower blood concentrations. In this study, a simple, sensitive, and rapid UPLC-MS/MS method was established and validated for the simultaneous quantification of 8 components in MXCHG in rat plasma and tissue samples. Modified Xiaochaihu granule was rapidly distributed and cleared from the rat tissues after a single oral administration. Moreover, the results showed that most of the targeted compounds were difficult to detect in the selected tissues of rats, except for the stomach and intestine. This study will offer some helpful information for further comprehensive preclinical research on MXCHG, and pharmacokinetic research on people with GUs.

Supplemental Material

Supplementary Material 1 - Supplemental material for Pharmacokinetics and Tissue Distribution Study of Modified Xiaochaihu Granules Against Gastric Ulcer Induced by Ethanol in Rats by UPLC-MS/MS

Supplemental material, Supplementary Material 1, for Pharmacokinetics and Tissue Distribution Study of Modified Xiaochaihu Granules Against Gastric Ulcer Induced by Ethanol in Rats by UPLC-MS/MS by Xin Chen, Yuan-Chun Ma, Mengling Yang, Pengtao You, Dan Liu, Xiaochuang Ye, Yang Yanfang, Aijun Zhou and Yanwen Liu in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work was supported by the National Natural Science Foundation of China (81573960), the Natural Science Foundation of Hubei Province (2016CFB359), the Program for Office of Science and Technology of Dongguan City (2014-130), and the School Supervision Subject of Hubei University of Chinese Medicine (5114-100714).

ORCID iD

Yuan-Chun Ma

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