MS

Accepted Manuscript Title: Pharmacokinetics study of Erhuang decoction extracts in rats by HPLC-MS/MS Authors: Jinglong Wang, Dandan Zheng, Yingzi Wang, Zhang Chao, Xiumei Sun PII: DOI: Reference:

S1570-0232(16)31334-4 http://dx.doi.org/doi:10.1016/j.jchromb.2017.05.019 CHROMB 20608

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

28-11-2016 20-5-2017 21-5-2017

Please cite this article as: Jinglong Wang, Dandan Zheng, Yingzi Wang, Zhang Chao, Xiumei Sun, Pharmacokinetics study of Erhuang decoction extracts in rats by HPLC-MS/MS, Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.05.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pharmacokinetics study of Erhuang decoction extracts in rats by HPLC-MS/MS Jinglong Wang a,*, Dandan Zheng a, Yingzi Wang b, Zhang Chao c, Xiumei Sun c a

College of Life Sciences, Zaozhuang University, Zaozhuang 277160, China

b

School of Chinese Materia Medica, Beijing University of Traditional Chinese Medicine, Beijing

100029, China c

College of pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250355,

China

*

Correspondence to: Jinglong Wang E-mail: [email protected] Tel: 0632-3786736 Fax: 0632-3786736

Abstract: To study the pharmacokinetics of Erhuang decoction extracts, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established for the determination of effective substances in rat plasma. The extracts prepared by water extraction (WE) method were given to rats by oral administration. After collected from the orbital venous plexus, plasma was treated by protein precipitation method. Then, the concentration of index components, including baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid, were determined by HPLC-MS/MS. Gradient elution mode was used to the chromatographic separation with an Inertsil ODS-SP column (100 mm×2.1 mm, 5 μm), with acetonitrile and 0.1% formic acid containing 10 mmol·L-1 ammonium acetate as the mobile phase. MS analysis was conducted by multiple reactions monitoring (MRM) with Electrospray Ionization (ESI). The extraction recoveries of the five active ingredients from plasma were greater than 86.04%, and the intra- and inter-day precisions were less than 16.57%. Results indicated that active ingredients in plasma of rats with oral administration of extracts showed certain difference in the pharmacokinetic parameters, which proved that the active ingredients were effectively absorbed. The established HPLC-MS/MS analytical method was sensitive and accurate, suitable for the pharmacokinetic study of active ingredients in Erhuang decoction. Keywords: Pharmacokinetics; Erhuang decoction; Active ingredients; HPLC-MS/MS

1. Introduction Erhuang decoction, consisting of Scutellaria Radix and Coptidis Rhizoma, has the functions of clearing away heat and toxic material, which could be used to treat the coke fire, big head swollen, swollen throat, ear nose mouth heart heat and boil the virus [1]. The effective substances of Erhuang decoction were flavonoids such as baicalin and liquiritin, alkaloids such as palmatine and berberine, as well as triterpenoid saponins such as glycyrrhizic acid. Those components exhibited the effects of anti-inflammatory [2-3], antioxygenation [4], hepatoprotective [5-6], and antitumor activity [7], etc. Chinese herbal medicines are rich in primary metabolites, including

polysaccharide, protein, tannin, resin and starch, which resulted that effective substances of the extractions prepared by traditional methods were difficult to concentrate. Researches have shown that the molecular weight of active components of traditional Chinese medicine is generally no more than 1000, while the molecular weight of inactive components is mostly more than 50 000 [8]. Therefore, hollow fiber ultrafiltration membrane (molecular weight cut-off was 10k, 5k or 1k, Millipore) was used in our previous studies to refine the extracts, aiming to achieve a high content of effective component [9]. The composition of traditional Chinese medicine compound was complex, and the multiple components often synergistically effect on the multiple systems or multiple targets. Pharmacokinetic parameters of single component could not represent the pharmacokinetics characteristics of traditional Chinese medicine compound [10]. After oral administration, the components absorpt into blood might be the prototype components, but also may be metabolites with lower blood concentrations. It has been reported that baicalin converted into blood was in the form of baicalin [11-13], and berberine and palmatine were in the form of a prototype in the blood [14], while glycyrrhizic acid metabolized by the flora, was mainly in the presence of glycyrrhetic acid [15-17]. Besides, liquiritin has two components in blood that is liquiritin and liquiritigenin [18]. Though the pharmacokinetic study of single or compound of Scutellariae Radix, Coptidis Rhizome and/or Glycyrrhizae Radix et Rhizoma have been reported, few research analyzed the five effective substance in vivo simultaneously. Therefore, baicalin, liquiritin, berberine, palmatine and glycyrrhetic acid were choosen as the indexes to investigate the pharmacokinetic of Erhuang decoction in this study, with <1000 Da extract as final product. And HPLC-MS/MS was used to determine the concentration of effective substance in plasma of rats. The results of pharmacokinetic parameters of multi index components could provide reference for clarifying the substance basis and mechanism of Traditional Chinese Medicine.

2 Material and Methods

2.1 Chemicals and Materials Scutellariae radix (root of Scutellaria baicalesis Georgi.), Coptidis rhizoma (root of Coptis chinensis Franch.) and Glycyrrhizae radix et rhizoma (whole grass of Glycyrrhiza uralensis Fisch.) were authenticated by Prof. Zhang (College of pharmacy, Shandong University of Traditional Chinese Medicine). Baicalin, liquiritin, berberine hydrochloride, glycyrrhetinic acid, baicalein, glycyrrhetic acid using as references, and hesperidin, nuciferin, ursolic acid using as internal standards (IS), were obtained from Shandong and National Institutes for Food and Drug Control, respectively.

Palmatine

Hydrochloride

was

bought

from

Nanchang

beta

Biotechnology Co. Ltd. Purity of all the references and internal standards was over 98%. Methanol and acetonitrile (Merck, Germany) were of high performance liquid chromatography (HPLC) grade, and water was purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA). Ammonium acetate and other chemicals and reagents were of analytical grade. Animals: Sprague–Dawley rats (body weight 200±20 g) which were used for pharmacokinetic studies, were obtained from the Experimental Animal Center of Shandong University of Traditional Chinese Medicine (Jinan, China). The animals were housed in cages for at least one week before experimentation, and fed with food and water ad libitum simultaneously. Prior to the experiments all the animals were kept under fasting but with free access to water overnight. The animal care and all the experimental procedures abided by the National Institutes of Health Guide for Care and Use of Laboratory Animals.

2.2 HPLC–MS/MS conditions The analyses were operated using an Agilent 1200 liquid chromatography system (Agilent Technologies, USA), equipped with Agilent 6410 Triple Quad MS system (Agilent Technologies, USA).

The separation was performed on an Inertsil ODS-SP column (100 mm×2.1 mm, 5μm) with mobile phase consisting of solvent A (acetonitrile) and solvent B (0.1% formic acid containing 10 mmol·L-1 ammonium acetate). Gradient elution was as follows: initial 0–6 min, linear change was from 20% A to 100% A (v/v); 6–13.5 min, linear change was 100% A (v/v); 13.5–13.7 min, linear change was from 100%A to 20% A (v/v); 13.7–20 min, linear change was 20% A (v/v). Other detection conditions were as follows: flow rate of the mobile phase: 0.6 mL·min-1; wavelength of the UV-detection: 265 nm; column temperature: 30 ℃; sample injection volume: 20 μL. Mass spectrometric detection was carried out on a triple quadrupole mass spectrometer with ESI source in positive and negative ionization switching mode, and quantification was performed with multiple reactions monitoring (MRM) mode. To separate and determine baicalin and berberine efficiently, the MS parameters were optimized: ion spray voltage: ±4500 V; ion source temperature: 350 ℃; desolvation gas flow 10 L·min-1; nebulizer gas pressure: 40 psi. Nitrogen served as the nebulizing and drying gas was of high purity. Selection of ionization pairs (m/z) was shown as follows: positive ion mode: baicalin: 447.2→271.1 (collision energy 25 V), baicalein: 271.1→123.1 (collision energy 25 V), berberine: 336.3→320.2 (collision energy 21 V), palmatine: 352.3→336.2 (collision energy 22 V), glycyrrhetinic acid: 471.4→189.3 (collision energy 35 V), nuciferin (IS): 295.8→265.9（collision energy 20 V); positive ion mode: baicalin: liquiritin: 417.2→135.2 (collision energy 22 V), hesperidin (IS): 608.5→608.5 (collision energy 0 V), ursolic acid (IS): 455.4→455.4 (collision energy 0 V).(Fig. 1).

2.3 Preparation of Erhuang decoction ≤1000Da extracts Scutellariae radix, Coptidis Rhizoma and Radix liquiritiae with the ratio of 1:1:1, were porphyrized and over 10-20 mesh sieve. Then, Scutellariae radix and Radix liquiritiae were decocted with neutral water for three times together, and decocting times were 2.0 h, 1.5 h and 1.5 h, respectively, while Coptidis Rhizoma was decocted separately. The combined concentrated decoctions were filtered by hollow fiber

membrane and centrifuged for 25 min at 4930 × g. Constant volume of the resulting mixture was 250 mL (containing 1 g·mL-1 ≤1000 Da extraction).

2.4 Preparation of calibration standards solutions, quality control (QC) solutions and internal standard solution Stock solutions were prepared by dissolving various accurate amounts of standards in methanol solution: 0.547 g·L-1 baicalin, 0.154 g·L-1 liquiritin, 0.200 g·L-1 berberine, 0.250 g·L-1 palmatine, 0.199 g·L-1 glycyrrhetinic acid, respectively. Appropriate amount of these primary stock solutions were combined and diluted with mobile phase (acetonitrile: 0.1% formic acid = 80:20, v/v), to prepare the working standard solutions at concentrations of 65.64, 164.10, 328.20, 656.40, 1641.00, 6564.00 and 16410 ng·mL-1 for baicalin, 4.62, 9.24, 23.10, 92.40, 231.00, 462.00 and 2310.00 ng·mL-1 for liquiritin, 1.50, 3.75, 15.00, 37.50, 150.00, 375.00 and 750.00 ng·mL-1 for berberine, 1.20, 3.00, 12.00, 30.00, 120.00, 300.00 and 600.00 ng·mL-1 for palmatine and 23.88, 59.70, 119.40, 298.50, 597.00, 1194.00 and 5970.00 ng·mL-1 for glycyrrhetinic acid. 0.1 mL of working standard solutions were placed in 1.5 mL centrifuge tubes, and 0.2 mL of blank rat plasma was added respectively, to obtain the assay calibration standard solutions containing 21.88-5470.00 ng/mL for baicalin, 1.54-770.00 ng/mL for liquiritin, 0.50-250.00 ng/mL for berberine, 0.40-200.00 ng/mL for palmatine and 7.96-1990.00 ng/mL for glycyrrhetinic acid. Quality control samples (QCs) were independently prepared as the same method of the assay calibration standard solutions at 54.70, 218.80 and 2188.00 ng·mL-1 of baicalin, 3.08, 30.80 and 154.00 ng·mL-1 of liquiritin, 1.25, 12.50 and 125.00 ng·mL-1 of berberine, 1.00, 10.00 and 200.00 ng·mL-1 of palmatine, and 19.90, 99.50 and 398.00 ng·mL-1 of glycyrrhetinic acid. The internal standard stock solutions were prepared by dissolving hesperidin, nuciferin, ursolic acid in methanol to 0.147 g·L-1, 0.17 g·L-1, 0.174 g·L-1. The hybrid internal standard solutions were prepared by combining the aliquots of each primary stock solution and diluting with methanol containing 147.00 ng·mL-1 hesperidin, 170

ng·mL-1 nuciferin, 174 ng·mL-1 ursolic acid before use. All the samples were stored at 4 ℃ until analysis.

2.5 Treatment of the plasma Protein precipitation method was used to dispose plasma samples. 0.2 mL of plasma was mixed with 0.1 mL internal standard solutions (containing 147.00 ng·mL-1 hesperidin, 170 ng·mL-1 nuciferin, 174 ng·mL-1 ursolic acid) and 20 μL ammonium acetate (the pH was adjusted to 4.0 with glacial acetic acid), and 0.8 mL methanol was added to precipitate protein by vortex for 2 min. The upper layer was centrifuged for 10 min at 14086 × g. The supernatant was evaporated under a light stream of nitrogen at 35◦C. Then, the residue was redissolved in 0.15 mL mobile phase followed by the treatments of vortex for 3 min and centrifuged for 10 min at 21130 × g. Afterwards, the supernatant layer was filtered through a 0.22 μm filter, and 20 μL of the filtrate was injected into HPLC column for analysis.

2.6 Method validation 2.6.1 Specificity assay The hybrid control solution with series concentrations of baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid was added into blank plasma, respectively. And the drug containing plasma was collected after administration of 15.0 g·kg-1 concentrate of Erhuang decoction to rats for 0.5 h (n = 6). All the samples were treated as the ‘treatment of the plasma’ and analyzed by the HPLC-LC/MS method.

2.6.2 Linearity and sensitivity The calibration curves for baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid were plotted using a 1/x2 weighted linear regression model of the peak–area ratio of each component to internal standard against the plasma concentrations of each component. The linear range applied to the validation

procedure. Analyte response at the level of LLOQ (the lower limit of quantification) should be at least five times of the blank plasma with acceptable accuracy within 20% deviation and precision between 80 and 120%.

2.6.3 Precision and accuracy The intra-day precision were analyzed with the control solutions (low, middle and high concentration in the linear range) in six replicates, which were determined in 24 h. The control solutions of low, middle and high concentrations in the linear range were prepared and determined for five continuous days to inter-day precision, respectively. Relative standard deviation (RSD%) was used to evaluate the intra- and inter-day precision. The relative error (RE%) was calculated according to the formula: RE% = [(assayed value − nominal value)/nominal value] × 100%.

2.6.4 Extraction recovery Three QC samples with low, medium and high concentrations were prepared and tested for the peak areas. Meanwhile, the peak areas of blank plasma extracted with methanol and then spiked with baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid standards with different concentrations was measured. Then, the extraction recoveries of five effective substances were determined by comparing the two kinds of peak areas (n = 6).

2.6.5 Matrix effects The matrix effect was determined by comparing the peak area of hybrid control solutions with extracted blank plasma samples spiked with analytes at three different concentrations (low, medium and high) concentration using three replicates.

2.6.6 Stability 2.6.6.1 Stability in 24 h and short term temperature stability In order to examine the stability of ingredients in plasma during measurement,

three standard plasma samples with low, medium and high concentrations were prepared, respectively. Each concentration of 6 replicates was measured immediately after treatment, and remeasured after 24 h preservation at 4 ℃. Short term temperature stability was evaluated using QC samples (low, medium and high concentrations) kept at room temperature for 6 h.

2.6.6.2 Repeated freezing and thawing stability and Long term stability The stability was investigated by analyzing twelve replicates of the samples at three QC levels (low, medium and high concentration) under different conditions. Six replicates of the samples were treated with 24 h storage at ambient 4 ℃, three freeze/thaw cycles, and the other six replicates were treated with 14 days storage at −20 ℃. The samples were considered stable after being thawed at room temperature.

2.7 Animals and pharmacokinetic study After administration of ≤1000Da WE extracts by intragastric gavage at a single dose of 15.0 g·kg-1, about 0.5 mL of blood sample of Sprague-Dawley rats was collected from the orbital venous plexus into heparinized 1.5 mL polythene tubes at predetermined time points (0.083, 0.167, 0.333, 0.5, 1, 2, 3, 4, 6, 8, 12, 24 and 48 h). After immediately centrifugation at 5634 × g for 10 min, the supernatant plasma was obtained and stored at −20 ℃ until analysis (n=6).

2.8 Data analysis Plasma concentrations of different time points in rats were analyzed by DAS 2.0 software, and the pharmacokinetic parameters of baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid were calculated using non-compartmental model.

3 Results and discussion

3.1 Optimization of chromatography conditions and MS conditions To analyze five substances of different chemical structures simultaneously,

hesperidin, nuciferin and ursolic acid were chosen as the internal standard of flavonoids (baicalin, liquiritin), alkaloids (berberine, palmatine) and ginsenosides (glycyrrhetinic acid), respectively. According to the longer peak rentention time of glycyrrhetinic

acid

and

ursolic

acid

compared

with

other

ingredients,

acetonitrile-water was selected as the mobile phase to satisfy the needs of large batch determination of biological samples. In addition, experiments of the effects of aqueous on each component certified that formic acid containing appropriate ammonium acetate could improve the chromatographic peak shape and the ionization efficiency. Therefore, acetonitrile and 0.1% formic acid containing 10 mmol·L-1 ammonium acetate was selected as the mobile phase, and all the analysis was performed successfully within only 20 min under this condition.

3.2 Method validation 3.2.1 Specificity The selectivity test was carried out by analyzing the blank plasma, blank biological matrix samples spiked with baicalin and berberine, and actual plasma after intragastrical administration of baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid, respectively. The five active ingredients were detected in 20 min without any endogenous interference. This method is fast and specific because of the high selectivity of MRM mode. Typical chromatograms of blank rat plasma, blank rat plasma spiked with baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid, plasma sample 2.5 h after intragastric gavage administration of Erhuang decoction are shown in Fig.2.

3.2.2 Linearity and sensitivity The linear ranges, typical calibration curves and regression coefficients were got by weighted least square method (W=1/X2) with concentration of baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid in plasma as the horizontal axis and the ratio of peak area of samples and internal standard as the vertical axis. As shown in supplementary information, the correlation coefficients (r) of the five active

ingredients were all higher than 0.9900, and the lower limit of quantification in rat plasma was 3.98 and 0.40 ng/mL, respectively. These results indicated that the present method was sufficiently sensitive for the pharmacokinetic study. 3.2.3 Precision and accuracy In Table 1, the results of intra- and inter-day precision and accuracy for QC samples at three different concentrations were displayed. The relative standard deviations (RSDs) of baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid were in the range of 2.30–8.04% for intra-day precision, and 4.00–10.25% for inter-day precision. The relative errors (REs) were within ±14.16% for the accuracy of intra-day precision and ±16.57% of intra-day precision, respectively, which indicated that the method was accurate and reproducible for the determination of five active ingredients in rat plasma.

3.2.4 Extraction recovery and matrix effects Extraction recovery could reveal if the methods of the study were acceptable. Table 2 showed that the extraction recoveries of baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid from rat plasma ranged from 86.04% to 95.81% with RSD% less than 6.75%, and the matrix effect values of five active ingredients ranged from 88.97 to 99.64% with RSD% less than 7.08%. The results showed that matrix effect was considered to be negligible.

3.2.5 Stability The results shown in Table 3 demonstrated that baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid were stable under various conditions that samples might experience. RE values of the stability test of five active ingredients in rat plasma at 4 ℃ for 24 h ranged from −4.32% to −13.65%. For the samples in a short term at room temperature for 6 h, the values were in a range of −1.53% to −10.25%. For the samples in a long term freezer at −20 ℃ for 14 days, the values were in a range of −6.27% to −15.08%, and after three freeze–thaw cycles, the RE values

ranged from −4.21% to −16.33%.

3.3 Pharmacokinetics study Plasma containing extracts (≤1000 Da) was absorbed and treated as the method described at‘2.5 Treatment of the plasma’. Then, the samples were analyzed by HPLC-MS/MS method to determine the concentration of baicalin, liquiritin, berberine, palmatine and glycyrrhetic acid. The concentration-time curves of five effective components were shown in Fig.3, and PK solver software was used to obtain the pharmacokinetic parameters in non-compartment model (Table 4). As shown in Table 4, the Tmax, Cmax and AUC0-t of baicalin were 0.083 h, 2.99 μg·mL-1 and 28.65 μg·h·mL-1, respectively. The good absorption effect might due to the co absorption of baicalin and baicalein in extracts. The concentration-time profile of baicalin in Fig.3-A displayed double peaks, which point out that the enterohepatic cycle play an important role in the metabolism of baicalin in vivo [19]. It has been reported that baicalein quickly converted to baicalin after transforming into the small intestine epithelial cells. Part of the baicalin was transported into blood by MRP3 protein (multidrug resistance-associated protein), and further metabolized in the liver through mesenteric vein. Another part of the baicalin was pumped back into the intestine by MRP2 protein [20]. In fact, the dynamic changes of baicalin in the blood resulted from the absorption of original components of baicalin after being hydrolyzed and reduced, as well as the presence of a certain amount of baicalein in the extracts directly absorbed through the reduction process into blood together. Fig.3-C and Fig.3-D showed that the concentration-time profiles of berberine and palmatine were also displayed double peaks, which implied that the enterohepatic cycle affect the metabolism of the two components. The Tmax of glycyrrhetic acid was 8 h, which was inconsistent with the reported 10~12 h [21-23]. The inconsistency might due to the relatively few sampling points, which resulted in a large difference in peak time. Besides, when glycyrrhizic acid was given to rats by oral administration, it was transformed into glycyrrhetic acid with physiological activity by intestinal flora, and almost undetectable in blood.

4 Conclusions The extract (≤1000Da) was taken as the final product, which reflected the features of traditional, comprehensive, objective, fuzzy and the dosage reducing compared with the crude extracts. A new HPLC-MS/MS method was established to determine baicalin, liquiritin, berberine, palmatine and glycyrrhetic acid in rat plasma with multiple internal standards, which could successfully separate the characteristic peaks of different substances. With the specific, sensitive and reliable method, the pharmacokinetics investigation of five active ingredients in rats was simultaneously performed for the first time. The study lays the foundation for further research of Erhuang decoction and has referential significance to the modernization of traditional Chinese medicine.

Acknowledgments This work was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (Project Name: Research on the key technology of Chinese herbal medicine "semi bionic extraction", approval number: 2010ZX09401).

References [1] Q. Wu, Golden Mirror of Medicine (photocopy), People's Medical Publishing House, Beijing, 1957. [2] D.U. Lee, Y.J. Kang, M.K. Park, Y.S. Lee, H.G. Seo, T.S. Kim, C.H. Kim, K.C. Chang, Effects of 13-alkyl-substituted berberine alkaloids on the expression of COX-II, TNF-alpha, iNOS, and IL-12 production in LPS-stimulated macrophages, Life Sci. 73 (2003) 1401-1412. [3] Y.M. Lee, P.Y. Cheng, L.S. Chim, C.W. Kung, S.M. Ka, M.T. Chung, J.R. Sheu. Baicalein, an active component of Scutellaria baicalensis Georgi, improves cardiac contractile function in endotoxaemic rats via induction of heme

oxygenase-1 and suppression of inflammatory responses, J. Ethnopharmacol. 135 (2011) 179-85. [4] Z.S. Markovic, J.M. Dimitric Markovic, D. Milenkovic, N. Filipovic, Mechanistic study of the structure–activity relationship for the free radical scavenging activity of baicalein, J. Mol. Model. 17 (2011) 2575-84. [5] T.C. Chou, L.P. Chang, C.Y. Li, C.S. Wong, S.P. Yang, The antiinflammatory and analgesic effects of baicalin in carrageenan-evoked thermal hyperalgesia, Anesth. Analg. 97 (2003) 1724. [6] S.I. Jang, H.J. Kim, K.M. Hwang, S.J. Jekal, H.O. Pae, B.M. Choi, Y.G. Yun, T.O. Kwon, H.T. Chung, Y.C. Kim, Hepatoprotective effect of baicalin, a major flavone from Scutellaria radix, on acetaminophen-induced liver injury in mice, Immunopharmacol. Immunotoxicol. 25 (2003) 585. [7] Q.Y. Zhang, M. Ye, Chemical analysis of the Chinese herbal medicine Gan-Cao (licorice). J. Chromatogr. A, 1216 (2009) 1954. [8] Y.B.Ji, Pharmacology and Application of Active Ingredients of Traditional Chinese Medicine, Heilongjiang Science and Technology Press, Harbin, 2004. [9] J.L. Wang, Y.Z. Wang, X.M. SUN, Z.W. Zhang, C. Zhang. Simultaneous determination of six components in the ≤1000 Da extract of Erhuang decoction by HPLC. Chin. J. Exp. Tradit. Med. Form. 20 (2014) 52. [10] C.X. Liu. Difficulty and hot-points on pharmacokinetics studies of traditional Chinese medicine. Acta Pharmaceutica Sinica, 40 (2005) 395-401. [11] J. Xing, X. Chen, D. Zhong. Absorption and enterohepatic circulation of baicalin in rats, Life Sci, 78(2005)140-146. [12] L. Zhang, G. Lin, Q. Chang, Z. Zou, Role of Intestinal first pass metabolism of baicalein in its absorption process, Pharma. Res. 22 (2005) 1050-1058. [13] T. Akao, Y. Sakashita, M. Hamada, H. Goto, Y. Shimada, K. Terasawa, Enteric excretionof baicalein, a flavone of scutellariae radix, via glucuronidation in rat:involvement of muhidrug resistance associated protein, Pharmal Res. 21 (2004) 2120-2126. [14] J.H. Chen, F.M. Wang, J. Liu, F.S. Lee, X. Wang, H. Yang, Analysis of alkaloids

in Coptis chinensis Franch by accelerated solvent extraction combined with ultra performance liquid chromatographic analysis with photodiodearray and tandem mass spectrometry detections, Analytica Chimica. Acta. 6 (2008) 184-195. [15] G. Cantelli-Forti, F. Maffei, P. Hrelia, F. Bugamelli, M. Bernardi, P. D'Intino, M. Maranesi, M.A. Raggi. Interaction of licorice on glycyrrhizin pharmacokinetics, Environ Health Persp. 102 (1994) 65-68. [16] Z. Wang, Y. Kurosaki, T. Nakayama, T. Kimura, Mechanism of gastrointestinal abso- rption of glycyrrhizin in rats, Biol. Pharm. Bull. 17 (1994) 1399-1403. [17] Y. Yamamura, T. Santa, H. Kotaki, K. Uchino, Y. Sawada, T. Iga, Administrationroute dependency of absorption of glycyrrhizin in rats: intraperitoneal administration dramatically enhanced bioavailability, Biol. Pharm. Bull. 18 (1995) 337-341. [18] J. Kameia, A. Saitoha, T. Asano, R. Nakamura, H. Ichiki, A. Iiduka, M. Kubo. Pharmacokinetic and pharmacodynamic profiles of the antitussive principles of Glycyrrhizae radix (licorice), a main component of the Kampo preparation Bakumondo-to (Mai-men-dong-tang), Eur. J. Pharmacol. 507 (2005) 163-168. [19] J. Xing, X. Chen, D. Zhong. Absorption and enterohepatic circulation of baicalin in rats, Life Sci. 78 (2005) 140-146. [20] T. Akao, Y. Sakashita, M. Hanada, H. Goto, Y. Shimada, K. Terasawa. Enteric excretion of baicalein, a flavone of Scutellariae Radix, via glucuronidation in rat: involvement of multidrug resistance-associated protein 2, Pharmal. Res. 21 (2004) 2120-2126. [21] G. Cantelli-Forti, F. Maffei, P. Hrelia, F. Bugamelli, M. Bernardi, P. D'Intino, M. Maranesi, MA. Raggi. Interaction of licorice on glycyrrhizin pharmacokinetics, Environ. Health Perspect, 102 (1994) 65-68. [22] Z. Wang, Y. Kurosaki, T. Nakayama, T. Kimura. Mechanism of gastrointestinal absorption of glycyrrhizin in rats, Biol. Pharm. Bull. 17 (1994) 1399-1403. [23] Y. Yamamura, T. Santa, H. Kotaki, K. Uchino，Y. Sawada, Administration-route dependency of absorption of glycyrrhizin in rats: intraperitoneal administration dramatically enhanced bioavailability, Biol. Pharm. Bull. 18 (1995) 337-341.

Figure Captions: Fig. 1. The chemical structures and mass spectra of baicalin, liquiritin, berberine, palmatine, glycyrrhetinic acid, nuciferine (IS), hesperidin (IS), ursolic acid (IS). Fig. 2. Representative MRM chromatograms of glycyrrhetinic acid(A), baicalin(B), palmatin(C), berberine(D), nuciferine(E, IS), hesperidin(F, IS), ursolic acid(G, IS), liquiritin(H). (Ⅰ)blank rat plasma; (Ⅱ)plasma sample of LLOQ; (Ⅲ) plasma sample 30min after oral administration of Erhuang Decoction. Fig. 3. Mean plasma concentration-time curve of baicalin (A), liquiritin (B), berberine (C), palmatine (D) and glycyrrhetinic acid (E) after oral administration of 15.0 g·kg-1 in rats (n=6, mean±SD). Fig. 1.

Fig. 2.

Fig 3

Table 1 Intra- and inter-assay precision, accuracy for the determining baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid in rat plasma (n = 6). Intra-assay precision Samples

Baicalin

Liquiritin

Berberine

Palmatine

Glycyrrhetinic acid

C (ng·mL-1)

C（ng·mL-1）

Inter-assay precision RE (%)

C（ng·mL-1）

X S

RSD (%)

54.7

47.08 ± 3.79

8.04

218.8

200.69 ± 11.69

2188.0

X S

RSD (%)

RE (%)

-13.92

46.42 ± 4.76

10.25

-15.14

5.83

-8.28

199.71 ± 13.32

6.67

-8.72

2095.29 ± 73.35

3.50

-4.24

2077.46 ± 83.58

4.02

-5.05

3.08

2.67 ± 0.13

4.77

-13.31

2.62 ± 0.20

7.73

-14.93

30.8

27.20 ± 1.21

4.44

-11.69

26.87 ± 1.70

6.32

-12.77

154.0

142.63 ± 4.43

3.10

-7.38

138.63 ± 6.53

4.71

-9.98

1.25

1.11 ± 0.06

5.26

-11.07

1.11 ± 0.06

5.80

-11.34

12.5

11.26 ± 0.52

4.62

-9.90

11.23 ± 0.56

5.01

-10.17

125.0

117.66 ± 3.08

2.62

-5.87

116.32 ± 4.65

4.00

-6.94

1.0

0.86 ± 0.04

5.01

-14.16

0.85 ± 0.05

5.65

-14.99

10.0

9.14 ± 0.34

3.67

-8.63

8.99 ± 0.57

6.29

-10.13

200.0

188.55 ± 5.40

2.86

-5.73

183.55 ± 10.03

5.46

-8.23

19.9

17.60 ± 0.81

4.63

-11.54

16.60 ± 1.21

7.29

-16.57

99.5

87.02 ± 3.15

3.62

-12.54

83.69 ± 7.06

8.44

-15.89

398.0

376.29 ± 8.64

2.30

-5.45

367.965 ± 19.13

5.20

-7.55

Table 2 Extraction recovery and matrix effects of baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid in rat plasma (n = 6).

Concentration

Extraction recovery/%

Matrix effect/%

ng·mL-1

X S

X S

54.7

86.04 ± 6.75

95.94 ± 3.74

218.8

91.76 ± 5.59

99.05 ± 3.80

2188

95.81 ± 3.25

99.64 ± 3.72

3.08

86.59 ± 3.98

92.12± 3.52

30.8

88.43 ± 4.26

95.50 ± 4.15

154

92.39 ± 3.13

91.72 ± 3.30

1.25

88.88 ± 4.65

92.15 ± 7.08

12.5

90.95 ± 4.58

92.52 ± 6.97

125

93.82 ± 2.99

88.97 ± 3.71

1

86.22 ± 4.28

94.57 ± 3.65

10

90.71 ± 3.91

94.54 ± 6.38

200

94.42 ± 2.84

92.45 ± 4.98

19.9

88.41 ± 4.04

92.25 ±4.02

99.5

88.00 ± 2.62

91.77 ± 4.92

398

94.40 ± 2.04

92.23 ±3.92

Samples

Baicalin

Liquiritin

Berberine

Palmatine

Glycyrrhetinic acid

Table 3 Stability of baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid in rat plasma (n = 6).

Samples

Baicalin

Liquiritin

Berberine

Palmatine

C

Short term stability C（ng·mL-1）

RSD

X S 54.7

24 h Stability RE

C（ng·mL-1）

RSD

(%)

(%)

X S

49.47±2.56

5.17

-9.56

218.8

209.97±6.65

3.17

2188.0

2089.83±138.04

3.08

Repeated freezing and thawing

Long term stability

stability RSD

RE

C（ng·mL-1）

RSD

RE

C（ng·mL-1）

(%)

(%)

X S

(%)

(%)

X S

(%)

47.25 ± 3.50

7.40

-13.62

48.42 ± 3.98

8.22

-11.49

46.52 ± 4.27

9.17

-14.96

-4.04

209.36 ± 3.99

1.91

-4.32

194.21 ± 9.34

4.81

-11.24

201.01 ± 12.17

6.05

-8.13

6.61

-4.49

2081.96 ± 55.60

2.67

-4.85

2050.79±79.20

3.86

-6.27

2095.91±76.47

3.65

-4.21

2.80±0.28

10.14

-9.15

2.72 ± 0.22

7.99

-11.70

2.62 ± 0.17

6.55

-15.08

2.66 ± 0.13

5.04

-13.73

30.8

27.64±1.59

5.74

-10.25

26.60 ± 1.45

5.47

-13.65

26.53 ± 1.70

6.42

-13.85

27.14 ± 1.57

5.77

-11.89

154.0

144.02±12.45

8.64

-6.48

140.30 ± 3.90

2.78

-8.90

136.84 ± 5.19

3.79

-11.14

140.41 ± 6.39

4.55

-8.83

1.25

1.15±0.09

7.51

-8.40

1.09 ± 0.10

9.47

-13.05

1.10 ± 0.08

7.64

-11.73

1.16 ± 0.10

9.60

-16.33

12.5

11.58±1.05

9.07

-7.39

11.25 ± 0.64

5.73

-10.03

11.27 ± 0.63

5.59

-9.82

11.20 ± 0.54

4.77

-9.89

125.0

123.09±6.88

5.59

-1.53

116.99 ± 3.69

3.15

-6.41

115.58 ± 4.27

3.69

-7.54

113.03 ± 3.59

3.06

-6.20

1.0

0.95±0.09

9.06

-5.00

0.87 ± 0.05

5.70

-13.31

0.87 ± 0.03

3.06

-12.86

0.85 ± 0.04

5.00

-14.53

10.0

9.33±0.82

8.76

-6.75

9.20 ± 0.34

3.73

-7.97

9.09 ± 0.42

4.63

-9.13

8.87 ± 0.35

3.97

-11.35

200.0

190.82±8.76

4.59

-4.59

189.55 ± 5.64

2.98

-5.23

182.55 ± 7.58

4.15

-8.73

177.02 ± 14.09

7.96

-11.49

19.9

18.82±0.91

4.84

-5.44

18.04 ± 0.49

2.69

-9.36

17.00 ± 1.17

6.90

-14.56

17.59 ± 1.31

7.42

-11.63

99.5

91.83±6.05

6.58

-7.71

87.36 ± 5.71

6.54

-12.20

87.02 ± 2.63

3.02

-12.54

86.71 ± 3.47

4.00

-12.85

398.0

371.03±18.35

4.95

-6.78

366.13 ± 21.50

5.87

-8.01

354.46 ± 20.72

5.85

-10.94

374.38 ± 9.59

2.56

-5.93

(ng·mL-1)

RE (%)

Glycyrrhetinic acid

Table 4 Non-compartmental pharmacokinetic parameters of baicalin, liquiritin, berberine, palmatine and glycyrrhetinic acid in rats after intravenous administration (n = 6, mean ± SD). Glycyrrhetinic

Parameters

Baicalin

Liquiritin

Berberine

Palmatine

AUC0-t(μg·h·mL-1)

28.65 ± 6.70

65.82 ± 7.79

285.11 ± 43.03

82.53 ± 7.56

4.70 ± 0.45

AUC0-inf_obs(μg·h·mL-1)

29.32 ± 6.66

84.54 ± 8.18

370.61 ± 55.81

110.99 ± 11.20

4.99 ± 0.40

Cmax(μg·mL-1)

2.99 ± 0.70

19.85 ± 3.26

201.93 ± 15.23

32.71 ± 4.55

0.38 ± 0.05

Tmax(h)

0.083 ± 0.00

0.33 ± 0.00

0.50 ± 0.00

0.50 ± 0.00

8.00 ± 0.00

t1/2(h)

8.36 ± 1.21

5.80 ± 0.99

23.97 ± 4.10

27.68 ± 5.06

9.78 ± 1.05

MRT0-t(h)

16.84 ± 0.60

7.99 ± 1.10

29.18 ± 4.37

33.36 ± 5.83

17.45 ± 1.29

Cl/F(L·kg-1·h-1)

3.73 ± 0.9

121.67 ± 12.08

378.50 ± 59.42

505.30 ± 51.06

7.66 ± 0.66

acid