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Pharmacokinetic study of four flavones of Glycyrrhiza in rat plasma using HPLC–MS
Journal of Ethnopharmacology 148 (2013) 266–270
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Pharmacokinetic study of four flavones of Glycyrrhiza in rat plasma using HPLC–MS Yin-Ping Wu 1, Xian-Sheng Meng n,1, Yong-Rui Bao, Shuai Wang School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China
art ic l e i nf o
a b s t r a c t
Article history: Received 23 October 2012 Received in revised form 12 April 2013 Accepted 13 April 2013 Available online 30 April 2013
Aim of the study: This study aimed to develop a specific HPLC–MS method for simultaneous quantification of four flavones of Glycyrrhiza in rat plasma after oral administration and to describe the pharmacokinetics of four flavones in rat plasma. Materials and methods: A simple, sensitive and selective method for simultaneous determination of four flavones of Glycyrrhiza in rat plasma, i.e., liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin, by high performance liquid chromatography–tandem mass spectrometry (HPLC–MS) with negative electrospray ionization mode, was developed and validated. The method was applied to investigate the pharmacokinetics of four flavones in rat plasma after oral administration of Glycyrrhiza flavones. Chromatographic separation was accomplished on an Agilent TC-C18 column (4.6 mm 250 mm, and 5 μm), with gradient elution by using a mixture of methanoic acid (A) and acetonitrile (B) as the mobile phase at a flow rate of 0.8 mL/min. Results: The calibration curves for four flavones had good linearity higher than 0.997 in the measured range. Relative standard deviations (RSDs) of the intra- and inter-day precision at different levels were all less than 4.8%. The pharmacokinetic profile of four flavones in rat plasma was fitted with a twocompartment model detected by a simple, rapid and accurate HPLC–MS method. Time (h) to reach peak concentration (μg/mL) of liquiritin (2.69 7 0.04), isoliquiritin (10.16 70.02), liquiritigenin (2.83 7 0.02), and isoliquiritigenin (0.28 70.01) was 2.027 0.23, 1.97 70.20, 0.48 7 0.02, and 1.93 70.36, respectively. The distribution and elimination half-life (h) and area under the concentration–time curve (μg/mL–h) from t ¼ 0 to last time of liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were 1.02 70.48/ 2.27 7 0.53/16.9770.43, 2.04 7 1.01/2.38 70.80/69.20 7 5.24, 0.3570.10/4.26 70.16/14.83 7 0.11, and 1.18 70.32/3.04 7 0.22/2.10 7 0.09, respectively. Isoliquiritin presented the phenomenon of double peaks and the others appeared together in a single and plateau absorption phase. Isoliquiritigenin had the lowest oral bioavailability because of Cmax and AUC0−∞. Liquiritigenin had the fastest absorption and distribution rate and the lowest elimination rate according to Tmax, t1/2α, and t1/2β. Conclusions: This paper first reported on identification and determination of four flavones of Glycyrrhiza in rat plasma and their respective pharmacokinetic characteristics. The results provided a meaningful basis for better understanding the absorption mechanism of Glycyrrhiza and evaluating the clinical application of this medicine. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Glycyrrhiza flavones Pharmacokinetics HPLC–MS
1. Introduction Glycyrrhiza, one of the most popular Traditional Chinese Medicines in the world which was officially listed in the Chinese Pharmacopoeia, has been widely used in China during the past millennia for its medical potential, in the treatment of antioxidation, anti-cancer, anti-ulcer, anti-inflammation, antivirus,
n Correspondence to: School of Pharmacy, Liaoning University of Traditional Chinese Medicine, No. 18 of DD5 Street, Dalian, Liaoning Province 116600, China. Tel./fax: +86 0411 8740 6496. E-mail address: [email protected] (X.-S. Meng). 1 The two authors have contributed equally to this work.
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.04.024
and liver function improvement (Ji et al., 2004; Aly et al., 2005; Fiore et al., 2007; Stickel and Schuppan 2007), decoction: 3–10 g, large dose: 30–60 g, born with qingrejiedu, grilled with buzhonghuanji (Yang 2008; Wang 1994). Phytochemical investigations reveal that flavones and triterpenes are regarded as the principal components responsible for the main pharmacological activities (Tu et al., 2010; Yutaka et al., 2003). However, many previous findings were obtained from in vitro studies. Whether those in vitro activities and mechanisms of Glycyrrhiza can be extended to in vivo situation remains to be clarified by pharmacokinetic study (Lin et al., 2009). To the best of our knowledge, several HPLC–MS methods are available for the determination of the main active constituents of
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267
2. Materials and methods
2.3.2. Stock solution, calibration standards and quality control (QC) samples Standard stock solutions of liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, and paeoniflorin were diluted to achieve a final concentration of 30.1, 401.0, 39.8, 3.3, and 4.3 μg/mL dissolved by methanol, respectively. Analytical standards for four flavones were prepared in methanol over a concentration range of 2.72–0.17 μg/mL for liquiritin, 36.48–2.28 μg/mL for isoliquiritin, 3.68–0.23 μg/mL for liquiritigenin and 0.32–0.02 μg/mL for isoliquiritigenin, by serial dilution, and the same concentration range for a calibration curve was prepared in the blank, and normal rat plasma. Quality control (QC) samples at four different concentration levels 0.17, 0.40, 1.50, and 2.40 μg/mL for liquiritin, 2.28, 4.00, 15.00, and 24.00 μg/mL for isoliquiritin, 0.23, 0.40, 1.50, and 2.40 μg/mL for liquiritigenin, 0.02, 0.04, 0.15, and 0.24 μg/mL for isoliquiritigenin (LLOQ, low, medium, high levels, respectively) were prepared in the same way as the plasma samples for calibration. All samples were stored in a −20 1C freezer until analysis.
2.1. Chemicals and reagents
2.4. Pretreatment of plasma samples
Glycyrrhiza was purchased from Liaoning Benxi three Medicine Co., Ltd. (Liaoning, China), which had been identified by Professor Yan-Jun Zhai (Liaoning University of Traditional Chinese Medicine) as dry roots and rhizomes of Leguminosae Glycyrrhiza uralensis Fisch., Glycyrrhiza inflate Bat. or Glycyrrhiza glabra L. The standards of liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, and paeoniflorin were purchased from Shanghai Forever Biotech Co., Ltd. (Shanghai, China). Methanol (HPLC grade) was obtained from Kermel Chemicals Ltd. (Tianjin, China). Acetonitrile (HPLC grade) was obtained from TEDIA (USA). Distilled water was purified by MilliQ system (Millipore Corp. Bedford, MA, USA). Other reagents were all of analytical grade.
Add 20 μL internal standard solution paeoniflorin and 800 μL methanol into 200 μL rat plasma samples, and then vortex-mix for 3 min and centrifuge at 8000g for 10 min at 4 1C. The upper organic layer was collected and blown to dryness under nitrogen gas in a 40 1C water-bath. The residue was dissolved into 100 μL methanol for analysis.
Glycyrrhiza in different biological samples; however, they are almost Glycyrrhiza triterpenes instead of Glycyrrhiza flavones (Gao et al., 2004; Xiang et al., 1999). In this paper, a simple, sensitive, precise and convenient HPLC–MS method for simultaneous determination of four flavones of Glycyrrhiza plasma concentration was established and used to determine the pharmacokinetics of four flavones. The overall analytical procedure was rapid and reproducible, which made it suitable for quantitative analysis of a large number of samples. The pharmacokinetics of four flavones in plasma was simultaneously investigated at the first time and the obtained results would be very helpful for evaluating safety and effective clinical application of this medicine (Tang et al., 2009; Ren et al., 2000; Makino et al., 2008; He et al., 2003; Zhao et al., 2008).
2.2. Instrumentation and analytical conditions The chromatographic separations were performed on an Agilent TC-C18 column (4.6 mm 250 mm, 5 μm) using a high performance liquid chromatography system (HPLC, Agilent, USA). The column was maintained at 25 1C. The mobile phases A and B consisted of methanoic acid and acetonitrile, respectively. The elution followed a linear gradient of 17–65% B over 0–28 min. Detector wavelengths were 276 nm and 362 nm. The eluent flow was 0.8 mL/min and a 20 mL injection of each sample was made onto the column. An Agilent MS system operated in negative electrospray ionization mode was employed in this study. Nitrogen was used as nebulization gas and was set to 10 L/min at a temperature of 350 1C. The capillary voltage was set at 3.5 kV. The neulizer pressure was 50 psig.
2.3. Preparation of samples 2.3.1. Glycyrrhiza flavones Take a moderate amount of Glycyrrhiza, crush, wash 4 times with 90% ethanol, extraction time is 1 h, soaking time is 5 h, extracting solution is added cold and then filtered, and condensed into per ml containing 1 g crude drug, use petroleum ether to extract or to remove small polar substances and then use ethyl acetate to extract 2 times, make the extraction sample solution into aqueous solution, adopt polyamide resin, pack column with wet method, 1.5 g herb:1 ml wet resin, wash with 3BV distilled water and then elute with 6BV 70% ethanol, collect ethanol eluent, evaporate to dryness and obtain Glycyrrhiza flavones enrichment; the purity is 88.3%.
2.5. Pharmacokinetic study in rat plasma Healthy SD rats weighing 200 720 g were obtained from Dalian Medical University Laboratory Animal Center (Dalian, China). After breeding in a controlled environment for 5 days, the rats orally administrated Glycyrrhiza flavones at a dose of 0.468 g/kg. For pharmacokinetic study, the blood samples of 6 rats were collected from the fossa orbitalis vein according to the specific schedule at times of 0, 0.083, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after dosing. The blood samples were immediately transferred to heparinized tubes and centrifuged at 3000g for 15 min. The separated plasma was frozen at −20 1C before assay. 3. Results 3.1. Method validation 3.1.1. Determination of Glycyrrhiza flavones in plasma Liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were identified by HPLC–MS in rat plasma after oral administration of Glycyrrhiza flavones. The target product ion mass spectra are shown in Fig. 1. 3.1.2. Selectivity The selectivity of the method was tested by comparing the chromatograms with HPLC–MS of blank plasma, spiked plasma and actual plasma samples after oral administration of Glycyrrhiza flavones. It was indicated that analytes were well separated and no interferences were detected from endogenous substances or metabolites. The representative chromatograms for determination of analytes in plasma are shown in Fig. 2. 3.1.3. Limit of detection and the lower limit of quantification The limits of detection (LOD, S/N ¼ 3) and the lower limits of quantification (LLOQ, S/N ¼10) for liquiritin, isoliquiritin, liquiritigenin and isoliquiritigenin were 0.057, 0.760, 0.077, 0.007 μg/mL and 0.17, 2.28, 0.23, 0.02 μg/mL respectively.
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Fig. 1. Product ion mass spectra of Glycyrrhiza flavones.
3.1.4. Linearity Add 20 mL standard stock solutions of different concentrations and 20 mL internal standard solution paeoniflorin into 200 mL blank plasma at five different concentration levels of 2.72, 1.36, 0.68, 0.34, and 0.17 μg/mL for liquiritin, 36.48, 18.24, 9.12, 4.56, and 2.28 μg/mL for isoliquiritin, 3.68, 1.84, 0.92, 0.46, and 0.23 μg/mL for liquiritigenin and 0.32, 0.16, 0.08, 0.04, and 0.02 μg/mL for isoliquiritigenin. The linear regression of the investigated flavones in rat plasma was constructed by plotting peak area with concentration of standard solutions. The regression equations were y¼62.73x+0.85 (R2 ¼0.997) (liquiritin), y¼4.178x+1.11 (R2 ¼0.999) (isoliquiritin), y¼50.74x−0.77 (R2 ¼0.998) (liquiritigenin), and y¼ 512.8x−1.36 (R2 ¼0.997) (isoliquiritigenin). The calibration curves showed good linearity over the concentration range in all bio-samples with a correlation coefficient (R2) larger than 0.997.
3.1.5. Precision and accuracy The precision and accuracy were assessed by determining QC samples at four concentration levels. Precision was expressed as a relative standard deviation (RSD) and accuracy was evaluated as (relative error, RE). The intra-day precision and accuracy were determined by repeatedly assaying QC samples on the same day (n ¼6), and inter-day precision and accuracy were determined
by repeated analysis during three successive days (n ¼6 series per day). RSDs were obtained below 4.8% for intra-day and inter-day precisions and RE was within 6.0% for intra-day and inter-day accuracies. The results indicated that overall reproducibility of the method was acceptable. 3.1.6. Stability The stability of four flavones in rat plasma was determined using the QC samples. Short-term stability was assessed by analyzing the samples kept at room temperature (15–25 1C) for 4 h after routine preparation, and long-term stability was determined by assaying the plasma samples after storage at −20 1C for 15 days. Freeze–thaw stability was determined for three freeze– thaw cycles (−20 1C/room temperature). The REs of short-term stability, long-term stability and freeze–thaw stability were less than 5.84%. The results suggested that all the analytes were stable under the indicated storage conditions. 3.1.7. Recovery Recovery¼peak area ratio from the spiked plasma after extraction/peak area ratio from the standard solutions in methanol at the same concentrations 100. The mean extraction recoveries of
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Fig. 3. Plasma concentration–time profile of four flavones after oral administration of Glycyrrhiza flavones.
Table 1 Pharmacokinetic parameters of four flavones after oral administration of Glycyrrhiza flavones. Parameters Fig. 2. Representative chromatograms with HPLC–MS of a blank plasma sample (A), a blank plasma sample spiked with four flavones (B), and an actual plasma sample of a rat taken 2 h after oral administration of Glycyrrhiza flavones (C). (1) Paeoniflorin, (2) liquiritin, (3) isoliquiritin, (4) liquiritigenin and (5) isoliquiritigenin.
the investigated flavones in plasma were found to be 92.5–98.7% with RSDs less than 5.0%. 3.2. Pharmacokinetic study The HPLC–MS method was successfully applied to the pharmacokinetic study of four flavones after oral administration of Glycyrrhiza flavones to rats. The mean plasma concentration–time profile is shown in Fig. 3. The pharmacokinetic model and the parameters were calculated by the practical pharmacokinetic program DAS2.0. It was found that they behaved as the twocompartment model for four flavones. The main pharmacokinetic parameters are summarized in Table 1. Values are mean7SD for n¼ 6 rats with Glycyrrhiza flavones. t1/2α, distribution half-life; t1/2β, elimination half-life; AUC0−∞, area under concentration–time curve from t¼0 to last time; Ka, absorption constant; Tmax, time to reach peak concentration; and Cmax, maximum plasma concentration.
Liquiritin
Isoliquiritin
Liquiritigenin Isoliquiritigenin
t1/2α (h) 1.02 7 0.48 2.047 1.01 0.357 0.10 t1/2β (h) 2.277 0.53 2.38 7 0.80 4.26 7 0.16 AUC0−∞ (μg/mL–h) 16.977 0.43 69.20 7 5.24 14.83 7 0.11 Ka (1/h) 2.60 7 0.11 1.82 7 0.32 23.26 7 1.89 Tmax (h) 2.02 7 0.23 1.977 0.20 0.487 0.02 Cmax (μg/mL) 2.69 7 0.04 10.167 0.02 2.83 7 0.02
1.187 0.32 3.047 0.22 2.107 0.09 0.667 0.07 1.93 7 0.36 0.28 7 0.01
using the HPLC–MS method and fitting the C–t curve. The pharmacokinetic parameters of four flavones of Glycyrrhiza were calculated in rats to assist in monitoring and understanding its absorption mechanism. Although liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were all flavones ingredients and had similar chemical construction, they had different pharmacokinetic characteristics. Isoliquiritin presented the phenomenon of double peaks and the others appeared together in a single and plateau absorption phase; it suggested that these components might have multiple absorption sites, regulation of enterohepatic circulation or the gastric emptying rate, or there was ingredient interaction (Shaw et al., 2012). Isoliquiritigenin had the lowest oral bioavailability, which was probably related to the degradation of intestinal flora. Liquiritigenin had the fastest absorption and distribution rate and the lowest elimination rate. The pharmacokinetic study of Glycyrrhiza flavones is a very useful reference for the researches of TCMs in clinical trials and applications, which should be a topic of study in the future.
4. Discussion Owing to the complexity of Traditional Chinese Medicines, many analogs may be co-eluted during analyses. In order to develop a sensitive and accurate HPLC–MS method for the determination of active flavones compositions of Glycyrrhiza in rat plasma, HPLC–MS is currently one of the most useful tools available for simultaneous quantification of herbal compounds because of its high sensitivity and selectivity (Qiu et al., 2011). To characterize the fragment ions of the investigated compounds, an electrospray interface with good sensitivity, fragmentation and linearity was optimized. Liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were identified via HPLC–MS by comparing retention times and main product ions. The two-compartment model of four flavones of Glycyrrhiza was determined by detecting concentration–time (C–t) values
5. Authors' statement Animal rights: The institutional and (inter)national guidelines for the care and use of laboratory animal were followed. See the ‘Application of method in pharmacokinetic study’ part for details.
Acknowledgments The authors gratefully acknowledge the financial supports by Liaoning Benxi Third Medicine Co. and Liaoning University of Traditional Chinese Medicine. This paper is also supported in part by famous and high quality Traditional Chinese Medicines large assortment qizhiweitong particle technical reform, eleventh five-
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year plan, Significant New Medicines Creation, Science and Technology Major Special Project (2010ZX09401-304-201); component of Traditional Chinese Medicines Compatibility Research Technology sub-platform, eleventh five-year plan, significant new medicines creation, science and technology major special project (2010ZX09401-304-105C); Liaoning province component of Traditional Chinese Medicines engineering technology research center (Liaoning science and technology development [2012] No. 37). References Aly, A.M., Al-Alousi, L., Salem, H.A., 2005. Licorice: a possible anti-inflammatory and anti-ulcer drug. AAPS PharmSciTech 6, E74–82. Fiore, C., Eisenhut, M., Krausse, R., Ragazzi, E., Pellati, D., Armanini, D., Bielenberg, J., 2007. Antiviral effects of Glycyrrhiza species. Phytotherapy Research 22, 141–148. Gao, Q.T., Chen, X.H., Bi, K.S., 2004. Comparative pharmacokinetic behavior of glycyrrhetic acid after oral administration of glycyrrhizic acid and Gancao-FuziTang. Biological and Pharmaceutical Bulletin 27, 226–228. He, J.X., Akao, T., Tani, T., 2003. Influence of co-administered antibiotics on the pharmacokinetic fate in rats of paeoniflorin and its active metabolite paeonimetabolin-I from Shaoyao-Gancao-tang. JPP 55, 313–322. Ji, Y.B., Jiang, W., Fan, Y.L., 2004. Advance in studies of Glycyrrhiza flavonoids. Chinese Traditional and Herbal Drugs 35, 5–6, fu. Lin, S.P., Tsai, S.Y., Hou, Y.C., Chao, P.D.L., 2009. Glycyrrhizin and Licorice significantly affect the pharmacokinetics of methotrexate in rats. Journal of Agricultural and Food Chemistry 57, 1854–1859. Makino, T., Ohtake, N., Watanabe, A., 2008. Down-regulation of a hepatic transporter multidrug resistance-associated protein 2 is involved in alteration of
pharmacokinetics of glycyrrhizin and its metabolites in a rat model of chronic liver injury. DMD 36, 1438–1443. Qiu, X.J., Huang, X., Chen, Z.Q., Ren, P., 2011. Pharmacokinetic study of the prokinetic compounds meranzin hydrate and ferulic acid following oral administration of Chaihu-Shugan-San to patients with functional dyspepsia. Journal of Ethnopharmacology 137, 205–213. Ren, P., Zhang, L., Wang, L.L., Huang, X., Li, Y.C., 2000. High-performance liquid chromatography used in determination of concentration of glycyrrhizin in plasma of rats with spleen deficiency and pharmacokinetics of glycyrrhizin. Journal of Nanjing University of Traditional Chinese Medicine 16, 345–346. Shaw, L.S., Lin, L.C., Tsai, T.H., 2012. HPLC–MS/MS analysis of a traditional chinese medical formulation of Bu-Yang-Huan-Wu-Tang and its pharmacokinetics after oral administration to rats. PLOS ONE 7, e43848. Stickel, F., Schuppan, D., 2007. Herbal medicine in the treatment of liver diseases. Digestive and Liver Disease 39, 293–304. Tang, J.C., Song, X.H., Zhu, M., Zhang, J.N., 2009. Study on the pharmacokinetics drug–drug interaction potential of Glycyrrhiza uralensis, a traditional Chinese medicine, with Lidocaine in rats. Phytotherapy Research 23, 603–607. Tu, J.H., He, Y.J., Chen, Y., 2010. Effect of glycyrrhizin on the activity of CYP3A enzyme in humans. European Journal of Clinical Pharmacology 66, 805–810. Wang, Y.J., 1994. The research of licorice dosage. Journal of Chinese Medicine 9, 4–5. Xiang, Q., Cheng, G., Chen, J.M., 1999. Determination of the concentration of glycyrrhetic acid in rat plasma by HPLC. Journal of Shenymag Pharmaceutical University 16, 107–109. Yang, Y.J., 2008. Discuss the clinical application of licorice. Chinese and Foreign Health Abstract Clinical Physicians 5, 44–45. Yutaka, Y., Takami, M., Ikuo, S., Tadato, T., 2003. Pharmaceutical evaluation of Glycyrrhiza uralensis roots cultivated in Eastern Nei-Meng-Gu of China. Biological and Pharmaceutical Bulletin 26, 1144–1149. Zhao, W.J., Wang, B.J., Wei, C.M., 2008. Determination of glycyrrhetic acid in human plasma by HPLC–MS method and investigation of its pharmacokinetics. Journal of Clinical Pharmacy and Therapeutics 33, 289–294.
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Pharmacokinetic study of four flavones of Glycyrrhiza in rat plasma using HPLC–MS Yin-Ping Wu 1, Xian-Sheng Meng n,1, Yong-Rui Bao, Shuai Wang School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China
art ic l e i nf o
a b s t r a c t
Article history: Received 23 October 2012 Received in revised form 12 April 2013 Accepted 13 April 2013 Available online 30 April 2013
Aim of the study: This study aimed to develop a specific HPLC–MS method for simultaneous quantification of four flavones of Glycyrrhiza in rat plasma after oral administration and to describe the pharmacokinetics of four flavones in rat plasma. Materials and methods: A simple, sensitive and selective method for simultaneous determination of four flavones of Glycyrrhiza in rat plasma, i.e., liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin, by high performance liquid chromatography–tandem mass spectrometry (HPLC–MS) with negative electrospray ionization mode, was developed and validated. The method was applied to investigate the pharmacokinetics of four flavones in rat plasma after oral administration of Glycyrrhiza flavones. Chromatographic separation was accomplished on an Agilent TC-C18 column (4.6 mm 250 mm, and 5 μm), with gradient elution by using a mixture of methanoic acid (A) and acetonitrile (B) as the mobile phase at a flow rate of 0.8 mL/min. Results: The calibration curves for four flavones had good linearity higher than 0.997 in the measured range. Relative standard deviations (RSDs) of the intra- and inter-day precision at different levels were all less than 4.8%. The pharmacokinetic profile of four flavones in rat plasma was fitted with a twocompartment model detected by a simple, rapid and accurate HPLC–MS method. Time (h) to reach peak concentration (μg/mL) of liquiritin (2.69 7 0.04), isoliquiritin (10.16 70.02), liquiritigenin (2.83 7 0.02), and isoliquiritigenin (0.28 70.01) was 2.027 0.23, 1.97 70.20, 0.48 7 0.02, and 1.93 70.36, respectively. The distribution and elimination half-life (h) and area under the concentration–time curve (μg/mL–h) from t ¼ 0 to last time of liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were 1.02 70.48/ 2.27 7 0.53/16.9770.43, 2.04 7 1.01/2.38 70.80/69.20 7 5.24, 0.3570.10/4.26 70.16/14.83 7 0.11, and 1.18 70.32/3.04 7 0.22/2.10 7 0.09, respectively. Isoliquiritin presented the phenomenon of double peaks and the others appeared together in a single and plateau absorption phase. Isoliquiritigenin had the lowest oral bioavailability because of Cmax and AUC0−∞. Liquiritigenin had the fastest absorption and distribution rate and the lowest elimination rate according to Tmax, t1/2α, and t1/2β. Conclusions: This paper first reported on identification and determination of four flavones of Glycyrrhiza in rat plasma and their respective pharmacokinetic characteristics. The results provided a meaningful basis for better understanding the absorption mechanism of Glycyrrhiza and evaluating the clinical application of this medicine. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Glycyrrhiza flavones Pharmacokinetics HPLC–MS
1. Introduction Glycyrrhiza, one of the most popular Traditional Chinese Medicines in the world which was officially listed in the Chinese Pharmacopoeia, has been widely used in China during the past millennia for its medical potential, in the treatment of antioxidation, anti-cancer, anti-ulcer, anti-inflammation, antivirus,
n Correspondence to: School of Pharmacy, Liaoning University of Traditional Chinese Medicine, No. 18 of DD5 Street, Dalian, Liaoning Province 116600, China. Tel./fax: +86 0411 8740 6496. E-mail address: [email protected] (X.-S. Meng). 1 The two authors have contributed equally to this work.
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.04.024
and liver function improvement (Ji et al., 2004; Aly et al., 2005; Fiore et al., 2007; Stickel and Schuppan 2007), decoction: 3–10 g, large dose: 30–60 g, born with qingrejiedu, grilled with buzhonghuanji (Yang 2008; Wang 1994). Phytochemical investigations reveal that flavones and triterpenes are regarded as the principal components responsible for the main pharmacological activities (Tu et al., 2010; Yutaka et al., 2003). However, many previous findings were obtained from in vitro studies. Whether those in vitro activities and mechanisms of Glycyrrhiza can be extended to in vivo situation remains to be clarified by pharmacokinetic study (Lin et al., 2009). To the best of our knowledge, several HPLC–MS methods are available for the determination of the main active constituents of
Y.-P. Wu et al. / Journal of Ethnopharmacology 148 (2013) 266–270
267
2. Materials and methods
2.3.2. Stock solution, calibration standards and quality control (QC) samples Standard stock solutions of liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, and paeoniflorin were diluted to achieve a final concentration of 30.1, 401.0, 39.8, 3.3, and 4.3 μg/mL dissolved by methanol, respectively. Analytical standards for four flavones were prepared in methanol over a concentration range of 2.72–0.17 μg/mL for liquiritin, 36.48–2.28 μg/mL for isoliquiritin, 3.68–0.23 μg/mL for liquiritigenin and 0.32–0.02 μg/mL for isoliquiritigenin, by serial dilution, and the same concentration range for a calibration curve was prepared in the blank, and normal rat plasma. Quality control (QC) samples at four different concentration levels 0.17, 0.40, 1.50, and 2.40 μg/mL for liquiritin, 2.28, 4.00, 15.00, and 24.00 μg/mL for isoliquiritin, 0.23, 0.40, 1.50, and 2.40 μg/mL for liquiritigenin, 0.02, 0.04, 0.15, and 0.24 μg/mL for isoliquiritigenin (LLOQ, low, medium, high levels, respectively) were prepared in the same way as the plasma samples for calibration. All samples were stored in a −20 1C freezer until analysis.
2.1. Chemicals and reagents
2.4. Pretreatment of plasma samples
Glycyrrhiza was purchased from Liaoning Benxi three Medicine Co., Ltd. (Liaoning, China), which had been identified by Professor Yan-Jun Zhai (Liaoning University of Traditional Chinese Medicine) as dry roots and rhizomes of Leguminosae Glycyrrhiza uralensis Fisch., Glycyrrhiza inflate Bat. or Glycyrrhiza glabra L. The standards of liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, and paeoniflorin were purchased from Shanghai Forever Biotech Co., Ltd. (Shanghai, China). Methanol (HPLC grade) was obtained from Kermel Chemicals Ltd. (Tianjin, China). Acetonitrile (HPLC grade) was obtained from TEDIA (USA). Distilled water was purified by MilliQ system (Millipore Corp. Bedford, MA, USA). Other reagents were all of analytical grade.
Add 20 μL internal standard solution paeoniflorin and 800 μL methanol into 200 μL rat plasma samples, and then vortex-mix for 3 min and centrifuge at 8000g for 10 min at 4 1C. The upper organic layer was collected and blown to dryness under nitrogen gas in a 40 1C water-bath. The residue was dissolved into 100 μL methanol for analysis.
Glycyrrhiza in different biological samples; however, they are almost Glycyrrhiza triterpenes instead of Glycyrrhiza flavones (Gao et al., 2004; Xiang et al., 1999). In this paper, a simple, sensitive, precise and convenient HPLC–MS method for simultaneous determination of four flavones of Glycyrrhiza plasma concentration was established and used to determine the pharmacokinetics of four flavones. The overall analytical procedure was rapid and reproducible, which made it suitable for quantitative analysis of a large number of samples. The pharmacokinetics of four flavones in plasma was simultaneously investigated at the first time and the obtained results would be very helpful for evaluating safety and effective clinical application of this medicine (Tang et al., 2009; Ren et al., 2000; Makino et al., 2008; He et al., 2003; Zhao et al., 2008).
2.2. Instrumentation and analytical conditions The chromatographic separations were performed on an Agilent TC-C18 column (4.6 mm 250 mm, 5 μm) using a high performance liquid chromatography system (HPLC, Agilent, USA). The column was maintained at 25 1C. The mobile phases A and B consisted of methanoic acid and acetonitrile, respectively. The elution followed a linear gradient of 17–65% B over 0–28 min. Detector wavelengths were 276 nm and 362 nm. The eluent flow was 0.8 mL/min and a 20 mL injection of each sample was made onto the column. An Agilent MS system operated in negative electrospray ionization mode was employed in this study. Nitrogen was used as nebulization gas and was set to 10 L/min at a temperature of 350 1C. The capillary voltage was set at 3.5 kV. The neulizer pressure was 50 psig.
2.3. Preparation of samples 2.3.1. Glycyrrhiza flavones Take a moderate amount of Glycyrrhiza, crush, wash 4 times with 90% ethanol, extraction time is 1 h, soaking time is 5 h, extracting solution is added cold and then filtered, and condensed into per ml containing 1 g crude drug, use petroleum ether to extract or to remove small polar substances and then use ethyl acetate to extract 2 times, make the extraction sample solution into aqueous solution, adopt polyamide resin, pack column with wet method, 1.5 g herb:1 ml wet resin, wash with 3BV distilled water and then elute with 6BV 70% ethanol, collect ethanol eluent, evaporate to dryness and obtain Glycyrrhiza flavones enrichment; the purity is 88.3%.
2.5. Pharmacokinetic study in rat plasma Healthy SD rats weighing 200 720 g were obtained from Dalian Medical University Laboratory Animal Center (Dalian, China). After breeding in a controlled environment for 5 days, the rats orally administrated Glycyrrhiza flavones at a dose of 0.468 g/kg. For pharmacokinetic study, the blood samples of 6 rats were collected from the fossa orbitalis vein according to the specific schedule at times of 0, 0.083, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after dosing. The blood samples were immediately transferred to heparinized tubes and centrifuged at 3000g for 15 min. The separated plasma was frozen at −20 1C before assay. 3. Results 3.1. Method validation 3.1.1. Determination of Glycyrrhiza flavones in plasma Liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were identified by HPLC–MS in rat plasma after oral administration of Glycyrrhiza flavones. The target product ion mass spectra are shown in Fig. 1. 3.1.2. Selectivity The selectivity of the method was tested by comparing the chromatograms with HPLC–MS of blank plasma, spiked plasma and actual plasma samples after oral administration of Glycyrrhiza flavones. It was indicated that analytes were well separated and no interferences were detected from endogenous substances or metabolites. The representative chromatograms for determination of analytes in plasma are shown in Fig. 2. 3.1.3. Limit of detection and the lower limit of quantification The limits of detection (LOD, S/N ¼ 3) and the lower limits of quantification (LLOQ, S/N ¼10) for liquiritin, isoliquiritin, liquiritigenin and isoliquiritigenin were 0.057, 0.760, 0.077, 0.007 μg/mL and 0.17, 2.28, 0.23, 0.02 μg/mL respectively.
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Fig. 1. Product ion mass spectra of Glycyrrhiza flavones.
3.1.4. Linearity Add 20 mL standard stock solutions of different concentrations and 20 mL internal standard solution paeoniflorin into 200 mL blank plasma at five different concentration levels of 2.72, 1.36, 0.68, 0.34, and 0.17 μg/mL for liquiritin, 36.48, 18.24, 9.12, 4.56, and 2.28 μg/mL for isoliquiritin, 3.68, 1.84, 0.92, 0.46, and 0.23 μg/mL for liquiritigenin and 0.32, 0.16, 0.08, 0.04, and 0.02 μg/mL for isoliquiritigenin. The linear regression of the investigated flavones in rat plasma was constructed by plotting peak area with concentration of standard solutions. The regression equations were y¼62.73x+0.85 (R2 ¼0.997) (liquiritin), y¼4.178x+1.11 (R2 ¼0.999) (isoliquiritin), y¼50.74x−0.77 (R2 ¼0.998) (liquiritigenin), and y¼ 512.8x−1.36 (R2 ¼0.997) (isoliquiritigenin). The calibration curves showed good linearity over the concentration range in all bio-samples with a correlation coefficient (R2) larger than 0.997.
3.1.5. Precision and accuracy The precision and accuracy were assessed by determining QC samples at four concentration levels. Precision was expressed as a relative standard deviation (RSD) and accuracy was evaluated as (relative error, RE). The intra-day precision and accuracy were determined by repeatedly assaying QC samples on the same day (n ¼6), and inter-day precision and accuracy were determined
by repeated analysis during three successive days (n ¼6 series per day). RSDs were obtained below 4.8% for intra-day and inter-day precisions and RE was within 6.0% for intra-day and inter-day accuracies. The results indicated that overall reproducibility of the method was acceptable. 3.1.6. Stability The stability of four flavones in rat plasma was determined using the QC samples. Short-term stability was assessed by analyzing the samples kept at room temperature (15–25 1C) for 4 h after routine preparation, and long-term stability was determined by assaying the plasma samples after storage at −20 1C for 15 days. Freeze–thaw stability was determined for three freeze– thaw cycles (−20 1C/room temperature). The REs of short-term stability, long-term stability and freeze–thaw stability were less than 5.84%. The results suggested that all the analytes were stable under the indicated storage conditions. 3.1.7. Recovery Recovery¼peak area ratio from the spiked plasma after extraction/peak area ratio from the standard solutions in methanol at the same concentrations 100. The mean extraction recoveries of
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Fig. 3. Plasma concentration–time profile of four flavones after oral administration of Glycyrrhiza flavones.
Table 1 Pharmacokinetic parameters of four flavones after oral administration of Glycyrrhiza flavones. Parameters Fig. 2. Representative chromatograms with HPLC–MS of a blank plasma sample (A), a blank plasma sample spiked with four flavones (B), and an actual plasma sample of a rat taken 2 h after oral administration of Glycyrrhiza flavones (C). (1) Paeoniflorin, (2) liquiritin, (3) isoliquiritin, (4) liquiritigenin and (5) isoliquiritigenin.
the investigated flavones in plasma were found to be 92.5–98.7% with RSDs less than 5.0%. 3.2. Pharmacokinetic study The HPLC–MS method was successfully applied to the pharmacokinetic study of four flavones after oral administration of Glycyrrhiza flavones to rats. The mean plasma concentration–time profile is shown in Fig. 3. The pharmacokinetic model and the parameters were calculated by the practical pharmacokinetic program DAS2.0. It was found that they behaved as the twocompartment model for four flavones. The main pharmacokinetic parameters are summarized in Table 1. Values are mean7SD for n¼ 6 rats with Glycyrrhiza flavones. t1/2α, distribution half-life; t1/2β, elimination half-life; AUC0−∞, area under concentration–time curve from t¼0 to last time; Ka, absorption constant; Tmax, time to reach peak concentration; and Cmax, maximum plasma concentration.
Liquiritin
Isoliquiritin
Liquiritigenin Isoliquiritigenin
t1/2α (h) 1.02 7 0.48 2.047 1.01 0.357 0.10 t1/2β (h) 2.277 0.53 2.38 7 0.80 4.26 7 0.16 AUC0−∞ (μg/mL–h) 16.977 0.43 69.20 7 5.24 14.83 7 0.11 Ka (1/h) 2.60 7 0.11 1.82 7 0.32 23.26 7 1.89 Tmax (h) 2.02 7 0.23 1.977 0.20 0.487 0.02 Cmax (μg/mL) 2.69 7 0.04 10.167 0.02 2.83 7 0.02
1.187 0.32 3.047 0.22 2.107 0.09 0.667 0.07 1.93 7 0.36 0.28 7 0.01
using the HPLC–MS method and fitting the C–t curve. The pharmacokinetic parameters of four flavones of Glycyrrhiza were calculated in rats to assist in monitoring and understanding its absorption mechanism. Although liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were all flavones ingredients and had similar chemical construction, they had different pharmacokinetic characteristics. Isoliquiritin presented the phenomenon of double peaks and the others appeared together in a single and plateau absorption phase; it suggested that these components might have multiple absorption sites, regulation of enterohepatic circulation or the gastric emptying rate, or there was ingredient interaction (Shaw et al., 2012). Isoliquiritigenin had the lowest oral bioavailability, which was probably related to the degradation of intestinal flora. Liquiritigenin had the fastest absorption and distribution rate and the lowest elimination rate. The pharmacokinetic study of Glycyrrhiza flavones is a very useful reference for the researches of TCMs in clinical trials and applications, which should be a topic of study in the future.
4. Discussion Owing to the complexity of Traditional Chinese Medicines, many analogs may be co-eluted during analyses. In order to develop a sensitive and accurate HPLC–MS method for the determination of active flavones compositions of Glycyrrhiza in rat plasma, HPLC–MS is currently one of the most useful tools available for simultaneous quantification of herbal compounds because of its high sensitivity and selectivity (Qiu et al., 2011). To characterize the fragment ions of the investigated compounds, an electrospray interface with good sensitivity, fragmentation and linearity was optimized. Liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were identified via HPLC–MS by comparing retention times and main product ions. The two-compartment model of four flavones of Glycyrrhiza was determined by detecting concentration–time (C–t) values
5. Authors' statement Animal rights: The institutional and (inter)national guidelines for the care and use of laboratory animal were followed. See the ‘Application of method in pharmacokinetic study’ part for details.
Acknowledgments The authors gratefully acknowledge the financial supports by Liaoning Benxi Third Medicine Co. and Liaoning University of Traditional Chinese Medicine. This paper is also supported in part by famous and high quality Traditional Chinese Medicines large assortment qizhiweitong particle technical reform, eleventh five-
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year plan, Significant New Medicines Creation, Science and Technology Major Special Project (2010ZX09401-304-201); component of Traditional Chinese Medicines Compatibility Research Technology sub-platform, eleventh five-year plan, significant new medicines creation, science and technology major special project (2010ZX09401-304-105C); Liaoning province component of Traditional Chinese Medicines engineering technology research center (Liaoning science and technology development [2012] No. 37). References Aly, A.M., Al-Alousi, L., Salem, H.A., 2005. Licorice: a possible anti-inflammatory and anti-ulcer drug. AAPS PharmSciTech 6, E74–82. Fiore, C., Eisenhut, M., Krausse, R., Ragazzi, E., Pellati, D., Armanini, D., Bielenberg, J., 2007. Antiviral effects of Glycyrrhiza species. Phytotherapy Research 22, 141–148. Gao, Q.T., Chen, X.H., Bi, K.S., 2004. Comparative pharmacokinetic behavior of glycyrrhetic acid after oral administration of glycyrrhizic acid and Gancao-FuziTang. Biological and Pharmaceutical Bulletin 27, 226–228. He, J.X., Akao, T., Tani, T., 2003. Influence of co-administered antibiotics on the pharmacokinetic fate in rats of paeoniflorin and its active metabolite paeonimetabolin-I from Shaoyao-Gancao-tang. JPP 55, 313–322. Ji, Y.B., Jiang, W., Fan, Y.L., 2004. Advance in studies of Glycyrrhiza flavonoids. Chinese Traditional and Herbal Drugs 35, 5–6, fu. Lin, S.P., Tsai, S.Y., Hou, Y.C., Chao, P.D.L., 2009. Glycyrrhizin and Licorice significantly affect the pharmacokinetics of methotrexate in rats. Journal of Agricultural and Food Chemistry 57, 1854–1859. Makino, T., Ohtake, N., Watanabe, A., 2008. Down-regulation of a hepatic transporter multidrug resistance-associated protein 2 is involved in alteration of
pharmacokinetics of glycyrrhizin and its metabolites in a rat model of chronic liver injury. DMD 36, 1438–1443. Qiu, X.J., Huang, X., Chen, Z.Q., Ren, P., 2011. Pharmacokinetic study of the prokinetic compounds meranzin hydrate and ferulic acid following oral administration of Chaihu-Shugan-San to patients with functional dyspepsia. Journal of Ethnopharmacology 137, 205–213. Ren, P., Zhang, L., Wang, L.L., Huang, X., Li, Y.C., 2000. High-performance liquid chromatography used in determination of concentration of glycyrrhizin in plasma of rats with spleen deficiency and pharmacokinetics of glycyrrhizin. Journal of Nanjing University of Traditional Chinese Medicine 16, 345–346. Shaw, L.S., Lin, L.C., Tsai, T.H., 2012. HPLC–MS/MS analysis of a traditional chinese medical formulation of Bu-Yang-Huan-Wu-Tang and its pharmacokinetics after oral administration to rats. PLOS ONE 7, e43848. Stickel, F., Schuppan, D., 2007. Herbal medicine in the treatment of liver diseases. Digestive and Liver Disease 39, 293–304. Tang, J.C., Song, X.H., Zhu, M., Zhang, J.N., 2009. Study on the pharmacokinetics drug–drug interaction potential of Glycyrrhiza uralensis, a traditional Chinese medicine, with Lidocaine in rats. Phytotherapy Research 23, 603–607. Tu, J.H., He, Y.J., Chen, Y., 2010. Effect of glycyrrhizin on the activity of CYP3A enzyme in humans. European Journal of Clinical Pharmacology 66, 805–810. Wang, Y.J., 1994. The research of licorice dosage. Journal of Chinese Medicine 9, 4–5. Xiang, Q., Cheng, G., Chen, J.M., 1999. Determination of the concentration of glycyrrhetic acid in rat plasma by HPLC. Journal of Shenymag Pharmaceutical University 16, 107–109. Yang, Y.J., 2008. Discuss the clinical application of licorice. Chinese and Foreign Health Abstract Clinical Physicians 5, 44–45. Yutaka, Y., Takami, M., Ikuo, S., Tadato, T., 2003. Pharmaceutical evaluation of Glycyrrhiza uralensis roots cultivated in Eastern Nei-Meng-Gu of China. Biological and Pharmaceutical Bulletin 26, 1144–1149. Zhao, W.J., Wang, B.J., Wei, C.M., 2008. Determination of glycyrrhetic acid in human plasma by HPLC–MS method and investigation of its pharmacokinetics. Journal of Clinical Pharmacy and Therapeutics 33, 289–294.