Journal of Ethnopharmacology 165 (2015) 243–250
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Research Paper
Investigating herb–herb interactions: The potential attenuated toxicity mechanism of the combined use of Glycyrrhizae radix et rhizoma (Gancao) and Sophorae flavescentis radix (Kushen) Lei Shi a, Xiuling Tang a, Xueliang Dang a, Qinhui Wang a, Xiangrui Wang b, Ping He b, Qingwei Wang a, Linna Liu a, XinYou Liu a, Yan Zhang a,n a b
Department of head!–td:Pharmacy,–>Pharmacy, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, PR China Department of Anesthesiology, Renji Hospital Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai 200127, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 20 January 2015 Accepted 9 February 2015 Available online 19 February 2015
Ethnopharmacological relevance: Glycyrrhizae radix et rhizoma (Gancao) is often prescribed together with Sophorae flavescentis radix (Kushen) in traditional Chinese medicinal practice to increase the efficacy on the treatment of hepatitis and hepatic fibrosis. Meanwhile, long-term single used Gancao can cause adverse reactions, lead to pseudohypercorticosteroidism especially. But the side effects of Gancao are significantly reduced when combined with Kushen; the reasons are still unknown. The aim of this study was to elucidate potential pharmacokinetic interaction between Kushen and Gancao, and to provide guidance for clinical medicine safety. Materials and methods: A specific and rapid HPLC–MS method was established to quantify the four main activity ingredients matrine (MT), oxymatrine (OMT), glycyrrhizin (GL) and glycyrrhetinic acid (GA) in rat plasma. In this study, the pharmacokinetic parameters and the pharmacokinetic differences of the four main activity ingredients MT, OMT, GL and GA in single herb and Kushen–Gancao couple were obtained. Results: Compared with oral administration of Gancao extract, K10 and Tmax of GA significantly increased to 0.43 h 1and 30 h after giving Kushen–Gancao (po0.05), but T1/2 and Vd were reduced to 0.73 L kg 1and 4.98 h (po 0.05). In addition, the AUC of GA was increased, and the other three activity ingredients all decreased. Conclusion: GA as the main factor leading to the sodium–water retention side effects of Gancao. The result found that the absorption of GA was significantly slowed down and the metabolism rate was accelerated in Kushen–Gancao than single herb. So the attenuated toxicity mechanism may be because the accumulation of GA reduced in vivo. The conclusion has important meaning to the compatibility of Chinese med. & 2015 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Matrine (Pubchem CID:91466) Oxymatrine (Pubchem CID:114850) Glycyrrhizin (Pubchem CID:14982) Glycyrrhetinic acid (Pubchem CID:10114) Keywords: Glycyrrhizae radix et rhizome Sophorae flavescentis radix Herb–herb interactions Attenuated toxicity
1. Introduction Traditional Chinese Medicine (TCM) is mostly prescribed in combination to obtain synergistic effects and reduce possible adverse reactions. Hence, the compatibility of Chinese medicinal herbs is an important theory in the combination of TCM. Herb couple, two herbs usually prescribed together to decrease toxicity and increase efficacy, is the basic unit which is commonly used in herbal formulae in TCM. They are much simpler than other complex formulae without altering the basic therapeutic features (Wang, et al., 2012; Wang, 2012). Generally, the law of compound compatibility was studied through pharmacodynamics and pharmacokinetics. Pharmacokinetic method was used in this study, which was helpful in understanding the action mechanism of drugs and the compatible priority of the entire prescription. n
Corresponding author.
http://dx.doi.org/10.1016/j.jep.2015.02.022 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Kushen–Gancao couple, a famous traditional herb couple, was recorded in Pu Ji Fang for thousands of years, and has been used as a TCM for treatment of hepatitis, chronic liver diseases, peptic ulcer and immunological disorders in China for a very long period ( Wagner, 2009). It also has been reported in various literatures, e.g. Handbook of Ning xia Chinese Herbal Medicine (Editorial Board of Hand book of Ningxia Chinese Herbal Medicine, 1971) and Journal of Shaanxi Traditional Chinese Medicine (China Academy of Chinese Medical Sciences, 1962), which are famous in the field of TCM. The traditional remedy is pill of Sophorae flavescentis radix (Kushen in Chinese), which is the root of Sophora flavescens Ait, and Glycyrrhizae radix et rhizoma (Gancao in Chinese), which was the root of Glycyrrhiza uralensis Fisch. Both the herbs are officially listed in the Chinese pharmacopeia (Pharmacopoeia of PR China, 2010). Sophorae flavescentis radix (Kushen) was widely used as TCM in the treatment of viral hepatitis and chronic liver diseases due to
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its wide spectrum of pharmacological actions including antiinflammatory, immune-inhibitory, anti-fibrotic, antiarrhythmic, anti-tumor and diuretic activities (Xu et al., 2011; Zhou et al., 2012; Zhi, 2009). Glycyrrhizae radix et rhizoma (Gancao) is prescribed in many Chinese traditional formulas for its medical potential effects in anti-inflammation (Fu et al., 2013), immunoloregulation (Lee et al., 2009), and anti-allergy nature (Shin et al., 2007). It also has been used for the treatment and prevention of hepatitis, chronic bronchitis, gastritis, tumor growth and immunological disorders (Miao et al., 2001; Kusano et al., 2003). Kushen–Gancao couple was widely used in the clinic treatment of viral hepatitis, chronic liver diseases, peptic ulcer and skin inflammations; furthermore, it obtained good curative effect (Xu, 1997; Li et al., 2010; Lin, 2011). It has been demonstrated that the treatment effect in hepatitis of Kushen–Gancao couple was significantly better than single herb; meanwhile, the adverse reactions caused by single use of Gancao was significantly decreased (Zhao et al., 2012). Matrine (MT) and Oxymatrine (OMT) are the main active components extracted from Kushen. Glycyrrhizin (GL) is the active component of Gancao. Glycyrrhetinic acid (GA) was the active metabolite in the body of the GL (Fig. 1). There were evidence from numerous clinical case reports and trials that conventional administration of GL may cause pseudohypercorticosteroidism, such as sodium and water retention, hypertension and hypokalaemia (Van Rossum et al., 2001; Kageyama et al., 1991; Shintani et al., 1992). Generally, the mechanism of this side effect was induced by GA competitive inhibition of hepatic microsomal 11B-typeII
hydroxysteroid dehydrogenase (11-HSD2) activity, which indirectly enhance cortical hormone concentration, showing the effect of steroids (Kratschmar et al., 2011). But these side effects significantly decreased when Gancao combined with Kushen (Wan et al. 2009). Some pharmacokinetic studies of these four active ingredients in single herbal drugs or other TCM formulas had been reported (Tang et al., 2013; Krähenbühl et al., 1994; Xu, et al., 2013). But the pharmacokinetic of these four active ingredients in Kushen–Gancao was not reported, the mechanism of attenuated toxicity of Gancao after co-administration with Kushen was unknown. The pharmacokinetic characteristics have an important significance for the interpretation of synergism and attenuation. So it is necessary to study the pharmacokinetic parameters of the four active ingredients in Kushen–Gancao and compare their possible pharmacokinetic differences after the administration of single-herb extracts to rats. So this paper will research the pharmacokinetic differences between single use of Sophorae flavescentis radix or Glycyrrhizae radix et rhizoma and combined use and investigate the potential herb–herb interaction, also reveal the attenuated toxicity mechanism of the combined use of Glycyrrhizae radix et rhizoma (Gancao) and Sophorae flavescentis radix In a previous study, we had developed a rapid and sensitive LC– MS/MS method to simultaneously determine Matrine (MT), Oxymatrine (OMT), Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) in rat plasma after oral administration of Kusheng–Gancao (Wang et al., 2014). In this study, the pharmacokinetic parameters and the pharmacokinetic differences of the four ingredients after the administration of single-herb extracts and Kushen–Gancao were
Fig. 1. Chemical structures of: (a) Oxymatrine (OMT), (b) Matrine (MT), (c) Glycyrrhizin (GL) and (d) Glycyrrhetinic acid (GA).
L. Shi et al. / Journal of Ethnopharmacology 165 (2015) 243–250
obtained. The results of our study would help us explore whether there were synergistic effects on the four ingredients in Kushen– Gancao. At the same time, these methods may provide solutions to study the reasonableness of the traditional medicine recipe.
2. Experimental 2.1. Materials and reagents The reference standards of Matrine, Oxymatrine, Glycyrrhizin and Glycyrrhetinic acid were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Aminopyrine as internal standard (I.S.) was purchased from Aladdin Chemistry Co. ltd (Shanghai, China). Methanol (HPLC grade) was purchased from Fisher Scientific (Fair Lawn, NJ, USA); Ammonium acetate (HPLC grade) was from Dikma (Richmond Hill, NY, USA); ultrapure water for HPLC analysis was prepared from a Milli-Q water purification system (Millipore, Milford, MA, USA); and all other reagents were of analytical grade. Kushen and Gancao were purchased from Xiaocao Medicinal Material Company (Xi’an, China) and authenticated by Professor Qing-Wei Wang (The Fourth Military Medical University, Xi’an, China).
2.2. Animals Male Sprague–Dawley rats (250 720 g) were supplied by the Lab Animal Center of Fourth Military Medical University. The experimental protocol was approved by the University Ethics Committee for the use of experimental animals and all animal studies were carried out according to the Guide for Care and Use of Laboratory Animals. The number of the Ethics Committee on Animal Experimentation was TDLL-20130502. These animals lived in suitable conditions: temperature-controlled room (22 7 2 1C) with a 12 h light-dark cycle and with free access to standard rat food and water.
2.3. Preparation of herb extract Sophorae flavescentis radix (Kushen, Sophora flavescens Ait, Leguminous, 100 g) and Glycyrrhizae radix et rhizoma (Gancao, Glycyrrhiza uralensis Fisch, Leguminous 100 g) were extracted three times by refluxing with water (1:8 w/v) for 1 h per time, respectively. The extraction solutions were filtered by a 120-mesh filter, and then vacuum condensed to 2 g/ml crude drug under 70 1C. Kushen and Gancao extracts were mixed at the ratio of 1:1 to constitute the prescription of KGP.
2.4. Liquid chromatography A Shimadzu liquid chromatography system (Shimadzu, Japan) with an LC-30AD binary pump, an on-line vacuum degasser, an autosampler, and a column oven were used for all analyses. The chromatographic separation was performed on Inertsil C18 analytical column (150 mm 4.6 mm i.d., 5 μm, Shimadzu, Japan), and the column temperature was maintained at 40 1C. The analyses were eluted with a gradient mixture of methanol (phase A) and ammonium acetate solution (5 mM, phase B). The gradient program was as follows: 0–5 min, 65%–65% A; 5–5.1 min, 65%–95% A; 95% methanol hold for 5 min, then 65% methanol held for 4 min. The flow rate was set at 0.8 ml/min. The auto-sampler was conditioned at 15 1C and the sample volume injected was 5 μl.
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2.5. Mass spectrometer Detection was performed with an API 4000 tandem mass spectrometer (Applied Biosystems/MDS SCIEX, concord, Ontario, Canada), equipped with an Electrospray Ionization (ESI) source. The electrospray ionization voltage in positive and negative mode was set at 5500 and 4500 V, respectively. The Turbo spray temperature was maintained at 550 1C. Nebulizer gas (gas 1) and heater gas (gas 2) were set as 40 and 50, respectively. The curtain gas was kept at 10 and the interface heater was on. The quantification determination was performed using the multiple reaction monitoring (MRM) method with the transitions of m/z 249.3148.2 for MT, m/z 232.2-113.2 for I.S, m/z 265.1-247.1 for OMT (positive mode), m/z 469.3-355.0 for GA, and m/z 821.4-351.1 for GL (negative mode). All data were collected and analyzed by Analyst software (Applied Biosystems/MDS SCIEX, version 1.6). 2.6. Assay validation The plasma (200 μl) was spiked with 10 μl of I.S. (aminopyrine, 400 ng/ml). The mixture was then extracted with 800 μl methanol by vortex for 1 min and centrifugation at 13000 rpm for 10 min. The supernatant was transferred into a clean Eppendorf tube, and evaporated to dryness under the stream of nitrogen. The residue was dissolved in 100 μl of methanol–water (65:35, v/v), vortexed and centrifuged at 13000 rpm for 10 min. The method was considered valid according to the following criteria: selectivity, linearity, accuracy, precision, percent recovery and stability. The plasma sample concentrations were calculated from the calibration curves. Intraand inter-day precision and accuracy were evaluated by assaying six replicates of each spiked QC sample at the low, middle, and high concentrations on 3 separate days. Precision is expressed as relative standard deviation (RSD). Accuracy was calculated as the percent error in the calculated mean concentration relative to the nominal concentrations (RE). Stability of the analyses in plasma was assessed by analyzing QC samples of three levels during the sample storage and processing procedures. Short-time stability was assessed by analyzing QC samples kept at room temperature for 4 h, which exceeded the routine preparation time of samples. Long-time stability was evaluated by keeping QC samples at 20 1C for 15 days. All stability testing QC samples were determined using the calibration curve which was freshly prepared. 2.7. Pharmacokinetic study The animals were acclimatized to the facilities for seven days with water and food allowed ad libitum, and then fasted with free access to water for 12 h prior to the experiment. The animals were randomly assigned into four groups (six in each group) and two of the groups orally administered with single Kushen and Gancao extracts separately and the rest administered with Kushen–Gancao. The single Kushen and Gancao extracts should be diluted with equal amounts of water before orally administered. Blood samples (0.3 ml) were collected in 1.5 ml heparinized polythene tubes before dosing and 0.083, 0.25, 0.5, 1, 2, 3, 5, 7, 10, 18, 24, and 48 h after dosing in single Kushen and Kushen–Gancao groups; Blood samples (0.3 ml) were collected at 0.5, 1, 2, 4, 6, 8, 10, 24, 30, 48 and 72 h after dosing in single Gancao and another Kushen– Gancao groups. The blood samples were immediately centrifuged at13,000 rpm for 10 min and the plasma was stored at 20 1C until analysis. 2.8. Pharmacokinetic data and statistical analysis Pharmacokinetic analysis was performed using the WinNonlin standard version 2.1 software (Pharsight Corp., Palo Alto, CA, USA).
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The area under the plasma concentration–time curve (AUC) was calculated using trapezoidal estimation and extrapolated to infinity. The pharmacokinetic parameters, such as maximum plasma concentration (Cmax) and time of maximum concentration (Tmax), were directly obtained from the plasma concentration– time plots. An independent samples t-test was carried out for statistical comparison between the experimental groups and the control group using the statistical software SPSS (version 18.0, USA), taking a value of p o0.05 as significant.
S. was 101.8% at the concentration of 400 ng/ml. The matrix effect values obtained for analytes ranged from 90.6% to 106.8%, and the matrix effect on I.S. was 96.3%. The results suggested that the matrix effects for analytes and I.S. were in acceptable range (Tables 2 and 3).The representative chromatograms of blank plasma, plasma sample spiked with analytes and I.S., and plasma sample are shown in Fig. 2.
3.3. Pharmacokinetic study 3. Results 3.1. The characterization of herb extraction Sophorae flavescentis radix (Kushen) and Glycyrrhizae radix et rhizoma (Gancao) extractions were detected by HPLC determination at 220 nm (Chinese Pharmacopoeia, 2010). Quantitation results indicated that the concentrations of each contained approximately 0.86 mg/mL (MT), 1.08 mg/mL (OMT) and 2.04 mg/mL (GL) and1.16 mg/mL (GA).
The validated method was successfully applied to the pharmacokinetic study of the MT, OMT, GL and GA in rat plasma after oral administration of Kushen, Gancao and Kushen–Gancao aqueous extract. The mean plasma concentration–time profiles are shown in Figs. 3 and 4. The pharmacokinetic parameters are shown in Tables 4 and 5. The differences of most pharmacokinetic parameters of each constituent between two groups were significant (po0.05), suggesting that drug–drug interactions occurred in this herb couple.
3.2. Methods validation In plasma assay, the linearity of calibration curves was evaluated using 1/x2 as the weighing factor and showed satisfactory linearity over the concentration range; the concentration range and the LLOQ are shown in Table 1.The precision evaluation revealed that all coelcients of variation were below 15% and the accuracy analysis showed that the relative errors to the true concentrations were below 10%. The results met the pertinent FDA guideline. The mean extraction recoveries of the investigated components in plasma at three different concentration levels were found to be 80.6–102.3% with RSD less than 5.7%. The recovery of I. Table 1 Results of linear regression of calibration curves, linear range and LLOQ of Oxymatrine (OMT), Matrine (MT), Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) in rat plasma. Analyte Linear range (ng/ml) Regression equation
R2
Table 3 Absolute matrix effect and extraction recovery data of Oxymatrine (OMT), Matrine (MT), Glycyrrhizin (GL), Glycyrrhetinic acid (GA) and I.S. in rat plasma (n¼ 6). Analyte Added (ng/ mL)
Recovery Mean (%)
Precision (R.S.D., %)
Mean (%)
Precision (R.S.D., %)
MT
86.2 84.1 85.9 87.4 80.3 78.9 79.3 82.4 88.3 87.5 84.9 87.8 87.2 86.5 84.5 83.6 88.5
5.7 14.8 5.0 10.7 7.7 5.2 2.5 10.3 9.2 14.7 11.2 7.3 8.2 13.4 3.9 6.9 5.35
102 96.6 106.8 94.4 95.7 94.5 90.6 103.6 101 96.6 102.4 104.6 94.7 92.3 105.6 102.9 96.3
3.17 4.31 6.35 2.12 4.28 3.21 8.6 1.85 5.31 6.32 3.92 5.81 6.22 4.52 9.72 5.75 4.0
OMT
GL
LLOQ (ng/ml) GA
MT OMT GL GA
10–5000 10–1000 10–1000 50–15,000
Y ¼ 0.00183X þ0.0391 Y ¼ 0.00342X þ 0.0029 Y ¼ 0.00021X þ0.0442 Y ¼ 0.000139X þ0.00297
0.9916 5 0.9928 5 0.9982 5 0.9976 20
I.S.
5.00 20.0 100 4000 5.00 20.0 100 800 5.00 20.0 100 800 20.0 100 1000 12,000 400
Matrix effect
Table 2 Summary of accuracy and precision of Oxymatrine (OMT), Matrine (MT), Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) in plasma (mean 7 SD, intra-day: 6 replicates at each concentration; inter-day: 6 replicates per day for 3 days). Analyte
Matrine (MT)
Oxymatrine (OMT)
Glycyrrhizin (GL)
Glycyrrhetinic acid (GA)
Added (ng/mL)
5.00 20.0 100 4000 5.00 20.0 100 800 5.00 20.0 100 800 20.0 100 1000 12,000
Intra-day (n¼ 6)
Inter-day (n¼ 18)
Mersured (ng/mL)
Accuracy (R.E., %)
Precision (R.S.D., %)
Measured (ng/mL)
Accuracy (R.E., %)
Precision (R.S.D., %)
5.18 70.35 21.20 72.0 104.8 78.8 3684 7167.6 5.16 70.22 21.82 72.0 102.3 72.6 748.8 743.0 4.92 70.35 19.3 72.0 105.3 79.1 742.4 770.3 20.48 71.35 96.2 74.4 942 750.6 11112 7809
3.60 6.10 4.8 7.9 3.2 9.1 2.3 6.4 1.6 3.7 5.3 7.2 2.4 3.8 5.8 7.4
6.70 9.43 8.39 4.55 4.26 9.15 2.54 5.74 7.1 10.36 8.64 9.47 6.59 4.57 5.37 7.28
5.16 70.27 20.57 72.41 102.1 79.0 3840 7205.8 5.14 70.17 20.84 72.32 105.4 75.6 765.8 748.7 5.06 70.29 19.42 72.20 105.5 713.7 750.4 773.4 23.4. 71.1 97.3 710.3 936.0 7111.6 12636 7860
3.2 2.83 2.05 4.03 2.8 4.15 5.40 4.27 1.2 2.9 5.5 6.2 1.7 2.7 6.4 5.3
5.23 11.72 8.81 5.36 3.31 11.13 5.31 6.36 5.73 11.33 13.00 9.78 4.70 10.58 11.92 6.81
L. Shi et al. / Journal of Ethnopharmacology 165 (2015) 243–250
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Fig. 2. Representative extract ion MRM chromatograms of m/z 249.3-148.2 for Matrine (MT), m/z 265.1-247.1 for Oxymatrine (OMT), m/z 821.4-351.1 for Glycyrrhizin (GL), m/z 469.3-355.0 for Glycyrrhetinic acid (GA), m/z 232.2-113.2 for I.S. (A) blank plasma; (B) blank plasma spiked with MT (5 ng mL 1), OMT (5 ng mL 1), GL (5 ng mL 1), GA (20 ng mL 1) and I.S; (C) plasma sample from a rat 2 h after oral administration of Kushen–Gancao at a dose of 15 mL/kg.
In Kushen group, MT and OMT reached maximum plasma concentration (Cmax) of 3148.7 and 74.2 ng/ml, respectively. While in Kushen–Gancao group, MT and OMT reached Cmax of 2115.4 (po0.05) and 64.7 (po0.05) ng/mL, respectively, the Cmax were reduced by
32.8% and 12.5% compared to Kushen group, AUC0 t values of MT (18969 ng min/mL, po0.05) and OMT (475.2 ng min/mL, po0.05) significantly decreased in Kushen–Gancao group, respectively, and reduced by 34.9% and 18.9%. The CL of MT and OMT were increased by
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Fig. 3. The mean plasma concentration–time curves of Oxymatrine (OMT) and Matrine (MT) following an oral administration of Kushen and Kushen–Gancao aqueous extracts to rats. Each point represents the mean 7 SD (n ¼6).
Fig. 4. The mean plasma concentration–time curves of Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) following oral administration of Gancao and Kushen–Gancao aqueous extracts to rats. Each point represents the mean 7 SD (n ¼6).
Table 4 The pharmacokinetic parameters of Oxymatrine (OMT) and Matrine (MT) after oral administration of Kushen and Kushen–Gancao aqueous extracts (n¼ 6). OMT
Kushen þGancao group
Kushen group
MT
Kushen þ Gancao group
Kushen group
AUC T1/2 Vd CL Cmax Tmax K10
475.217 35.23a 5.127 0.23a 240.6 7 23.1 32.6 73.3a 64.77 5.8 3 1.45 7 0.21a
585.95 7 48.67 8.09 7 0.21 281.6 7 32.3 24.137 2.1 74.2 7 6.9 3 0.2127 0.07
AUC T1/2 Vd CL Cmax Tmax K10
18969 7 1554a 5.95 7 0.26 5.79 7 1.24a 0.677 0.17 2115.4 7 156.4a 3 0.247 0.08
29142 72313 5.377 0.23 3.25 7 0.81 0.42 7 0.12 3148.7 7212.2 3 0.277 0.10
Oxymatrine (OMT), Matrine (MT) AUC0-t: area under the curves from time 0 to 48 h (μg /L h). T1/2: half-life (h) V/F: apparent volume of distribution (L kg 1). CL/F: clearance (L kg 1 h 1). Cmax: peak plasma concentration (μg/ml). Tmax: peak time (h) K10: the elimination rate (h 1). a
p o 0.05.
6.08% (p40.05) and 35.1% (po0.05), respectively, compared to Kushen group; T1/2 of OMT was significantly decreased by 36.7% (po0.05). In Gancao group, GL and GA reached maximum plasma concentration (Cmax) of 318.2 and 105333 ng/mL, respectively. While in
Kushen–Gancao group, GL and GA reached Cmax of 278.17 and 12565 ng/mL (p40.05); compared to Gancao group, AUC0 t values of GA (306831 ng min/mL, po0.05) significantly increased in Kushen–Gancao group, by contrast GL (2461 ng min/mL, po0.05) significantly decreased. T1/2 of GA has significant differences in the
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Table 5 The pharmacokinetic parameters of Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) after oral administration of Gancao and Kushen–Gancao aqueous extracts (n¼ 6). GA
Kushen þGancao group
Gancao group
GL
Kushen þ Gancao group
Gancao group
AUC T1/2 Vd CL Cmax Tmax K10
3068317 4523a 4.98 70.23a 0.737 0.11a 0.102 70.06 12565 7 1052 30 0.43 70.11a
288284 7 3217 17.317 0.31 2.52470.79 0.1017 0.04 10533.37 966 10 0.02 70.01
AUC T1/2 Vd CL Cmax Tmax K10
2461 789.5a 14.2 7 0.26 287.4 733.7 13.2 7 2.1 278.177 23.6 2 0.16 70.03
27317 101 13.9 7 0.18 266.2 7 27.2 12.7 71.6 318.2 7 34.8 2 0.14 70.02
Glycyrrhizin (GL), Glycyrrhetinic acid (GA) AUC0-t: area under the curves from time 0 to 72 h (μg /L h). T1/2: half-life (h) V/F: apparent volume of distribution (L kg 1). CL/F: clearance (L U kg 1 h 1). Cmax: peak plasma concentration (μg/ml). Tmax: peak time (h) K10: the elimination rate (h 1). a
p o 0.05.
two groups (Kushen–Gancao group, 4.98 h; Gancao group 17.31 h). The elimination rate of GA was significantly increased.
4. Discussion Glycyrrhizic acid (GL), one of the main active components of Glycyrrhizae radix et rhizome, has anti-inflammatory, anti-allergic, anti-ulcer, anti-virus and liver detoxification activities. GL was hydrolyzed into 3-monoglucuronyl-glycyrrhetinic acid (3MGA) by β-glucuronidase in the gastrointestinal microflora and then into GA after being absorbed. Meanwhile, a small amount of GA was catalyzed into 3-keto GA by 3β-hydroxy steroid dehydrogenase, finally into 3A-hydroxy GA by 3A-hydroxy GA dehydrogenase and then absorbed. GA and 3A-hydroxy GA can conjugate with sulfuric acid to form conjugates within the liver, and also can be combined with glucuronic acid to form 3MGA and GL (Akao et al., 1991). These conjugates participate in the enterohepatic circulation into the intestine by the bile. That is the reason why GL shows concentration time-curve of two peaks. 3MGA is a powerful 11β-hydroxysteroid dehydrogenase inhibitor, which suppresses in vivo hydrocortisone cortisone into inactive, resulting in the increase of hydrocortisone and showing mineralocorticoid hormone-like effects (Ohtake et al., 2007). So 3MGA accumulation is the reason why long time application of GL cause pseudohyperaldosteronism. Meanwhile, the other research found that NAD þ dependent 11β-dehydrogenaseactivity in rat kidney microsomal fraction was decreased in a GA dose-dependent manner and that 11β-hydroxysteroid dehydrogenase type 2 proteins and mRNA expression were concomitantly decreased in the GA group (Tanahashi et al., 2002). Therefore, GA can also lead to sodium and water retention. The Tmax of GA in Gancao group was 10 h, but the Tmax was 30 h in Kushen–Gancao group, the Tmax was three times than the single group. The result showed that combination with Kushen can significantly slow down the absorption process of GA. While the key point which limits GA absorption is the formation of 3MGA, the GA absorption rate changes may be due to the formation of 3MGA slowing down. Through the above results, we can speculate that coadministration of Kushen may slow down the formation of 3MGA, reducing the accumulation of 3MGA in the body. On the other hand, our study found that when rats were given a single Glycyrrhizae radix et rhizome (Gancao) decoction, the T1/2 of GA in Gancao group was 17.3 h; however when combined with Sophorae flavescentis radix. (Kushen), the T1/2 of GA significantly reduced. The Vd was also significantly reduced almost three times.
The result indicated that combination with Kushen may accelerate the excretion of GA. In vitro studies suggest that GA mainly metabolizes through hepatic CYP3A4, and the study found that matrine can induce hepatic CYP3A enzymes (Ueng et al., 2009). So this may be the reason for the excretion of GA acceleration when Gancao was combined with Kushen. As we know, the accumulation of 3MGA and GA is the reason why Gancao causes water–sodium retention side effects. This pharmacokinetic study showed that combination with Kushen can significantly influence the in vivo metabolism process of Gancao. When combined with Kushen, the absorption process of GA slowed down and the excretion of GA accelerated. This may be the reason why the water–sodium retention side effects of Gancao were decreased when combined with Kushen. The pharmacokinetics study found the AUC0-t of matrine and oxymatrine is significantly reduced when combined with Gancao. The metabolic mechanism of matrine and oxymatrine is not clear. Studies showed that oxymatrine metabolized to active metabolites of matrine by in vivo bacteria, but matrine was not metabolized by CYP and UGT (Yang et al., 2010). So combination with Gancao may accelerate the metabolism of the two substances by gastrointestinal micro-flora, but the exact mechanism remains to be studied. In addition, it has been demonstrated that the treatment effect in hepatitis was significantly increased when Kushen combined with Gancao (Zhao et al. 2012). But the pharmacokinetic data showed that only the AUC of GA increased, the others declined. So the antihepatitis efficacy increase when combined with two herbs was not due to the increase of AUC. This result shows that the two drugs in the body may play different roles in achieving therapeutic effect. However, the onset mechanism remains to be studied.
5. Conclusions In conclusion, significant effects were observed on the pharmacokinetics of GA in rats after the simultaneous coadministration of Sophorae flavescentis radix. (Kushen) and Glycyrrhizae radix et rhizoma. (Gancao).The potential attenuated mechanism of the combined use of these two herbs was induced by the herb–herb interaction. It is important to be aware that this work only provides a non-clinical proof of the effects of the herb–herb interaction of Sophorae flavescentis radix and Glycyrrhizae radix et rhizoma. Thus, before aiming at confirming these results in human a thorough clinical trial is required.
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Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 81274171 and 81173639). References Akao, T., Hattori, M., Kanaoka, M., Yamamoto, K., Namba, T., Kobashi, K., 1991. Hydrolysis of glycyrrhizin to 18 beta-glycyrrhetylmonoglucuronide by lysosomalbeta-D-glucuronidase of animal livers. Biochemical Pharmacology 41, 1025–1029. China Academy of Chinese Medical Sciences, 1962. Journal of Shaanxi Traditional Chinese Medicine. Committee of the Pharmacopoeia of PR China, 2010. Pharmacopoeia of PR China, Part I. China Medical Science Press, pp. 285–286 127–128. Editorial Board of Handbook of Ningxia Chinese Herbal Medicine, 1971. Handbook of Ningxia Chinese Herbal Medicine. Ningxia People's Publishing House, Ningxia, China. Fu, Y., Chen, J., Li, Y.J., Zheng, Y.F., Li, P., 2013. Antioxidant and anti-inflammatory activities of six flavonoids separated from licorice. Food Chemistry 141, 1063–1071. Kageyama, Y., Suzuki, H., Saruta, T., 1991. Renin-dependency of glycyrrhizin induced pseudoaldosteronism. Endocrinologia Japonica 38, 103–108. Krähenbühl, S., Hasler, F., Krapf, R., 1994. Analysis and pharmacokinetics of glycyrrhizic acid and glycyrrhetinic acid in humans and experimental animals. Steroids 59, 121–126. Kratschmar, D.V., Vuorinen, A., Da, Cunha, T., Wolber, G., Classen-Houben, D., Doblhoff, O., Schuster, D., Odermatt, A., 2011. Characterization of activity and binding mode of glycyrrhetinic acid derivatives inhibiting 11β-hydroxysteroid dehydrogenase type 2. Journal of Steroid Biochemistry and Molecular Biology 125, 129–142. Kusano, G., Shibano, M., Watanabe, H., 2003. Pharmaceutical botanical studies on some Glycyrrhiza species. Yakugaku Zasshi 123, 619–631. Lee, J.Y., Lee, J.H., Park, J.H., Kim, S.Y., Choi, J.Y., Lee, S.H., Kim, Y.S., Kang, S.S., Jang, E.C., Han, Y., 2009. Liquiritigenin, a licorice flavonoid, helps mice resist disseminated candidiasis due to Candida albicans by Th1 immune response, whereas liquiritin, its glycoside form, does not. International Immunopharmacology 9, 632–638. Li, J.H., Li, H.Y., Li, H.B., 2010. Clinical treatment of 30 cases of peptic ulcer with Gancao decoction and Kushen decoction. Journal of Practical Medicine Department of Internal Medicine 2, 23–25. Lin, Y., 2011. Clinical treatment of 32 cases of Behcet's disease with Gancao decoction and Kushen decoction. Modern Traditional Chinese Medicine 31, 21–22. Miao, K.L., Zhang, J.Z., Dong, Y., Xi, Y.F., 2001. Research progress on the chemical compounds and pharmacology of Sophora flavescens. Natural Product Research Development 13, 69–73. Ohtake, N., Kido, A., Kubota, K., Tsuchiya, N., Morita, T., Kase, Y., Takeda, S., 2007. A possible involvement of 3-monoglucuronyl-glycyrrhetinic acid, a metabolite of glycyrrhizin (GL), in GL-induced pseudoaldosteronism. Life Sciences 80, 1545–1552. Shin, Y.W., Bae, A.E., Lee, B., Kim, J.A., Kin, Y.S., Kim, D.H., 2007. In vitro and in vivo antiallergic effects of Glycyrrhiza glabra and its components. Planta Medica 73, 257–261.
Shintani, S., Murase, H., Tsukagoshi, H., Shiigai, T., 1992. Glycyrrhizin (licorice)induced hypokalemic myopathy. Report of 2 cases and review of the literature. European Neurology 32, 44–51. Tanahashi, T., Mune, T., Morita, H., Tanahashi, H., Isomura, Y., Suwa, T., Daido, H., Gomez-Sanchez, C.E., Yasuda, K., 2002. Glycyrrhizic acid suppresses type 2 11βhydroxysteroid dehydrogenase expression in vivo. Journal of Steroid Biochemistry and Molecular Biology 80, 441–447. Tang, L., Dong, L.N., Peng, X.J., Li, Y., Shi, J., Zhou, F.Y., Liu, Z.Q., 2013. Pharmacokinetic characterization of oxymatrine and matrine in rats after oral administration of radix Sophoraetonkinensis extract and oxymatrine by sensitive and robust UPLC–MS/MS method. Journal of Pharmaceutical and Biomedical Analysis 83, 179–185. Ueng, Y.F., Chen, C.C., Tsai, C.C., Soucek, P., 2009. Differential inductive profiles of hepatic cytochrome P450s by the extracts of Sophora flavescens in male and female C57BL/6JNarl mice. Journal of Ethnopharmacology 126, 437–446. Van Rossum, T.G., de Jong, F.H., Hop, W.C., Boomsma, F., Schalm, S.W., 2001. Pseudoaldosteronism’induced by intravenous glycyrrhizin treatment of chronic hepatitis C patients. Journal of Gastroenterology and Hepatology 16, 789–795. Wagner, H., 2009. Appendix: Quality proof of herbal drugs from Traditional Chinese Medicine (TCM drugs), A Materia Medica for Chinese Medicine, pp. 993–1004. Wan, X.Y., Luo, M., Li, X.D., He, P., 2009. Hepatoprotective and antihepatocarcinogenic effects of glycyrrhizin and matrine. Chemico-biological Interactions 181, 15–19. Wang, J.H., 2012. Traditional Chinese medicine and the positive correlation with homeostatic evolution of human being: based on medical perspective. Chinese Journal of Integrative Medicine 18, 629–634. Wang, Q.H., Shi, L., Tang, X.L., Wang, Q.W., Dang, X.L., Zhang, Y., 2014. Pharmacokinetic study of multiple active constituents from Kushen–Gancao Decoction after oral administration in rat by HPLC-MS/MS. Journal of Chromatography B 965, 19–26. Wang, S., Hu, Y., Tan, W., Wu, X., Chen, R., Cao, J., Chen, M., Wang, Y., 2012. Compatibility art of traditional Chinese medicine: from the perspective of herb pairs. Journal of Ethnopharmacology 143, 412–423. Xu, C.H., Wang, P., Wang, Y., Yang, Y.a.n., Li, D.H., Li, H.F., Sun, S.Q., Wu, X.Z., 2013. Pharmacokinetic comparisons of two different combinations of Shaoyao– Gancao Decoction in rats: competing mechanisms between paeoniflorin and glycyrrhetinic acid. Journal of Ethnopharmacology 149, 443–452. Xu, W., 1997. Clinical observation on the treatment of 31 cases of viral cholestatic hepatitis with Kushen decoction. Journal of Zhejiang College of Traditional Chinese Medicine 21, 51–52. Xu, W., Lin, H., Zhang, Y., Chen, X., Hua, B., Hou, W., Qi, X., Pei, Y., Zhu, X., Zhao, Z., Yang, L., 2011. Compound Kushen injection suppresses human breast cancer stem-like cells by down-regulating the canonical Wnt/β-catenin pathway. Journal of Experimental & Clinical Cancer Research 28, 103. Yang, Z., Gao, S., Yin, T., Kulkarni, K.H., Teng, Y., You, M., Hu, M., 2010. Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC–MS/MS method. Journal of Pharmaceutical and Biomedical Analysis 51, 1120–1127. Zhao, J., Wan, X.Y., Luo, M., Chen, T.S., He, P., 2012. Antifibrotic effects of glycyrrhizin and matrine in vitro and in vivo. Biomedicine & Preventive Nutrition 2, 132–137. Zhi, X.Y., 2009. Traditional Chinese medicine diagnosis and treatment of type 2 diabetes in Tianjin urban population. Zhong Xi Yi Jie He Xue Bao 7, 823–826. Zhou, S.K., Zhang, R.L., Xu, Y.F., Bi, T.N., 2012. Antioxidant and immunity activities of Fufang Kushen injection liquid. Molecules 17, 6481–6490.
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Research Paper
Investigating herb–herb interactions: The potential attenuated toxicity mechanism of the combined use of Glycyrrhizae radix et rhizoma (Gancao) and Sophorae flavescentis radix (Kushen) Lei Shi a, Xiuling Tang a, Xueliang Dang a, Qinhui Wang a, Xiangrui Wang b, Ping He b, Qingwei Wang a, Linna Liu a, XinYou Liu a, Yan Zhang a,n a b
Department of head!–td:Pharmacy,–>Pharmacy, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, PR China Department of Anesthesiology, Renji Hospital Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai 200127, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 20 January 2015 Accepted 9 February 2015 Available online 19 February 2015
Ethnopharmacological relevance: Glycyrrhizae radix et rhizoma (Gancao) is often prescribed together with Sophorae flavescentis radix (Kushen) in traditional Chinese medicinal practice to increase the efficacy on the treatment of hepatitis and hepatic fibrosis. Meanwhile, long-term single used Gancao can cause adverse reactions, lead to pseudohypercorticosteroidism especially. But the side effects of Gancao are significantly reduced when combined with Kushen; the reasons are still unknown. The aim of this study was to elucidate potential pharmacokinetic interaction between Kushen and Gancao, and to provide guidance for clinical medicine safety. Materials and methods: A specific and rapid HPLC–MS method was established to quantify the four main activity ingredients matrine (MT), oxymatrine (OMT), glycyrrhizin (GL) and glycyrrhetinic acid (GA) in rat plasma. In this study, the pharmacokinetic parameters and the pharmacokinetic differences of the four main activity ingredients MT, OMT, GL and GA in single herb and Kushen–Gancao couple were obtained. Results: Compared with oral administration of Gancao extract, K10 and Tmax of GA significantly increased to 0.43 h 1and 30 h after giving Kushen–Gancao (po0.05), but T1/2 and Vd were reduced to 0.73 L kg 1and 4.98 h (po 0.05). In addition, the AUC of GA was increased, and the other three activity ingredients all decreased. Conclusion: GA as the main factor leading to the sodium–water retention side effects of Gancao. The result found that the absorption of GA was significantly slowed down and the metabolism rate was accelerated in Kushen–Gancao than single herb. So the attenuated toxicity mechanism may be because the accumulation of GA reduced in vivo. The conclusion has important meaning to the compatibility of Chinese med. & 2015 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Matrine (Pubchem CID:91466) Oxymatrine (Pubchem CID:114850) Glycyrrhizin (Pubchem CID:14982) Glycyrrhetinic acid (Pubchem CID:10114) Keywords: Glycyrrhizae radix et rhizome Sophorae flavescentis radix Herb–herb interactions Attenuated toxicity
1. Introduction Traditional Chinese Medicine (TCM) is mostly prescribed in combination to obtain synergistic effects and reduce possible adverse reactions. Hence, the compatibility of Chinese medicinal herbs is an important theory in the combination of TCM. Herb couple, two herbs usually prescribed together to decrease toxicity and increase efficacy, is the basic unit which is commonly used in herbal formulae in TCM. They are much simpler than other complex formulae without altering the basic therapeutic features (Wang, et al., 2012; Wang, 2012). Generally, the law of compound compatibility was studied through pharmacodynamics and pharmacokinetics. Pharmacokinetic method was used in this study, which was helpful in understanding the action mechanism of drugs and the compatible priority of the entire prescription. n
Corresponding author.
http://dx.doi.org/10.1016/j.jep.2015.02.022 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Kushen–Gancao couple, a famous traditional herb couple, was recorded in Pu Ji Fang for thousands of years, and has been used as a TCM for treatment of hepatitis, chronic liver diseases, peptic ulcer and immunological disorders in China for a very long period ( Wagner, 2009). It also has been reported in various literatures, e.g. Handbook of Ning xia Chinese Herbal Medicine (Editorial Board of Hand book of Ningxia Chinese Herbal Medicine, 1971) and Journal of Shaanxi Traditional Chinese Medicine (China Academy of Chinese Medical Sciences, 1962), which are famous in the field of TCM. The traditional remedy is pill of Sophorae flavescentis radix (Kushen in Chinese), which is the root of Sophora flavescens Ait, and Glycyrrhizae radix et rhizoma (Gancao in Chinese), which was the root of Glycyrrhiza uralensis Fisch. Both the herbs are officially listed in the Chinese pharmacopeia (Pharmacopoeia of PR China, 2010). Sophorae flavescentis radix (Kushen) was widely used as TCM in the treatment of viral hepatitis and chronic liver diseases due to
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its wide spectrum of pharmacological actions including antiinflammatory, immune-inhibitory, anti-fibrotic, antiarrhythmic, anti-tumor and diuretic activities (Xu et al., 2011; Zhou et al., 2012; Zhi, 2009). Glycyrrhizae radix et rhizoma (Gancao) is prescribed in many Chinese traditional formulas for its medical potential effects in anti-inflammation (Fu et al., 2013), immunoloregulation (Lee et al., 2009), and anti-allergy nature (Shin et al., 2007). It also has been used for the treatment and prevention of hepatitis, chronic bronchitis, gastritis, tumor growth and immunological disorders (Miao et al., 2001; Kusano et al., 2003). Kushen–Gancao couple was widely used in the clinic treatment of viral hepatitis, chronic liver diseases, peptic ulcer and skin inflammations; furthermore, it obtained good curative effect (Xu, 1997; Li et al., 2010; Lin, 2011). It has been demonstrated that the treatment effect in hepatitis of Kushen–Gancao couple was significantly better than single herb; meanwhile, the adverse reactions caused by single use of Gancao was significantly decreased (Zhao et al., 2012). Matrine (MT) and Oxymatrine (OMT) are the main active components extracted from Kushen. Glycyrrhizin (GL) is the active component of Gancao. Glycyrrhetinic acid (GA) was the active metabolite in the body of the GL (Fig. 1). There were evidence from numerous clinical case reports and trials that conventional administration of GL may cause pseudohypercorticosteroidism, such as sodium and water retention, hypertension and hypokalaemia (Van Rossum et al., 2001; Kageyama et al., 1991; Shintani et al., 1992). Generally, the mechanism of this side effect was induced by GA competitive inhibition of hepatic microsomal 11B-typeII
hydroxysteroid dehydrogenase (11-HSD2) activity, which indirectly enhance cortical hormone concentration, showing the effect of steroids (Kratschmar et al., 2011). But these side effects significantly decreased when Gancao combined with Kushen (Wan et al. 2009). Some pharmacokinetic studies of these four active ingredients in single herbal drugs or other TCM formulas had been reported (Tang et al., 2013; Krähenbühl et al., 1994; Xu, et al., 2013). But the pharmacokinetic of these four active ingredients in Kushen–Gancao was not reported, the mechanism of attenuated toxicity of Gancao after co-administration with Kushen was unknown. The pharmacokinetic characteristics have an important significance for the interpretation of synergism and attenuation. So it is necessary to study the pharmacokinetic parameters of the four active ingredients in Kushen–Gancao and compare their possible pharmacokinetic differences after the administration of single-herb extracts to rats. So this paper will research the pharmacokinetic differences between single use of Sophorae flavescentis radix or Glycyrrhizae radix et rhizoma and combined use and investigate the potential herb–herb interaction, also reveal the attenuated toxicity mechanism of the combined use of Glycyrrhizae radix et rhizoma (Gancao) and Sophorae flavescentis radix In a previous study, we had developed a rapid and sensitive LC– MS/MS method to simultaneously determine Matrine (MT), Oxymatrine (OMT), Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) in rat plasma after oral administration of Kusheng–Gancao (Wang et al., 2014). In this study, the pharmacokinetic parameters and the pharmacokinetic differences of the four ingredients after the administration of single-herb extracts and Kushen–Gancao were
Fig. 1. Chemical structures of: (a) Oxymatrine (OMT), (b) Matrine (MT), (c) Glycyrrhizin (GL) and (d) Glycyrrhetinic acid (GA).
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obtained. The results of our study would help us explore whether there were synergistic effects on the four ingredients in Kushen– Gancao. At the same time, these methods may provide solutions to study the reasonableness of the traditional medicine recipe.
2. Experimental 2.1. Materials and reagents The reference standards of Matrine, Oxymatrine, Glycyrrhizin and Glycyrrhetinic acid were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Aminopyrine as internal standard (I.S.) was purchased from Aladdin Chemistry Co. ltd (Shanghai, China). Methanol (HPLC grade) was purchased from Fisher Scientific (Fair Lawn, NJ, USA); Ammonium acetate (HPLC grade) was from Dikma (Richmond Hill, NY, USA); ultrapure water for HPLC analysis was prepared from a Milli-Q water purification system (Millipore, Milford, MA, USA); and all other reagents were of analytical grade. Kushen and Gancao were purchased from Xiaocao Medicinal Material Company (Xi’an, China) and authenticated by Professor Qing-Wei Wang (The Fourth Military Medical University, Xi’an, China).
2.2. Animals Male Sprague–Dawley rats (250 720 g) were supplied by the Lab Animal Center of Fourth Military Medical University. The experimental protocol was approved by the University Ethics Committee for the use of experimental animals and all animal studies were carried out according to the Guide for Care and Use of Laboratory Animals. The number of the Ethics Committee on Animal Experimentation was TDLL-20130502. These animals lived in suitable conditions: temperature-controlled room (22 7 2 1C) with a 12 h light-dark cycle and with free access to standard rat food and water.
2.3. Preparation of herb extract Sophorae flavescentis radix (Kushen, Sophora flavescens Ait, Leguminous, 100 g) and Glycyrrhizae radix et rhizoma (Gancao, Glycyrrhiza uralensis Fisch, Leguminous 100 g) were extracted three times by refluxing with water (1:8 w/v) for 1 h per time, respectively. The extraction solutions were filtered by a 120-mesh filter, and then vacuum condensed to 2 g/ml crude drug under 70 1C. Kushen and Gancao extracts were mixed at the ratio of 1:1 to constitute the prescription of KGP.
2.4. Liquid chromatography A Shimadzu liquid chromatography system (Shimadzu, Japan) with an LC-30AD binary pump, an on-line vacuum degasser, an autosampler, and a column oven were used for all analyses. The chromatographic separation was performed on Inertsil C18 analytical column (150 mm 4.6 mm i.d., 5 μm, Shimadzu, Japan), and the column temperature was maintained at 40 1C. The analyses were eluted with a gradient mixture of methanol (phase A) and ammonium acetate solution (5 mM, phase B). The gradient program was as follows: 0–5 min, 65%–65% A; 5–5.1 min, 65%–95% A; 95% methanol hold for 5 min, then 65% methanol held for 4 min. The flow rate was set at 0.8 ml/min. The auto-sampler was conditioned at 15 1C and the sample volume injected was 5 μl.
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2.5. Mass spectrometer Detection was performed with an API 4000 tandem mass spectrometer (Applied Biosystems/MDS SCIEX, concord, Ontario, Canada), equipped with an Electrospray Ionization (ESI) source. The electrospray ionization voltage in positive and negative mode was set at 5500 and 4500 V, respectively. The Turbo spray temperature was maintained at 550 1C. Nebulizer gas (gas 1) and heater gas (gas 2) were set as 40 and 50, respectively. The curtain gas was kept at 10 and the interface heater was on. The quantification determination was performed using the multiple reaction monitoring (MRM) method with the transitions of m/z 249.3148.2 for MT, m/z 232.2-113.2 for I.S, m/z 265.1-247.1 for OMT (positive mode), m/z 469.3-355.0 for GA, and m/z 821.4-351.1 for GL (negative mode). All data were collected and analyzed by Analyst software (Applied Biosystems/MDS SCIEX, version 1.6). 2.6. Assay validation The plasma (200 μl) was spiked with 10 μl of I.S. (aminopyrine, 400 ng/ml). The mixture was then extracted with 800 μl methanol by vortex for 1 min and centrifugation at 13000 rpm for 10 min. The supernatant was transferred into a clean Eppendorf tube, and evaporated to dryness under the stream of nitrogen. The residue was dissolved in 100 μl of methanol–water (65:35, v/v), vortexed and centrifuged at 13000 rpm for 10 min. The method was considered valid according to the following criteria: selectivity, linearity, accuracy, precision, percent recovery and stability. The plasma sample concentrations were calculated from the calibration curves. Intraand inter-day precision and accuracy were evaluated by assaying six replicates of each spiked QC sample at the low, middle, and high concentrations on 3 separate days. Precision is expressed as relative standard deviation (RSD). Accuracy was calculated as the percent error in the calculated mean concentration relative to the nominal concentrations (RE). Stability of the analyses in plasma was assessed by analyzing QC samples of three levels during the sample storage and processing procedures. Short-time stability was assessed by analyzing QC samples kept at room temperature for 4 h, which exceeded the routine preparation time of samples. Long-time stability was evaluated by keeping QC samples at 20 1C for 15 days. All stability testing QC samples were determined using the calibration curve which was freshly prepared. 2.7. Pharmacokinetic study The animals were acclimatized to the facilities for seven days with water and food allowed ad libitum, and then fasted with free access to water for 12 h prior to the experiment. The animals were randomly assigned into four groups (six in each group) and two of the groups orally administered with single Kushen and Gancao extracts separately and the rest administered with Kushen–Gancao. The single Kushen and Gancao extracts should be diluted with equal amounts of water before orally administered. Blood samples (0.3 ml) were collected in 1.5 ml heparinized polythene tubes before dosing and 0.083, 0.25, 0.5, 1, 2, 3, 5, 7, 10, 18, 24, and 48 h after dosing in single Kushen and Kushen–Gancao groups; Blood samples (0.3 ml) were collected at 0.5, 1, 2, 4, 6, 8, 10, 24, 30, 48 and 72 h after dosing in single Gancao and another Kushen– Gancao groups. The blood samples were immediately centrifuged at13,000 rpm for 10 min and the plasma was stored at 20 1C until analysis. 2.8. Pharmacokinetic data and statistical analysis Pharmacokinetic analysis was performed using the WinNonlin standard version 2.1 software (Pharsight Corp., Palo Alto, CA, USA).
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The area under the plasma concentration–time curve (AUC) was calculated using trapezoidal estimation and extrapolated to infinity. The pharmacokinetic parameters, such as maximum plasma concentration (Cmax) and time of maximum concentration (Tmax), were directly obtained from the plasma concentration– time plots. An independent samples t-test was carried out for statistical comparison between the experimental groups and the control group using the statistical software SPSS (version 18.0, USA), taking a value of p o0.05 as significant.
S. was 101.8% at the concentration of 400 ng/ml. The matrix effect values obtained for analytes ranged from 90.6% to 106.8%, and the matrix effect on I.S. was 96.3%. The results suggested that the matrix effects for analytes and I.S. were in acceptable range (Tables 2 and 3).The representative chromatograms of blank plasma, plasma sample spiked with analytes and I.S., and plasma sample are shown in Fig. 2.
3.3. Pharmacokinetic study 3. Results 3.1. The characterization of herb extraction Sophorae flavescentis radix (Kushen) and Glycyrrhizae radix et rhizoma (Gancao) extractions were detected by HPLC determination at 220 nm (Chinese Pharmacopoeia, 2010). Quantitation results indicated that the concentrations of each contained approximately 0.86 mg/mL (MT), 1.08 mg/mL (OMT) and 2.04 mg/mL (GL) and1.16 mg/mL (GA).
The validated method was successfully applied to the pharmacokinetic study of the MT, OMT, GL and GA in rat plasma after oral administration of Kushen, Gancao and Kushen–Gancao aqueous extract. The mean plasma concentration–time profiles are shown in Figs. 3 and 4. The pharmacokinetic parameters are shown in Tables 4 and 5. The differences of most pharmacokinetic parameters of each constituent between two groups were significant (po0.05), suggesting that drug–drug interactions occurred in this herb couple.
3.2. Methods validation In plasma assay, the linearity of calibration curves was evaluated using 1/x2 as the weighing factor and showed satisfactory linearity over the concentration range; the concentration range and the LLOQ are shown in Table 1.The precision evaluation revealed that all coelcients of variation were below 15% and the accuracy analysis showed that the relative errors to the true concentrations were below 10%. The results met the pertinent FDA guideline. The mean extraction recoveries of the investigated components in plasma at three different concentration levels were found to be 80.6–102.3% with RSD less than 5.7%. The recovery of I. Table 1 Results of linear regression of calibration curves, linear range and LLOQ of Oxymatrine (OMT), Matrine (MT), Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) in rat plasma. Analyte Linear range (ng/ml) Regression equation
R2
Table 3 Absolute matrix effect and extraction recovery data of Oxymatrine (OMT), Matrine (MT), Glycyrrhizin (GL), Glycyrrhetinic acid (GA) and I.S. in rat plasma (n¼ 6). Analyte Added (ng/ mL)
Recovery Mean (%)
Precision (R.S.D., %)
Mean (%)
Precision (R.S.D., %)
MT
86.2 84.1 85.9 87.4 80.3 78.9 79.3 82.4 88.3 87.5 84.9 87.8 87.2 86.5 84.5 83.6 88.5
5.7 14.8 5.0 10.7 7.7 5.2 2.5 10.3 9.2 14.7 11.2 7.3 8.2 13.4 3.9 6.9 5.35
102 96.6 106.8 94.4 95.7 94.5 90.6 103.6 101 96.6 102.4 104.6 94.7 92.3 105.6 102.9 96.3
3.17 4.31 6.35 2.12 4.28 3.21 8.6 1.85 5.31 6.32 3.92 5.81 6.22 4.52 9.72 5.75 4.0
OMT
GL
LLOQ (ng/ml) GA
MT OMT GL GA
10–5000 10–1000 10–1000 50–15,000
Y ¼ 0.00183X þ0.0391 Y ¼ 0.00342X þ 0.0029 Y ¼ 0.00021X þ0.0442 Y ¼ 0.000139X þ0.00297
0.9916 5 0.9928 5 0.9982 5 0.9976 20
I.S.
5.00 20.0 100 4000 5.00 20.0 100 800 5.00 20.0 100 800 20.0 100 1000 12,000 400
Matrix effect
Table 2 Summary of accuracy and precision of Oxymatrine (OMT), Matrine (MT), Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) in plasma (mean 7 SD, intra-day: 6 replicates at each concentration; inter-day: 6 replicates per day for 3 days). Analyte
Matrine (MT)
Oxymatrine (OMT)
Glycyrrhizin (GL)
Glycyrrhetinic acid (GA)
Added (ng/mL)
5.00 20.0 100 4000 5.00 20.0 100 800 5.00 20.0 100 800 20.0 100 1000 12,000
Intra-day (n¼ 6)
Inter-day (n¼ 18)
Mersured (ng/mL)
Accuracy (R.E., %)
Precision (R.S.D., %)
Measured (ng/mL)
Accuracy (R.E., %)
Precision (R.S.D., %)
5.18 70.35 21.20 72.0 104.8 78.8 3684 7167.6 5.16 70.22 21.82 72.0 102.3 72.6 748.8 743.0 4.92 70.35 19.3 72.0 105.3 79.1 742.4 770.3 20.48 71.35 96.2 74.4 942 750.6 11112 7809
3.60 6.10 4.8 7.9 3.2 9.1 2.3 6.4 1.6 3.7 5.3 7.2 2.4 3.8 5.8 7.4
6.70 9.43 8.39 4.55 4.26 9.15 2.54 5.74 7.1 10.36 8.64 9.47 6.59 4.57 5.37 7.28
5.16 70.27 20.57 72.41 102.1 79.0 3840 7205.8 5.14 70.17 20.84 72.32 105.4 75.6 765.8 748.7 5.06 70.29 19.42 72.20 105.5 713.7 750.4 773.4 23.4. 71.1 97.3 710.3 936.0 7111.6 12636 7860
3.2 2.83 2.05 4.03 2.8 4.15 5.40 4.27 1.2 2.9 5.5 6.2 1.7 2.7 6.4 5.3
5.23 11.72 8.81 5.36 3.31 11.13 5.31 6.36 5.73 11.33 13.00 9.78 4.70 10.58 11.92 6.81
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Fig. 2. Representative extract ion MRM chromatograms of m/z 249.3-148.2 for Matrine (MT), m/z 265.1-247.1 for Oxymatrine (OMT), m/z 821.4-351.1 for Glycyrrhizin (GL), m/z 469.3-355.0 for Glycyrrhetinic acid (GA), m/z 232.2-113.2 for I.S. (A) blank plasma; (B) blank plasma spiked with MT (5 ng mL 1), OMT (5 ng mL 1), GL (5 ng mL 1), GA (20 ng mL 1) and I.S; (C) plasma sample from a rat 2 h after oral administration of Kushen–Gancao at a dose of 15 mL/kg.
In Kushen group, MT and OMT reached maximum plasma concentration (Cmax) of 3148.7 and 74.2 ng/ml, respectively. While in Kushen–Gancao group, MT and OMT reached Cmax of 2115.4 (po0.05) and 64.7 (po0.05) ng/mL, respectively, the Cmax were reduced by
32.8% and 12.5% compared to Kushen group, AUC0 t values of MT (18969 ng min/mL, po0.05) and OMT (475.2 ng min/mL, po0.05) significantly decreased in Kushen–Gancao group, respectively, and reduced by 34.9% and 18.9%. The CL of MT and OMT were increased by
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Fig. 3. The mean plasma concentration–time curves of Oxymatrine (OMT) and Matrine (MT) following an oral administration of Kushen and Kushen–Gancao aqueous extracts to rats. Each point represents the mean 7 SD (n ¼6).
Fig. 4. The mean plasma concentration–time curves of Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) following oral administration of Gancao and Kushen–Gancao aqueous extracts to rats. Each point represents the mean 7 SD (n ¼6).
Table 4 The pharmacokinetic parameters of Oxymatrine (OMT) and Matrine (MT) after oral administration of Kushen and Kushen–Gancao aqueous extracts (n¼ 6). OMT
Kushen þGancao group
Kushen group
MT
Kushen þ Gancao group
Kushen group
AUC T1/2 Vd CL Cmax Tmax K10
475.217 35.23a 5.127 0.23a 240.6 7 23.1 32.6 73.3a 64.77 5.8 3 1.45 7 0.21a
585.95 7 48.67 8.09 7 0.21 281.6 7 32.3 24.137 2.1 74.2 7 6.9 3 0.2127 0.07
AUC T1/2 Vd CL Cmax Tmax K10
18969 7 1554a 5.95 7 0.26 5.79 7 1.24a 0.677 0.17 2115.4 7 156.4a 3 0.247 0.08
29142 72313 5.377 0.23 3.25 7 0.81 0.42 7 0.12 3148.7 7212.2 3 0.277 0.10
Oxymatrine (OMT), Matrine (MT) AUC0-t: area under the curves from time 0 to 48 h (μg /L h). T1/2: half-life (h) V/F: apparent volume of distribution (L kg 1). CL/F: clearance (L kg 1 h 1). Cmax: peak plasma concentration (μg/ml). Tmax: peak time (h) K10: the elimination rate (h 1). a
p o 0.05.
6.08% (p40.05) and 35.1% (po0.05), respectively, compared to Kushen group; T1/2 of OMT was significantly decreased by 36.7% (po0.05). In Gancao group, GL and GA reached maximum plasma concentration (Cmax) of 318.2 and 105333 ng/mL, respectively. While in
Kushen–Gancao group, GL and GA reached Cmax of 278.17 and 12565 ng/mL (p40.05); compared to Gancao group, AUC0 t values of GA (306831 ng min/mL, po0.05) significantly increased in Kushen–Gancao group, by contrast GL (2461 ng min/mL, po0.05) significantly decreased. T1/2 of GA has significant differences in the
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Table 5 The pharmacokinetic parameters of Glycyrrhizin (GL) and Glycyrrhetinic acid (GA) after oral administration of Gancao and Kushen–Gancao aqueous extracts (n¼ 6). GA
Kushen þGancao group
Gancao group
GL
Kushen þ Gancao group
Gancao group
AUC T1/2 Vd CL Cmax Tmax K10
3068317 4523a 4.98 70.23a 0.737 0.11a 0.102 70.06 12565 7 1052 30 0.43 70.11a
288284 7 3217 17.317 0.31 2.52470.79 0.1017 0.04 10533.37 966 10 0.02 70.01
AUC T1/2 Vd CL Cmax Tmax K10
2461 789.5a 14.2 7 0.26 287.4 733.7 13.2 7 2.1 278.177 23.6 2 0.16 70.03
27317 101 13.9 7 0.18 266.2 7 27.2 12.7 71.6 318.2 7 34.8 2 0.14 70.02
Glycyrrhizin (GL), Glycyrrhetinic acid (GA) AUC0-t: area under the curves from time 0 to 72 h (μg /L h). T1/2: half-life (h) V/F: apparent volume of distribution (L kg 1). CL/F: clearance (L U kg 1 h 1). Cmax: peak plasma concentration (μg/ml). Tmax: peak time (h) K10: the elimination rate (h 1). a
p o 0.05.
two groups (Kushen–Gancao group, 4.98 h; Gancao group 17.31 h). The elimination rate of GA was significantly increased.
4. Discussion Glycyrrhizic acid (GL), one of the main active components of Glycyrrhizae radix et rhizome, has anti-inflammatory, anti-allergic, anti-ulcer, anti-virus and liver detoxification activities. GL was hydrolyzed into 3-monoglucuronyl-glycyrrhetinic acid (3MGA) by β-glucuronidase in the gastrointestinal microflora and then into GA after being absorbed. Meanwhile, a small amount of GA was catalyzed into 3-keto GA by 3β-hydroxy steroid dehydrogenase, finally into 3A-hydroxy GA by 3A-hydroxy GA dehydrogenase and then absorbed. GA and 3A-hydroxy GA can conjugate with sulfuric acid to form conjugates within the liver, and also can be combined with glucuronic acid to form 3MGA and GL (Akao et al., 1991). These conjugates participate in the enterohepatic circulation into the intestine by the bile. That is the reason why GL shows concentration time-curve of two peaks. 3MGA is a powerful 11β-hydroxysteroid dehydrogenase inhibitor, which suppresses in vivo hydrocortisone cortisone into inactive, resulting in the increase of hydrocortisone and showing mineralocorticoid hormone-like effects (Ohtake et al., 2007). So 3MGA accumulation is the reason why long time application of GL cause pseudohyperaldosteronism. Meanwhile, the other research found that NAD þ dependent 11β-dehydrogenaseactivity in rat kidney microsomal fraction was decreased in a GA dose-dependent manner and that 11β-hydroxysteroid dehydrogenase type 2 proteins and mRNA expression were concomitantly decreased in the GA group (Tanahashi et al., 2002). Therefore, GA can also lead to sodium and water retention. The Tmax of GA in Gancao group was 10 h, but the Tmax was 30 h in Kushen–Gancao group, the Tmax was three times than the single group. The result showed that combination with Kushen can significantly slow down the absorption process of GA. While the key point which limits GA absorption is the formation of 3MGA, the GA absorption rate changes may be due to the formation of 3MGA slowing down. Through the above results, we can speculate that coadministration of Kushen may slow down the formation of 3MGA, reducing the accumulation of 3MGA in the body. On the other hand, our study found that when rats were given a single Glycyrrhizae radix et rhizome (Gancao) decoction, the T1/2 of GA in Gancao group was 17.3 h; however when combined with Sophorae flavescentis radix. (Kushen), the T1/2 of GA significantly reduced. The Vd was also significantly reduced almost three times.
The result indicated that combination with Kushen may accelerate the excretion of GA. In vitro studies suggest that GA mainly metabolizes through hepatic CYP3A4, and the study found that matrine can induce hepatic CYP3A enzymes (Ueng et al., 2009). So this may be the reason for the excretion of GA acceleration when Gancao was combined with Kushen. As we know, the accumulation of 3MGA and GA is the reason why Gancao causes water–sodium retention side effects. This pharmacokinetic study showed that combination with Kushen can significantly influence the in vivo metabolism process of Gancao. When combined with Kushen, the absorption process of GA slowed down and the excretion of GA accelerated. This may be the reason why the water–sodium retention side effects of Gancao were decreased when combined with Kushen. The pharmacokinetics study found the AUC0-t of matrine and oxymatrine is significantly reduced when combined with Gancao. The metabolic mechanism of matrine and oxymatrine is not clear. Studies showed that oxymatrine metabolized to active metabolites of matrine by in vivo bacteria, but matrine was not metabolized by CYP and UGT (Yang et al., 2010). So combination with Gancao may accelerate the metabolism of the two substances by gastrointestinal micro-flora, but the exact mechanism remains to be studied. In addition, it has been demonstrated that the treatment effect in hepatitis was significantly increased when Kushen combined with Gancao (Zhao et al. 2012). But the pharmacokinetic data showed that only the AUC of GA increased, the others declined. So the antihepatitis efficacy increase when combined with two herbs was not due to the increase of AUC. This result shows that the two drugs in the body may play different roles in achieving therapeutic effect. However, the onset mechanism remains to be studied.
5. Conclusions In conclusion, significant effects were observed on the pharmacokinetics of GA in rats after the simultaneous coadministration of Sophorae flavescentis radix. (Kushen) and Glycyrrhizae radix et rhizoma. (Gancao).The potential attenuated mechanism of the combined use of these two herbs was induced by the herb–herb interaction. It is important to be aware that this work only provides a non-clinical proof of the effects of the herb–herb interaction of Sophorae flavescentis radix and Glycyrrhizae radix et rhizoma. Thus, before aiming at confirming these results in human a thorough clinical trial is required.
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