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Simultaneous determination and pharmacokinetics of sixteen Angelicae dahurica coumarins in vivo by LC-ESI-MS/MS following oral delivery in rats.

ABSTRACT

Background: The roots of Angelica dahurica cv. Qibaizhi is frequently used in clinical practice as a traditional Chinese medicine. However, a comprehensive study of the pharmacokinetics of this medicine has not been carried out.

Method: A sensitive and specific liquid chromatographic-tandem mass (LC-MS/MS) spectrometric method was established to investigate pharmacokinetics of sixteen coumarins of Angelicae dahuricae Radix (ADR) in rat plasma, including xanthotoxol (1), oxypeucedanin hydrate (2), 5-hydroxy-8-methoxypsoralen (3), (-)-marmesin (4), byakangelicin (5), columbianetin (6), psoralen (7), xanthotoxin (8), neobyakangelicol (9), isoimpinellin (10), bergapten (11), heraclenin (12), oxypeucedanin ethanolate (13), imperatorin (14), phellopterin (15), isoimperatorin (16). Detection was performed on a triple quadrupole mass spectrometer in multiple-reaction-mode (MRM).

Results: The method established in this assay was successfully applied to the pharmacokinetic study of the selected coumarins in rat plasma after oral administration of the extract of ADR, and the pharmacokinetic characteristics of sixteen coumarins were clearly elucidated.

Conclusion: This pharmacokinetic identification of multiple coumarins of ADR in rats provides a significant basis for better understanding the metabolic mechanism of the herb medicine.

Keywords:

LC-ESI-MS/MS

Traditional Chinese Medicine

Angelicae Dahuricae Radix

Coumarins

Pharmacokinetics

Introduction

Herb medicines have been used to treat ailments for thousands of years. Today, about 80% of medicines and 25% share of the pharmaceutical arsenal around the world are directly or indirectly derived from plants (Bhattaram et al. 2002). Angelicae Dahuricae Radix (ADR), the dried roots of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. or A. dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. var. formosana (Boiss.) Shan et Yuan, is used as a food additive and traditional medicine in China, Korea, and Japan. The cultivar of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. in Hebei province of China was botanically named Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. ex Franch. et Sav. cv. Qibaizhi Yuan et Shan (A. dahurica cv. Qibaizhi) and used as one of its substitutes in clinical practice (Editorial Board of Flora of China of Chinese Academy of Sciences 1992). As a well-known traditional Chinese medicine (TCM), ADR is frequently used for the treatment of the common cold, and headache, toothaches, asthma, coryza, hypertension, vitiligo, psoriasis, acne, herpes zoster virus, and freckles (Chinese Pharmacopoeia Commission 2015; He et al. 2008; Wu et al. 2009). As previously described, the main constituents of ADR are coumarins (Zhao et al. 2012). To date, more than 70 coumarins have been isolated and identified from ADR. Among them, imperatorin, phellopterin, isoimperatorin, and oxypeucedanin hydrate, etc. are the major active ingredients. As a class of natural products, coumarins have attracted considerable interest for their various pharmacological activities in recent years. Imperatorin has anti-cancer activity as well as anticonvulsant potential due to increasing level of gamma-aminobutyric acid (GABA) in vitro (Choi et al. 2005; Luszczki et al. 2010; Kleiner et al. 2001; Yang et al. 2006). Isoimperatorin demonstrates various pharmacological effects of anti-inflammatory, analgesic, antispasmodic and anticancer activities (Chen et al. 1995; Moon et al. 2011; Moon et al. 2008; Wang et al. 2010). Isoimperatorin affects both the peripheral and central nervous systems and significantly suppresses the smooth muscle spasm of rabbit's isolate intestine induced by Ba[Cl.sub.2]. Isoimperatorin also inhibits the reproduction of several tumor cells lines, such as HELA, P388, HL-60, and A549, in vitro. Byakangelicin shows potent inhibition on human acetylcholinesterase and butylcholinesterase (Seo et al. 2003). Phellopterin, as well as bergapten, byakangelicin, xanthotoxin, and neobyakangelicin, showed antiproliferative activity of B16F10 melanoma cells and causes G2/M arrest through an increase in the level of Chkl phosphorylation and decrease in the level of cdc2 (Tyr 161) phosphorylation (Maho et al. 2014). As the brain GABA type A receptor modulators, imperatorin, isoimperatorin, phellopterin, oxypeucedanin possessed modulative effect of GABA-induced chloride currents on recombinant [alpha]1[beta]2[gamma]2S [GABA.sub.A] receptors expressed in Xenopus laevis (African clawed frog) oocytes (Singhuber et al. 2011). These studies suggest that coumarins may be the main bioactive components contributing to the pharmacological efficacy of ADR.

With increasing knowledge of illative active constituents of TCM and rapid development of analytical methods, pharmacokinetic studies of complex mixtures of compounds in TCM have become available in the last decade. So far, several analytical methods for the determination of coumarins in ADR in vivo have been reported. Xie et al (Xie et al. 2007) developed an HPLC-UV method to study the pharmacokinetics of oxypeucedanin hydrate and byakangelicin in mongrel dog plasma after oral administration of ADR extracts. Zhao et al (Zhao et al. 2013) established a GC-MS method for the quantitative determination of eight coumarins including coumarin, isopsoralen, psoralen, xanthotoxin, bergapten, osthole, imperatorin, and oxypeucedanin in rat plasma after oral administration of a mixture of compounds at a dose of 10 mg/kg of each analyte. Wan et al (Wan et al. 2013) published an HPLC-MS/MS method to analyze the plasma and brain pharmacokinetics of three ingredients including imperatorin, isoimperatorin, and cnidilin in mice after oral administration of ADR extract at a dose of 800 mg/kg. Though these studies could not fully reflect the complex pharmacokinetic characteristics of ADR in the body, the methods provided a foundation for the metabolic study of ADR in vivo. Therefore, it is necessary to establish a more appropriate analysis method to characterize the pharmacokinetics of ADR in vivo.

In the present study, we developed a sensitive, rapid, and selective LC-MS/MS method for simultaneous determination of sixteen active ADR ingredients (Fig. 1) in rat plasma following oral administration of the extract of the roots of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. ex Franch. et Sav. cv. Qibaizhi Yuan et Shan (ERADQ). It was anticipated that this study would provide data for mechanism of action and pharmacological effects of ADR in vivo. To our knowledge, it is the first study with detailed pharmacokinetic characterizations of the sixteen coumarins in rat plasma.

Materials and methods

Chemicals and reagents

LC-MS grade acetonitrile (ACN) and methanol (MeOH) were obtained from J. T. Baker (Phillipsburg, USA). HPLC-grade formic acid was purchased from Dikma Tech. Inc. (Beijing, China). Water (H20) was purified by a Milli-Q system (Millipore, Billerica, MA, USA) in our laboratory. Other reagents were of analytical grade.

The standards of xanthotoxol (1), oxypeucedanin hydrate (2), 5-hydroxy-8-methoxypsoralen (3), (-)-marmesin (4), byakangelicin (5), columbianetin (6), psoralen (7), xanthotoxin (8), neobyakangelicol (9), isoimpinellin (10), bergapten (11), heraclenin (12), oxypeucedanin ethanolate (13), imperatorin (14), phellopterin (15), isoimperatorin (16) were isolated and identified from the roots of A. dahurica cv. Qibaizhi and A. dahurica Benth. et Hook. f. ex Franch. et Sav. (Zhao et al. 2012; Zhao et al. 2014). The purities of all standards were more than 98.5%, making them suitable for LC-MS/MS analysis. Daidzein (internal standard, IS) was purchased from National Institutes for Food and Drug (Beijing, China) with purity > 99.0%.

The roots of A. dahurica cv. Qibaizhi were collected from Anguo city of Hebei province, China, in 2008 and identified by Prof. Wang Wen-quan in Beijing University of Chinese Medicines. Voucher specimen (No. 20081025Q) was deposited at the State Key Laboratory of Natural & Biomimetic Drugs, Peking University (Beijing, China).

Preparation of Angelica dahurica extract and the solutions for pharmacokinetic study

The dried roots of A. dahurica cv. Qibaizhi (1 kg) were refluxed with 31 70% ethanol for four times. The solution was filtrated and evaporated under reduced pressure in a Buchi R-210 rotary evaporator (Buchi Ltd., Labortechnik AG, Switzerland). Subsequently, the concentrated extract was lyophilized using a lyophilizer. (Four-ring Science Instrument Plant Beijing Co., LTD., Beijing, China). The lyophilized powder was then dissolved in [H.sub.2]O to form a 1.2g/ml (crude drug 2.4 g/ml) suspension.

Apparatus and chromatographic conditions

Detection and quantification of the analytes were performed on a LC-MS system. The analytical DIONEX Ultimate 3000 HPLC system equipped with an Ultimate 3000 Pump, a DIONEX Ultimate 3000 Autosampler and a DIONEX Ultimate 3000 Compartment. The chromatograph was connected online to a 4000QTRAP triple quadrupole tandem mass spectrometer (Applied Biosystems/MDS Sciex, Canada) equipped with an electrospray ionization (ESI) source for the mass analysis and detection. Analyst 1.5.1 software (Applied Biosystems/MDS Sciex, Canada) was used for data collection and analysis. The separation was performed on a Diamonsil ODS [C.sub.18] column (250 x 4.6 mm i.d., 5 [micro]m; Dikma, China). The mobile phase was a mixture of [H.sub.2]O containing 0.1% formic acid (v/v) (A) and ACN (B). The gradient program of mobile phase was carried out as follows: 0-5 min, 10-40% B; 5-10 min, 40-50% B; 10-15 min, 50-70% B; 15-20 min, 70-80% B; 20-20.1 min, 80-10% B; 20.1-27 min, 10% B. The flow rate was set at l.Oml/min and the injection volume was 5 [micro]l. Ionization was performed in the positive electrospray mode and the turbo ionspray source was set as follows; capillary voltage 4.5 kV, source temperature 600[degrees]C, collision activated dissociation (CAD) 2.0 with nitrogen as collision gas. Nitrogen was also used as nebulizing gas, curtain gas and heater gas with pressures of 60, 15, and 50 psi, respectively. Declustering potential (DP) and collision energy (CE) were optimized by infusing the standard solution of each individual compound into the mass spectrometer separately with a syringe pump at a flow rate of 10 [micro]l/min. The quantification was carried out with multiple reactions monitoring (MRM) mode.

Preparation of samples

20 [micro]l IS solution (20ng/ml) and 900 [micro]l ethyl acetate (EtOAc) were added into 300 [micro]l rat plasma sample. The mixture was vortexed for 1.0 min and then centrifuged at 16,000 g for 10 min. The supernatant was transferred into a clean tube and evaporated to dryness under a gentle stream of [N.sub.2] at 40[degrees]C. Finally, the residue was dissolved in 200 [micro]l MeOH with vortex-mixing for 1.0 min. After centrifugation at 16,000 x g for 10 min, 5 [micro]l of aliquot was injected into the LC-MS/MS system for analysis.

Preparation of calibration standards and quality control samples

Standard stock solutions for the sixteen coumarins (1-16) and IS were prepared in MeOH at a concentration of 1.0 mg/ml, respectively. Appropriate aliquots of the coumarin stock solutions were diluted to prepare a mixed stock solution. The mixed solution was then serially diluted with MeOH to obtain working standard solutions of desired concentrations. IS solution was diluted with MeOH to a final concentration of 20 ng/ml. All of the solutions were stored at 4[degrees]C and brought to room temperature before use.

Calibration standards were prepared by combining 20 [micro]1 of working standard solution, 20 [micro]l IS, and 900 [micro]l EtOAc to a 300 [micro]l aliquot of drug-free plasma. The mixture was handled as described in "Preparation of samples" section. Each calibration curve was obtained with six concentrations. Quality control (QC) samples were independently prepared to give three levels of low, medium and high concentrations for each analyte. The solutions and samples were kept at -20[degrees]C before use.

Method validation

The method was validated in terms of specificity, linearity, lower limit of detection (LLOD), lower limit of quantification (LLOQ), accuracy and precision, extraction recovery, stability, and matrix effects according to US Food and Drug Administration for bioanalytical method validation (US Food and Drug Administration 2001).

The specificity of the method was determined from the lack of potential interfering peaks of endogenous substances within the range of the retention time of each analyte. The linearity of each calibration curve was determined by plotting the peak area ratio (analyte/IS) versus the nominal concentration of the analyte. The LLOD and LLOQ were defined as the lowest concentration of a signal-noise (S/N) ratio of 3:1 and 10:1, respectively. The accuracy and precision were evaluated by analyzing QC samples in six replicates at low, medium, and high concentrations on one day and then for three consecutive days. The precision was expressed as relative standard deviations (RSD) and the accuracy was presented as percentage rate of the calculated and preparative concentration.

The extraction recoveries were measured by comparing the peak areas obtained from extracted blank plasma spiked with the analytes to those of the same amounts dissolved in MeOH at three corresponding concentrations. Matrix effects were investigated by comparing corresponding peak areas of the post-extraction spiked samples to those of the analytes directly dissolved in MeOH at three concentration levels. The stability of analytes in rat plasma was examined by analyzing three replicates of QC samples during the sample storage and processing procedures. The blank plasma samples spiked with the analytes and IS were stored at -20[degrees]C for 24 h, and then were thawed at room temperature for 12 h. The analysis was performed after the freeze-thaw cycle was repeated three times.

Pharmacokinetic study

Male Sprague-Dawley rats (200 [+ or -] 20g) were supplied by the Laboratory Animal Center of Peking University Health Science Center (Beijing, China). All of the experimental procedures were in compliance with the guidelines defined by the Peking University Committee on Animal Care and Use for the use of experimental animals. Animals were bred in a breeding room with temperature of 24 [+ or -] 2[degrees]C, humidity of 60 [+ or -] 5%. Water and food were supplied ad libitum and the animals were fasted with free access to water for 12 h prior to experiments.

The time schedule included 12 time-points, five experimental rats were sacrificed at each time-point, and 60 experimental rats were used in this study all together. Each rat was given ERADQ at a dose of 6.0g/kg by oral administration (equivalent to crude drug 12g/kg). Blood samples (0.6 ml) were collected from the sub-orbital vein into heparinized tubes at 0.083, 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, and 24.0 h after delivery. All blood samples were immediately centrifuged for 15 min at 16,000 x g and the supernatants were stored at -20[degrees]C until analysis.

Data analysis

All statistical analyses were calculated with SPSS 17.0 software. Major pharmacokinetic parameters of the sixteen coumarins were calculated by the Drug and Statistics (DAS) 2.0 software supplied by Chinese Mathematical Pharmacology Society, and the data were expressed in the form of mean [+ or -] standard deviation (SD) for each parameter.

Results

Liquid chromatographic conditions

Due to the amount of the analytes, the first step of this study was to develop a suitable liquid condition to achieve good separation of the analytes in a relatively short period of time. Finally, Diamonsil ODS [C.sub.18] column was choosen for its good resolution in the same analysis time. Mobile phases were selected to optimize peak shapes and separation. MeOH and ACN, the two mostly used organic solvents in reversed-phase liquid chromatography, were evaluated and ACN was selected for its better separation effect over MeOH. The mobile phase was a mixture of [H.sub.2]O containing 0.1% formic acid (v/v) (A) and ACN (B), the gradient program was as follows: 0-5 min, 10-40% B; 5-10 min, 40-50% B; 10-15 min, 50-70% B; 15-20 min, 70-80% B; 20-20.1 min, 80-10% B; 20.1-27 min, 10% B. The flow rate was 1.0 ml/min. In order to improve the sensitivity of this method, we evaluated the influence of different sampling volume to the sensitivity and separation of the analytes. Injection volumes ranging from 3 to 10 [micro]l were compared and 5 [micro]l was finally selected. The typical chromatograms of the analytes in rat plasma are shown in Fig. 2.

Mass spectrometric analysis

To optimize the number of precursor and product ions of the analytes and IS in MRM mode, the standard solutions of each individual compound were infused into the mass spectrometer separately by syringe pump infusion with a flow rate of 10 [micro]l/min. ESI interface in positive mode was selected because of its higher sensitivity. The mass parameters such as DP and CE were also optimized (see Table SI). Finally, the quantification was carried out using MRM mode by positive electrospray ionization at m/z 203.1 [right arrow] 147.1 (1), m/z 305.2 [right arrow] 203.1 (2), m/z 233.2 [right arrow] 218.0 (3), m/z 247.1 [right arrow] 175.1 (4), m/z 335.2 [right arrow] 231.1 (5), m/z 247.2 [right arrow] 175.0 (6), m/z 187.1 [right arrow] 131.1 (7), m/z 217.1 [right arrow] 202.1 (8), m/z 317.2 [right arrow] 231.2 (9), m/z 247.3 [right arrow] 217.2 (10), m/z 217.1 [right arrow] 202.1 (11), m/z 287.1 [right arrow] 203.1 (12), m/z 333.1 [right arrow] 203.1 (13), m/z 271.1 [right arrow] 203.0 (14), m/z 301.1 [right arrow] 233.1 (15), m/z 271.1 [right arrow] 203.1 (16), and m/z 255.0 [right arrow] 199.2 (IS).

Linearity of calibration curves and lower limits of detection and quantification

The calibration curve for each analyte was obtained by injecting 5 [micro]l of different combined standard solutions into the LC-MS system. As shown in Table 1, all of the calibration curves exhibited good linearity in the concentration ranges with correlation coefficients (r) [greater than or equal to] 0.9903. The LLOQ. and LLOD which were summarized in Table 1 are sufficient to support the pharmacokinetic study of the sixteen compounds.

Method validation

Validation of specificity, precision, accuracy, extract recovery, stability, and Matrix effects was executed according to the US Food and Drugs Administration (FDA) guidelines for bioanalytical assay validation (US Food and Drug Administration 2001).

The specificity of this method was determined by comparing the chromatograms of blank and medicated plasma samples. As shown in Fig. 2, no interfering peaks from endogenous substances were observed at the retention times of each analyte. It suggested that the LC-MS/MS conditions in this study provided sufficient selectivity for the analytes. Retention times of the relative compounds were 9.53 (1), 9.56 (2), 9.67 (3), 9.68 (4), 9.69 (5), 10.20 (6), 13.18 (7), 13.49 (8), 14.31 (9), 14.61 (10), 14.75 (11), 14.91 (12), 16.52 (13), 18.73 (14), 19.55 (15), 20.60 (16), and 9.29 (IS) min.

To evaluate the precision and accuracy of this method, the intra- and inter-day precisions and accuracy were assessed at three concentration levels of each analyte. The intra- and inter-day precisions (expressed as the relative standard deviation; RSDs) for the investigated compounds were all less than 15%. The intra-and inter-day accuracies were in the range of 85.07% - 114.7% (Table S2). The results indicated that an acceptable precision and accuracy of the method was established for the determination of the sixteen compounds in rat plasma.

The extract recoveries of the sixteen compounds were determined at low, middle and high concentration levels. The mean overall recoveries of the sixteen analytes were in the range of 72.56% - 98.80% with the precision less than 15.65% (Table S2). After three freeze-thaw cycles, the corresponding relative standard deviations for low, middle and high concentrations of the sixteen compounds were <15% (Table S2). Therefore, the results herein were in the range of acceptable limits during the entire validation.

Due to the influence of other components from the blank matrices on the specific analytes' ionization process, there may be ion suppression or amplification effects. For the sixteen analytes at three QC concentrations in rat plasma, the accuracy of ion suppression were in the range of 87.42% - 102.1% (Table S2), indicating that there were minimal matrix effects on the ionization of the analytes under the analytical conditions.

Application to pharmacokinetic study

The present LC-MS/MS method was successfully applied to the pharmacokinetic study of the selected coumarins in rat plasma after oral administration of ERADQ. Plasma concentrations of the sixteen components were remained in the range of calibration curves and determined up to 24 h throughout the study. The mean plasma concentration-time profiles were illustrated in Fig. 3. The pharmacokinetic parameters were calculated by non-compartment model and summarized in Table 2.

Discussion

After oral administration of ERADQ, all of the tested coumarins were absorbed from rat gastrointestinal tract and detected at 5 min in plasma. The absorption of (-)-marmesin and columbianetin with maximum concentration ([T.sub.max]) occurred at 7.0 and 6.0 h, respectively, which was significantly different from other compounds ([T.sub.max] < 3 h). The result was in line with previous research (Luo et al. 2013), when Luo et al. found that the [T.sub.max] of columbianetin reached at 6.29 h after oral administration of Angelica pubescens extract to rats. The parameter was extended compared to oral administration of pure columbianetin. In this study, the extension of [T.sub.max] for columbianetin and (-)-marmesin may be related to the conversion of other ingredients in ERADQ. The [T.sub.max] values of oxypeucedanin hydrate, byakangelicin, psoralen, xanthotoxin, bergapten, imperatorin, and isoimperatorin were 2.2 h, 2.3 h, 1.9 h, 2.0 h, 2.4 h, 1.8 h, and 2.8 h, respectively, which were similar with the previous reports (Gu et al. 2009; Xie et al. 2007; Xing et al. 2013; Zhao et al. 2013; Zhang et al. 2009). The results suggested that this experiment veritably reflected the pharmacokinetic characteristics of these compounds in rats. Compared with chemical drugs, ingredients of plant medicine are complex and have synergistic activity. In this experiment, we found that most of the ingredients reached their maximum concentrations in about 2 hours after oral administration of ERADQ. This phenomenon might allow ERADQ to exert its best pharmacological activity in clinical application. A large number of studies have shown that furanocoumarins are inhibitors of the CYP450 enzyme. Thus, the co-existence of a variety of coumarins in ERADQ may lead to metabolic interaction and a change in pharmacokinetics. As shown in Table 2, the elimination half-lives ([T.sub.1/2]) of columbianetin, imperatorin, and isoimperatorin were 2.25 h, 2.96 h, and 5.42 h. These values were extended compared with administration of pure compounds (Luo et al. 2013; Wang et al. 2007; Zhang et al. 2009). The plasma concentration-time profiles (Fig. 3) of oxypeucedanin hydrate, 5-hydroxy-8-methoxypsoralen, (-)-marmesin, byakangelicin, psoralen, bergapten, and phellopterin exhibits steeper slopes at the last time points of the concentration-time curves. This characteristic may point to nonlinear pharmacokinetics of these compounds. Saturation of components in the system is typically a reason for nonlinear pharmacokinetics which can be divided into three types (Ludden 1991). The saturation of metabolic enzymes and drug transporters may play a role in enlarging the AUC and prolonging the [T.sub.1/2]. However, the sources, weights, and housing environment all might influence the metabolic behaviors of the same drugs. So the clear-cut reason should be further verified in the future study. Due to the similar chemical composition and structure, a common phenomenon in TCM is the adient pharmacokinetic behavior and double peaks, which is prone to producing enterohepatic circulation to maintain effective plasma concentrations and show longer pharmacological efficacy. In this study, some compounds show double peaks in the time-course curves. The main reason is a possible enterohepatic recirculation, however, this hypothesis needs to be further confirmed. Anyhow, the change of pharmacokinetic parameters may increase the bioavailability of ingredients, which may also lead to an increase in adverse reactions besides better exert their effects and we should pay attention to the dosage of ERADQ in long-term clinical application.

A simple, sensitive and accurate LC/MS/MS method was established in this study for the simultaneous determination of sixteen coumarins in rat plasma. Our results show that this method is efficient for the bioanalysis and application to the pharmacokinetic study of multiple coumarins of ERADQ in rats. To our knowledge, little research has been reported on the pharmacokinetics of so many ingredients in TCM. In previous reports, other analytical methods, including HPLC (Xie et al. 2007), GC-MS (Zhao et al. 2013), and LC-MS (Xing et al. 2013; Zhang et al. 2009), have been applied to evaluate the pharmacokinetic profiles of coumarins of ERADQ in vivo. Due to limitations of the analysis method or the numbers of the components, the above methods could not fully reflect the pharmacokinetic characteristics of ERADQ. TCM is a complex whole, its effect to ameliorate the disease is the synergistic action of multiple ingredients in it. Sometimes, TCM as a whole even exhibits better pharmacological potency than single compound. In comparison to previous studies, we found that the coexistence of ingredients in ERADQ could alter the pharmacokinetic behavior of single compound significantly, improving bioavailability and prolonging the time in system circulation. Thus, the novelty of this research not only lies in building simple and reliable multi-components analysis method, but also in comprehensively revealing the pharmacokinetic behavior of ERADQ. The present data believed to be valuable for determining the effective components of ERADQ and guiding its rational clinical application.

http://dx.doi.org/10.1016/j.phymed.2016.06.015

ARTICLE INFO

Article history:

Received 13 November 2015

Revised 20 April 2016

Accepted 14 June 2016

Conflict of interest

The authors declared no conflict of interest.

Acknowledgments

The authors thank the NIH Fellows Editorial Board (NIH, Bethesda, USA) for editing this manuscript. This project was partly supported by the National Key Technology R&D Program of China (2011BAI07B08, 2012BAI29B02), the National Natural Science Foundation of China (81473321), Beijing Municipal Natural Science Foundation of China (7152086), Beijing Municipal Special-purpose Science Foundation of China (Z0004105040311).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.06.015.

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Ai-Hong Zhao (a,b,1), You-Bo Zhang (a,c,1), Xiu-Wei Yang (a,*)

(a) State Key Laboratory of Natural and Biomimetic Drugs and Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, 100191, Beijing, China

(b) School of Life Science and Engineering, Lanzhou University of Technology, 730050, Lanzhou, China

(c) Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, United States

Abbreviations: LC-MS/MS, liquid chromatographic-tandem mass; ADR, Angelicae dahuricae Radix; MRM, multiple-reaction-mode; TCM, traditional Chinese medicine; GABA, gamma-aminobutyric acid; ERADQ, extract of roots of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. f. ex Franch. et Sav. cv. Qibaizhi Yuan et Shan; ACN, acetonitrile; MeOH, methanol; IS, internal standard; CAD, collision activated dissociation; DP, Declustering potential; CE, collision energy; EtOAc, ethyl acetate; QC, quality control; LLOD, lower limit of detection; LLOQ, lower limit of quantification; S/N, signal-noise ratio; RSD, relative standard deviations; FDA, food and drugs administration.

* Corresponding author. Fax: +86-10-82802724.

E-mail address: xwyang@bjmu.edu.cn (X.-W. Yang).

(1) These authors contributed equally to this work.

Table 1
Calibration curve, correlation coefficient (r) and linear
range of the sixteen analytes from extract of the roots of
A. dahurica cv. Qibaizhi in rat plasma samples.

                                             Linear range
Sample     Calibration curves    [R.sup.2]   (ng/ml)

1         y = 0.0190x - 0.8544   0.9966      1.164-11,640
2         y = 0.0338x + 2.5990   0.9908      1.212-121,200
3         y = 0.1306x - 0.4229   0.9914      1.200-12,000
4         y = 0.0130x + 0.2158   0.9988      3.104-31,040
5         y = 0.0263x + 1.4435   0.9907      0.315-63,000
6         y = 0.0171x - 0.0686   0.9919      0.880-8800
7         y = 0.0179x - 0.3556   0.9937      0.840-840
8         y = 0.0344x - 0.8740   0.9958      0.368-3680
9         y = 0.1689x + 0.0462   0.9982      0.352-1760
10        y = 0.1014x - 0.2169   0.9994      0.317-3168
11        y = 0.0344x - 0.4044   0.9947      2.760-27,600
12        y = 0.2927x - 1.3739   0.9966      0.309-618
13        y = 0.0559x - 0.0372   0.9990      1.856-37,120
14        y = 0.2063x - 6.5907   0.9918      0.135-27,040
15        y = 0.2982X + 4.5902   0.9903      0.114-11,400
16        y = 0.0890x - 3.0877   0.9913      0.144-12,096

          LLOQ      LLOD
Sample    (ng/ml)   (ng/ml)

1         1.164     0.388
2         1.212     0.404
3         1.200     0.400
4         3.104     1.034
5         0.315     0.105
6         0.880     0.293
7         0.840     0.280
8         0.368     0.123
9         0.352     0.117
10        0.317     0.106
11        2.760     0.920
12        0.309     0.103
13        1.856     0.618
14        0.270     0.045
15        0.114     0.038
16        0.144     0.048

Table 2
Pharmacokinetic parameters of the sixteen analytes in
rat plasma after oral administration of extract of the roots
of A. dahurica cv. Qibaizhi (mean [+ or -] SD, n = 5).

Parameters       Units                   1

AUC(0-t)         ng * h/ml    19169.1 [+ or -] 3264.1
AUC(0-           ng * h/ml    20107.6 [+ or -] 3634.4
  [infinity])
AUMC(0-t)                    147443.3 [+ or -] 36043.4
MRT(0-t)         h              7.634 [+ or -] 0.782
[T.sub.1/2z]     h              5.044 [+ or -] 1.664
[T.sub.max]      h              2.000 [+ or -] 0.000
CLz/F            L/h/kg         1.089 [+ or -] 0.189
Vz/F             L/kg           7.716 [+ or -] 1.897
[C.sub.max]      ng/ml         2679.5 [+ or -] 640.9

Parameters                    2                           3

AUC(0-t)         339596.5 [+ or -] 80,940.5   46120.9 [+ or -] 3443.5
AUC(0-           342188.3 [+ or -] 81157.6    46154.5 [+ or -] 3454.1
  [infinity])
AUMC(0-t)       2403911.0 [+ or -] 739988.1  316680.2 [+ or -] 37828.4
MRT(0-t)            7.004 [+ or -] 0.508        6.850 [+ or -] 0.406
[T.sub.1/2z]        3.303 [+ or -] 0.347        2.212 [+ or -] 0.215
[T.sub.max]         2.200 [+ or -] 0.447        2.000 [+ or -] 0.000
CLz/F               0.693 [+ or -] 0.136        0.111 [+ or -] 0.009

Vz/F                3.303 [+ or -] 0.704        0.354 [+ or -] 0.027
[C.sub.max]      52,803.0 [+ or -] 7978.0      6808.5 [+ or -] 1355.1

Parameters                   4                           5

AUC(0-t)         43882.4 [+ or -] 2953.1    195397.9 [+ or -] 39341.3
AUC(0-           63481.5 [+ or -] 45831.2   196448.4 [+ or -] 39199.9
  [infinity])
AUMC(0-t)       355845.5 [+ or -] 48148.3  1318164.3 [+ or -] 286542.8
MRT(0-t)           8.081 [+ or -] 0.559        6.729 [+ or -] 0.214
[T.sub.1/2z]       3.566 [+ or -] 4.655        2.874 [+ or -] 0.484
[T.sub.max]        7.000 [+ or -] 2.236        2.300 [+ or -] 0.975
CLz/F              0.120 [+ or -] 0.045        0.736 [+ or -] 0.141
Vz/F               0.381 [+ or -] 0.183        3.070 [+ or -] 0.824
[C.sub.max]       4703.3 [+ or -] 623.4     30,765.0 [+ or -] 8979.6

Parameters                  6                        7

AUC(0-t)         16775.5 [+ or -] 588.1    901.7 [+ or -] 113.8
AUC(0-           16837.0 [+ or -] 675.5   1260.4 [+ or -] 357.1
  [infinity])
AUMC(0-t)       128323.9 [+ or -] 5902.4  4468.8 [+ or -] 783.1
MRT(0-t)           7.649 [+ or -] 0.225    4.944 [+ or -] 0.463
[T.sub.1/2z]       2.252 [+ or -] 0.583    5.572 [+ or -] 3.306
[T.sub.max]        6.000 [+ or -] 0.000    1.900 [+ or -] 0.224
CLz/F              0.208 [+ or -] 0.008    8.930 [+ or -] 2.514
Vz/F               0.672 [+ or -] 0.147   63.824 [+ or -] 24.94
[C.sub.max]       2314.9 [+ or -] 69.65    173.3 [+ or -] 39.87

Parameters                  8                       9

AUC(0-t)         9184.9 [+ or -] 1353.6   1505.7 [+ or -] 185.3
AUC(0-           9272.1 [+ or -] 1348.7   1508.9 [+ or -] 182.8
  [infinity])
AUMC(0-t)       51420.1 [+ or -] 8127.7   8207.6 [+ or -] 1172.0
MRT(0-t)          5.597 [+ or -] 0.235     5.441 [+ or -] 0.148
[T.sub.1/2z]      3.468 [+ or -] 0.304     2.456 [+ or -] 0.518
[T.sub.max]       2.000 [+ or -] 0.000     2.000 [+ or -] 0.000
CLz/F             3.228 [+ or -] 0.498     18.42 [+ or -] 2.377
Vz/F              16.24 [+ or -] 3.308     66.57 [+ or -] 23.635
[C.sub.max]      2064.8 [+ or -] 501.6     335.8 [+ or -] 51.63

Parameters                  10                        11

AUC(0-t)          7773.3 [+ or -] 1689.5    39255.8 [+ or -] 3758.0
AUC(0-            7936.9 [+ or -] 1800.4    39680.7 [+ or -] 3817.5
  [infinity])
AUMC(0-t)        45275.2 [+ or -] 14234.7  262233.0 [+ or -] 34450.5
MRT(0-t)           5.723 [+ or -] 0.978       6.667 [+ or -] 0.338
[T.sub.1/2z]       3.349 [+ or -] 1.554       3.129 [+ or -] 0.813
[T.sub.max]        2.400 [+ or -] 0.894       2.400 [+ or -] 0.894
CLz/F              0.612 [+ or -] 0.149       3.794 [+ or -] 0.379
Vz/F               2.809 [+ or -] 0.958       17.11 [+ or -] 4.508
[C.sub.max]       1550.2 [+ or -] 591.9      5664.8 [+ or -] 867.5

Parameters                 12                       13

AUC(0-t)          9071.7 [+ or -] 867.6   13189.6 [+ or -] 377.9
AUC(0-            9085.4 [+ or -] 867.8   13247.6 [+ or -] 390.3
  [infinity])
AUMC(0-t)        61785.7 [+ or -] 8863.2  71966.9 [+ or -] 3925.8
MRT(0-t)           6.794 [+ or -] 0.436     5.459 [+ or -] 0.333
[T.sub.1/2z]        2.44 [+ or -] 0.126     2.816 [+ or -] 0.217
[T.sub.max]        2.000 [+ or -] 0.000     1.900 [+ or -] 0.224
CLz/F              0.366 [+ or -] 0.034     0.208 [+ or -] 0.006
Vz/F               1.288 [+ or -] 0.136     0.844 [+ or -] 0.063
[C.sub.max]       1484.3 [+ or -] 314.6    2532.1 [+ or -] 386.9

Parameters                   14                         15

AUC(0-t)          71480.9 [+ or -] 8374.0     6642.7 [+ or -] 1248.6
AUC(0-            72095.9 [+ or -] 9067.5     6694.5 [+ or -] 1224.4
  [infinity])
AUMC(0-t)        450183.8 [+ or -] 110071.8  35928.2 [+ or -] 13290.0
MRT(0-t)            6.229 [+ or -] 1.008       5.277 [+ or -] 1.229
[T.sub.1/2z]        2.963 [+ or -] 0.809       2.734 [+ or -] 1.398
[T.sub.max]         1.800 [+ or -] 0.274       1.700 [+ or -] 0.274
CLz/F               19.11 [+ or -] 2.451       122.6 [+ or -] 21.726
Vz/F                80.13 [+ or -] 14.623      488.2 [+ or -] 272.6
[C.sub.max]      12,921.0 [+ or -] 2258.4     1615.4 [+ or -] 218.0

Parameters                  16

AUC(0-t)         25607.5 [+ or -] 3534.7
AUC(0-           27166.9 [+ or -] 3507.3
  [infinity])
AUMC(0-t)       195706.4 [+ or -] 27386.2
MRT(0-t)           7.642 [+ or -] 0.098
[T.sub.1/2z]       5.416 [+ or -] 0.571
[T.sub.max]        2.800 [+ or -] 1.095
CLz/F              14.11 [+ or -] 2.148
Vz/F               111.5 [+ or -] 29.28
[C.sub.max]       3069.2 [+ or -] 583.6


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Title Annotation:Original article
Author:Zhao, Ai-Hong; Zhang, You-Bo; Yang, Xiu-Wei
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Sep 15, 2016
Words:6809
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