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Journal of Ethnopharmacology 151 (2014) 191–197
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Pharmacokinetic study on costunolide and dehydrocostuslactone after oral administration of traditional medicine Aucklandia lappa Decne. by LC/MS/MS Jingze Zhang a,1, Xiao Hu b,1, Wenyuan Gao c,n, Zhuo Qu c, Huimin Guo c, Zhen Liu c, Changxiao Liu d a
Department of Pharmacy, Logistics College of Chinese People's Armed Police Forces, Tianjin 300162, China Tianjin Key Laboratory for Prevention and Control of Occupational and Environmental Hazard. Logistics College of Chinese People’s Armed Police Forces, Tianjin 300162, China c School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China d The State Key Laboratories of Pharmacodynamics and Pharmacokinetics, Tianjin 300193, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 30 May 2013 Received in revised form 30 July 2013 Accepted 7 October 2013 Available online 8 November 2013
Ethnopharmacological relevance: Radix Aucklandiae (RA), a well known traditional Chinese medicine, is widely used for treating various problems in digestive system. A selective and sensitive highperformance liquid chromatography coupled with mass spectrometry method was first developed and validated for simultaneous quantification of costunolide and dehydrocostuslactone in rat plasma with diazepam as internal standard after oral administration of RA extraction. Materials and methods: Plasma samples were extracted via solid-phase extraction and detected by multiple-reaction monitoring mode under positive electrospray. Chromatographic separation was accomplished on an Agilent C18 column (2.1 mm 150 mm, 5 mm), with 0.1% formic acid and acetonitrile (1:1) as the mobile phase at a flow rate of 0.5 mL/min. Results: The quantification was performed using the transitions of m/z 233/187 for costunolide, m/z 231/ 185 for dehydrocostuslactone and m/z 285/193 for diazepam, respectively. Calibration curves were linear over the concentration range of 0.7–769.7 ng/mL for costunolide and 0.9–956.0 ng/mL for dehydrocostuslactone. The intra-day and inter-day precisions (RSD%) for two compounds was less than 8.76% and 9.70% and the accuracy (RE%) range from 6.14% to 5.35%. The time to reach the maximum plasma concentration (Tmax) was 10.46 h for costunolide, 12.39 h dehydrocostuslactone. The elimination halftime (t1/2) of costunolide and dehydrocostuslactone was 5.547 0.81 and 4.32 70.71 (h). The AUC of costunolide and dehydrocostuslactone was 308.83 and 7884.51 respectively (ng h/mL). Conclusions: It was the first report for the study of pharmacokinetic profile of costunolide and dehydrocostuslactone in rat plasma after oral administration of RA extract. These results provided a meaningful basis for better understanding the absorption of traditional medicine, RA, and provide useful scientific data for clinical application. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sesquiterpene lactones Radix Aucklandiae Pharmacokinetic HPLC–MS
1. Introduction Radix Aucklandiae (RA), known as Muxiang in Chinese, is derived from the dried root of Aucklandia lappa Dence. belonging to the family Asteraceae. It is one of the commonly used traditional Chinese medicines which listed in Chinese Phaemacopoeia (Committee of National Pharmacopoeia, 2010). Costunolide (1) and dehydrocostuslactone (2) with a guaiane skeleton belonging to sesquiterpene lactones are the major active constituents in n
Corresponding author. Tel./fax: þ 86 22 87401895. E-mail addresses: [email protected], [email protected] (W. Gao). 1 These two authors contributed equally to this work.
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.10.024
RA (Li et al., 2005). In Chinese Phaemacopoeia, as the makers to control herb quality, the total content of costunolide and dehydrocostuslactone was no less than 1.8%. In recent years, researches on RA have been mainly focused on the chemical constituent (Chhabra et al., 1998; Hou et al., 1998) and pharmacology (Pandey et al., 2007; Parekh and Chanda, 2007; Yu et al., 2007). However, there is no literature available for the pharmacokinetic of main constituents in RA. In previous study of the chemical constituents, Shum et al. (2007) reported that ninety chemicals can be detected in the essential oil of Aucklandia lappa and among them 28 were unique to RA. Isodihydrocostunolide, a new sesquiterpene lactone from the roots of Saussurea lappa, exhibited good activity on cancer cell lines and IC50 values (Robinson et al., 2008). The
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effects of eighteen sesquiterpenes isolated from Saussurea lappa on the proliferation of six human cancer cell lines were evaluated by MTT assay (Wang et al., 2008). RA has been widely used for the treatment of various problems in digestive system, including loss of appetite, indigestion, diarrhea, and abdominal pain (Chang and But, 1986). In addition, it possesses activities of antispasmodic (Shoji et al., 1986) antiinflammatory (Damre et al., 2003; Choi et al., 2012), anticancer (Yang et al., 2005), anti-ulcer (Taniguchi, et al., 1995; Yamahara, et al., 1985) and cholagogic (Mitra et al., 1996). Several analytical techniques have been published for the analysis of costunolide and dehydrocostuslactone in the herb by high performance liquid chromatography (HPLC) (Wang et al., 2000), gas chromatography with mass detection (GC–MS) (Qiu. et al., 2001). Hu et al. established a sensitive UPLC-MS/MS for the quantification of costunolide and dehydrocostuslactone in rat plasma after oral administration of the mixture of two monomers (Hu et al., 2011). However, no study focused on the pharmacokinetic profile of costunolide and dehydrocostuslactone after administration of the herb extract of RA. Prior published reports showed the pharmacokinetic parameters were significant differently after oral administration of one active component, herb or its prescription. The different pharmacokinetic parameters of paeoniflorin were obtained after oral administration of Cortex Moutan or Shuang-Dan decoction (Wu et al., 2009), while the significant changes were obviously observed following administration of pure baicalin, Radix scutellariae extract and Huang-Lian-Jie-Du-Tang (Lu et al., 2007). Therefore comparing with the pharmacokinetic parameters of the pure monomer, those may change after oral administration of the herb. In present study, the pharmacokinetic parameters were studied by a simple, rapid and sensitive HPLC–MS/MS method for quantification of costunolide and dehydrocostuslactone in the rat plasma after oral administration of the herb extract of RA. The preliminary pharmacokinetic behavior of two sesquiterpene lactones in rat plasma after oral administration of the dried root of Aucklandia lappa Dence. was firstly elucidated.
2. Materials and methods 2.1. Chemicals HPLC grade acetonitrile was purchased from Fisher (USA). Water was purified by a Milli-Q water purification system (Millipore, USA). Methanol of analytical grade was purchased from Guangfu Technology Limited Company (Tianjin, China).
Standards including costunolide, dehydrocostuslactone and diazepam (internal standard, IS) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All the reference compounds have over 98% purity. Aucklandia lappa Decne. was provided by Tianjin Zhongxin Pharmaceutical Group Co., Ltd. (Tianjin, China) and identified by Professor Wenyuan Gao from School of Pharmaceutical Science and Technology, Tianjin University, China. The voucher specimens (Voucher no. MX100108) are available in the herbarium of Research Center of Tianjin Zhongxin Pharmaceuticals. 2.2. Instrumentation The LC-MS/MS system consisted of an Agilent 1200 mod. liquid chromatography system (Agilent, MA, USA) equipped with a binary solvent delivery system, an autosampler, a column compartment and a 6410 triple quadrupole mass spectrometer with electrospray ionization (ESI) source. Data were analyzed by MassHunter software (Agilent Corporation, MA, USA). The analytical column was a Agilent C18 (150 mm 4.6 mm, 5 μm). A bionary mobile phase system consisted of (A) acetonitrile and (B) 0.1% formic acid (1:1, v/v) at a flow rate of 0.5 mL/min. 2.3. Preparation of the extracts In the present study, twenty grams of Aucklandia lappa Decne. were powdered and extracted with 200 mL methanol for 2 h in a reflux condenser. The filtrate was collected and the residue was reextracted with 200 mL methanol. Then the solvent was removed under reduced pressure in a rotary evaporator. Costunolide and dehydrocostuslactone (the structures given in Fig. 1) were main components in Aucklandia lappa Decne. and the contents of two constituents in the extract checked by HPLC methods (Wu et al., 2009) were 0.79% and 3.59% respectively. 2.4. Animals Healthy male Wistar rats, weighing about 250–280 g were used in the experiments. The animals were purchased from the Experimental Animal Center, Chinese Academy of Medical Sciences, Peking (SCXK-2007-004). The animals were housed in an environmentally (t¼ 25 1C) and air humidity (60%) controlled room with a 12 h light-dark (07:00–19:00 h and 19:00–07:00 h) cycle, kept on a standard laboratory diet and drinking water ad libitum. This study was carried out in accordance with the Regulation for the Administration of Affairs Concerning Experimental Animals (State Council of China, 1988).
Fig. 1. Chemial structures of costunolide, dehydrodehydrocostus and diazepam (IS).
J. Zhang et al. / Journal of Ethnopharmacology 151 (2014) 191–197
2.5. Rat plasma sample preparation A 100 μL aliquot of rat plasma sample and 10 μL of diazepam (internal standard, 100 ng/mL) were added to a 1.5 mL heparinized eppendorf tube. After vortexing for 3 min, the sample was centrifuged at 10,000 rpm for 5 min and then the supernatant was loaded onto the preconditioned SPE (i.e., preconditioned with 1 mL methanol followed by 2 mL purified water). The cartridge was rinsed twice with 1 mL of water and the constituents were eluted with 1 mL methanol. The methanol fraction was dried with nitrogen at 45 1C and the dry residue was reconstituted with 50 μL of methanol. After centrifugation at 10,000 rpm for 5 min, 10 μL of the sample was directly injected into the HPLC–MS system for analysis. 2.6. Preparation of standard solution and quality control samples The linearity was determined by analysis of standard solutions at nine different concentrations. Known amounts of costunolide, dehydrocostuslactone and IS were added into 100 mL of blank plasma to prepare the following series of standards. The standard sets of costunolide had good linearity with its own linearity range at 0.70, 6.01, 12.03, 24.05, 48.11, 96.21, 192.42, 384.85 and 769.70 ng/mL. And the standard sets of dehydrocostuslactone ranged at 0.96, 7.47, 14.94, 29.88, 59.75, 119.50, 239.00, 478.00 and 956.00 ng/mL. The quality control (QC) samples were prepared at three different concentrations 6.01, 48.11, 384.85 ng/mL for costunolide and 7.47, 59.75, 478.00 ng/mL for dehydrocostuslactone. 2.7. Method validation Validation was performed by establishing the within-batch and between-batch accuracy, precision, recovery and stability of the method on quality control (QC) samples. 2.7.1. Selectivity Spiked blank rat plasma with costunolide, dehydrocostuslactone and IS, blank plasma sample and the mixed standard (costunolide, dehydrocostuslactone and IS) were tested. Chromatographic peaks of the analytes were identified based on their retention times and MRM responses. 2.7.2. Linearity, limit of detection (LOD) and limit of quantification (LLOQ) Calibration curves were constructed by the analysis of plasma samples spiked with nine different concentrations of standards. The LOD was determined using the signal-to-noise ratio (S/N) of 3:1 by comparing test results from samples with known concentrations of analyte with blank samples. The LLOQ was defined as the lowest concentration point of the calibration curve at which can be detected with acceptable precision and accuracy. 2.7.3. Precision and accuracy The intra-day and inter-day precisions were determined by the assay of standard solutions at three concentrations on a single day and three continuous days. Five determinations of each concentration level of costunolide and dehydrocostuslactone were analyzed and precision and accuracy were expressed as relative standard deviation (RSD%) and deviation from theoretical value. 2.7.4. Stability The stability of costunolide and dehydrocostuslactone in rat plasma was tested by analysis of freeze-thawing three times and stored in room temperature for 24 h. Spiked plasma samples were
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repeated in triplicate (n ¼3) at three concentration levels of each standard. 2.7.5. Recovery and matrix effect The extraction efficiency of costunolide, dehydrocostuslactone and IS was analyzed by the determination of QC samples of three concentration levels. Five replicates of each sample were spiked with the analytes prior to and after extraction and injected into the analytical column. Recovery of costunolide and dehydrocostuslacton was calculated using the following equation: recovery ¼peak area after extraction/peak area direct injectionn100%. The matrix effect was measured by comparing the peak area of samples spiked post-extraction with those of standard solution evaporated and reconstituted in mobile phase. The recovery and matrix effect of two components were detected at three levels of QC samples in five replicates. 2.8. Applications in pharmacokinetics studies The extract was suspended in 0.5% CMC and six rats were orally administrated the herb at the dose of 2 g/kg body weight, equal to the content of costunolide 15.7 mg/kg and dehydrocostuslactone 71.9 mg/kg. Blood samples of 100 μL were collected in heparinized eppendorf tubes via angular vein before dosing and subsequently at 0.083, 0.33, 0.67, 1.33, 2, 3, 4, 6, 8, 10, 12, 24, 36 and 48 h after oral administration. The rats were given free access to water during the experiment. Blood samples were immediately heparinized and centrifuged at 10,000 rpm for 5 min and the supernatants were placed into 1.5 mL polypropylene tubes and stored at 20 1C for the further analysis. The plasma concentrations of costunolide and dehydrocostuslactone were evaluated using the equation from the standard curves that were run with each batch of samples. The plasma concentration-time data were analyzed with the 3P97 software. The following pharmacokinetic parameters were calculated: maximum plasma concentration (Cmax) and the time to reach maximum (Tmax) were determined directly from the maximum measured data; the terminal elimination rate constant (ke) was determined by linear least-squares regression of the terminal portion of the plasma concentration–time curve, and the elimination half-life (t1/2) was calculated as 0.693/ke; the area under the plasma concentration–time curve from time 0 to the time of the last measurable concentration (AUC0-t) was calculated by the trapezoidal rule.
3. Result and discussion 3.1. Optimization of analysis conditions Chromatographic analysis of the analytes and IS was initiated under isocratic conditions aiming to develop a simple separation process with good resolution of adjacent peaks in a short run time. HPLC analytical parameters including separation column, mobile phase and its elution mode were all investigated. A simple and accurate analysis method for the determination of the two sesquiterpenes in rat serum was performed with a C18 column (Agilent, 2.1 mm 150 mm, i.d., 5 mm) and a C18 guard column (4.6 mm 7.5 mm, i.d., 5 mm). And the flow rate was 0.5 mL/min with the mobile phase composed of 0.1% formic acid and acetonitrile (1:1) and the column temperature was maintained at 351 C. No endogenous interference was observed at retention times of costunolide (10.6 min), dehydrocostuslactone (11.7 min) and IS (3.6 min) because of the high selectivity of MRM mode. Typical
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Fig. 2. Representative of A, B and C stand for the blank plasma, blank plasma spiked with the standards and the plasma sample after 1 h oral administration of RA. a: MRM spectrums of costunolide; b: MRM spectrums of dehydrocostuslactone; c: MRM spectrums of IS.
chromatograms of blank plasma, spiked plasma and subject samples were shown in Fig. 2. The analytes and IS standard solutions by direct full scan method indicated that the sensitive signals were got from electrospray ionization source in positive mode. The electrospray ionization of costunolide and dehydrocostuslactone produced the abundant protonated molecular ions [Mþ H] þ at m/z 231 and 233 which using different collision energies of 40 ev and 45 ev. 3.2. Sample preparation In order to obtain the optimism method of rat sample preparation, three methods of extraction including liquid–liquid extraction with ethyl acetate, protein precipitation with methanol or acetonitrile, and C18 SPE cartridges were used for the preparation of plasma samples. According to the results of high recovery and low endogenous matrix inference, deproteinization with C18 SPE cartridges was used in the present experiment. In order to reduce the matrix effect of the sample and improve sensitivity to the constituents, interfering compounds can be removed by deionized water. Diazepam was selected as IS because it behaves very reproducibly under the optimized extraction procedure and it completely resolved from two sesquiterpene lactones and endogenous compounds. 3.3. Method validation 3.3.1. Selectivity Typical chromatograms of drug-free rat plasma, blank plasma spiked with costunolide, dehydrocostuslactone and IS, and plasma samples obtained from 10 h after oral administration of the herb extract were displayed in Fig. 2. As shown that costunolide and dehydrocostuslactone gave retention times of 10.60 and 11.74 min, respectively, and the retention of time of IS was at 3.22 min. In the run time of 15 min, two sesquiterpene lactones and IS in biological samples got a good separation and no interference from endogenous substances were observed at the time of the analytes and IS. In order to obtain a sensitive mass response of the analytes, the transitions of the targets and the main parameters, such as cone, capillary and collision voltages, desolvation and source temperatures were investigated carefully. All MS parameters were optimized to find the most abundant and characteristic molecular and product ions. Full scan product ion spectra of sesquiterpene lactones and IS were investigated with MRM mode. Compared with the previous method for the determination of costunolide and dehydrocostuslactone in rat plasma, the present HPLC–MS/MS method added diazepam into the plasma sample as the internal standard. For costunolide, typical fragment ion of m/z 187.1 was observed and for dehydrocostuslactone, the fragment was m/z 185. The characteristic fragment ion of m/z 193.1 for IS was
selected. Their fragmentation pathways are shown in Fig. 3. No significant interference in the MRM channels at the relevant retention times was observed during the study. 3.3.2. Calibration curves and LLOQ Two sesquiterpene lactones calibration curves were constructed by plotting peak area ratio (y) of costunolide or dehydrocostuslactone to the internal standard, versus their concentration (x). Linearity was assessed by weighted (1/x) linear regression of calibration curves generated in triplicate on three consecutive days using analyte-internal standard peak area ratios. Typical equations of the calibration curves were y¼6.754x 4.568 for costunolide, and y¼0.792x 0.109 for dehydrocostuslactone. Good linearity was obtained over the concentration range from 0.7 to 769.7 ng/mL for costunolide with correlation coefficients (r2) ¼ 0.9978, and from 0.9 to 956.0 ng/mL for dehydrocostuslactone with r2 ¼0.9979. The results of LLOQ of two sesquiterpene lactones were established at 0.7 ng/ml for costunolide and 0.9 ng/ml for dehydrocostuslactone, which were sensitive enough for pharmacokinetic study after oral administration of herb extract. The results of limit of detection (LOD), defined as a signal to noise ratio of 3:1, were 0.2 and 0.3 ng/mL respectively. 3.3.3. Precision and accuracy The intra- and inter-batch precision and accuracy of the assay were assessed by analyzing QC samples which were prepared at three different concentrations 6.01, 48.11, 384.85 ng/mL for costunolide and 7.47, 59.75, 478.00 for dehydrocostuslactone. In this assay, the intra- and inter-batch precisions were less than 8.76% and 9.70%, and the accuracy was within 6.14% and 5.35% for two sesquiterpene lactones. The results, summarized in Table 1, demonstrated that the values of precision and accuracy were within the acceptable range and indicated that the method was accurate and precise. 3.3.4. Recovery and matrix effect The extraction recovery was calculated by comparing the peak areas of the plasma sample spiked with standards with those of the standard solutions. Average recovery of costunolide from rat plasma was 76.23%, 77.74% and 77.39% for low, middle and high level of QC samples, while 82.85%, 82.57% and 80.04% for dehydrocostuslactone respectively. The results of recovery and matrix detection of costunolide, dehydrocostuslactone and IS were displayed in Table 2. 3.3.5. Stability The stability of costunolide and dehydrocostuslactone in rat plasma was investigated using three concentration levels of QC samples. The stability results showed that all analytes were stable
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Fig. 3. MS spectrum of costunolide, dehydrocostuslactone and IS, the quantifier ions m/z transitions for costunolide, dehydrocostuslactone and IS were 233-187, 231-185 and 285-193, respectively.
in plasma samples after three freeze-thaw cycles, 24 h at room temperature and 30 days kept frozen at 20 C. Stability data are summarized in Table 3.
3.3.6. Pharmacokinetic studies Mean plasma concentration–time profiles of costunolide and dehydrocostuslactone after oral administration of the extract of
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Table 1 Precision and accuracy of the determination of costunolide and dehydrocostuslactone. Components
Spiked concentration (lg/mL)
Intra-day (n¼6)
Inter-day (n¼ 6)
Measured concentration (lg/mL, mean 7S.D.)
RSD (%)
RE (%)
Measured concentration (lg/mL, mean 7S.D.)
RSD (%)
RE (%)
Costunolide
6.01 48.11 384.85
5.96 47.41 375.72
7.15 5.50 3.52
0.92 1.45 2.37
5.80 47.15 365.55
8.86 6.82 7.28
3.44 2.00 5.01
Dehydrocostuslactone
7.47 59.75 478.00
7.93 59.98 470.78
5.28 8.76 6.93
6.14 0.38 1.51
7.87 60.37 467.68
4.46 9.70 8.92
5.35 1.04 2.16
Table 2 Extract recovery and matrix effect of costunolide and dehydrocostuslactone in rat plasma. Components
Spiked concentration (lg/mL)
Extract recovery (n¼ 6) RSD(%)
Matrix effect (n¼ 6) RSD(%)
Costunolide
6.01 48.11 384.85
76.23 7 3.07 77.74 7 6.79 77.39 7 3.98
92.22 7 3.78 93.20 7 2.42 92.677 4.59
Dehydrocostuslactone
7.47 59.75 478.00
82.85 7 2.79 82.57 7 2.95 80.04 7 3.13
93.147 2.15 91.34 7 3.60 91.23 7 3.60
Table 3 Stability of costunolide and dehydrocostuslactone in rat plasma. Components
Concentration added (lg/mL)
Accuracy (%, mean7 S.D.) 24 h at room temperature
Freeze-thaw cycles 3 times
30 days storage at 20 1C
Costunolide
6.01 48.11 384.85
97.84 7 6.48 97.93 7 3.49 99.89 7 5.42
99.89 7 5.00 98.45 7 4.62 100.767 7.13
92.01 7 8.78 104.36 7 9.46 98.18 7 6.99
Dehydrocostuslactone
7.47 59.75 478
101.03 7 5.41 99.85 7 3.64 100.52 7 4.42
100.09 7 5.78 98.80 7 7.32 98.007 5.67
99.06 7 4.74 99.85 7 3.64 100.52 7 4.42
Fig. 4. Time–concentration curve for costunolide (A) and dehydrocostuslactone (B) after a single dose of oral administration of RA.
Aucklandia lappa Decne. were shown in Fig. 4. The main pharmacokinetic parameters were listed in Table 4. The results showed that the concentrations of the anlytes in rat plasma were detected at least 48 h using the validated analytical method in present study. The dose of 2 g/kg herb extract which containing 15 mg costunolide and 72 mg dehydrocostuslactone was orally administrated to rats. After 5 min of oral administration, costunolide and dehydrocostuslactone
were detected in rat plasma, while the plasma concentrations of two sesquiterpene lactones within 48 h were analyzed. The time to reach peak concentration (Tmax) of costunolide and dehydrocostuslactone was the same at 12 h and peak concentration (Cmax) attained to 19.84 and to 493.00 ng/mL respectively. As the mean plasma concentration–time profiles showed that costunolide and dehydrocostuslactone had similar pharmacokinetic
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Table 4 Pharmacokinetic parameters of costunolide (Co) and dehydrocostuslactone (De) after i.g. administration of RA. Parameter
Co
De
Ke(1/h) Ka(1/h) t1/2(ka) (h) t1/2(ke) (h) Tmax(peak) (h) Cmax(ng/ml) AUC((ng/ml)nh) CL/F(s) (mg/h/(ng/ml)) V/F(c) ((mg)/(ng/ml))
0.071 0.13 5.54 9.74 10.46 19.84 308.83 0.0032 0.046
0.13 0.22 4.32 5.33 12.39 493.00 7884.51 0.032 0.019
behaviors and the pharmacokinetic parameters were obvious different to those after oral administration of the monomers in previously report. The pharmacokinetic parameters of dehydrocostuslactone displayed the significant changes that comparison to that following oral administration of the monomers the Tmax was delayed and Cmax was increased. Except that the literature survey indicated that sesquiterpene lactones were rich in costus roots (3% of fresh weight), of which costunolide and dehydrocostuslactone were the chief constituent, several other sesquiterpene lactones were separated from RA, such as β-cyclocostunolide, dihydro costunolide, dehydro costunolide and isodihydrocostunolide et al. Owing to the interaction of these chemical constituent in the herb, it may lead to the change of the pharmacokinetic behavior in vivo. Xu et al. studied on the pharmacokinetic comparisons of schizandrin after oral administration of schizandrin monomer and Fructus Schisandrae aqueous extract (Xu et al., 2008). Their report indicated that some parameters such as T1/2 and AUC displayed significant differences between the groups of the monomer and the herb extract. The similar phenomenon found in the studied of the pharmacokinetic comparisons of baicalin, pure paeoniflorin (Wu et al., 2009; Lu et al., 2007). The results indicated that pharmacokinetic behavior were differences between the monomer and the herb. Except for sesquiterpene lactones, monoterpene and triterpene also detected in the extract of RA. Sesquiterpene lactones were the main activity components and their pharmacokinetic profiles may be influenced by other constituents. 4. Conclusion A sensitive, specific and accurate method with estazolam as IS was the first time to describe for the determination of costunolide and dehydrocostuslactone in rat plasma by LC–MS/MS in positive electrospray ionization mode using MRM and fully validated according to commonly accepted criteria. In terms of high selectivity, low LLOQ and wide linear range, the method exhibited excellent performance by deproteinization with C18 SPE cartridges. The results of validation indicated that the method suited for the studied on the pharmacokinetic of two sesquiterpene lactones after oral administration of the herb extract of RA at a single dose of 2 g/kg. Different from the pharmacokinetic parameters following oral administration of the monomer, it is most important to provide the data for the reaches of the pharmacokinetic of traditional Chinese medicine. Acknowledgement The work was supported by the Natural Science Foundation of Tianjin, China (No.13JCQNJC13000).
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A new sesquiterpene lactone from the roots of Saussurea lapp: structure-anticancer activity study. Bioorg. Med. Chem. Lett. 18, 4015–4017. Shoji, N., Umeyama, A., Saits, N., Takemoto, T., 1986. Vasoactive substances from Saussurea lappa. J. Nat. Prod. 49, 1112–1113. Shum, K.C., Chen, F., Li, S.L., Wang, J., But, P.P.H., Shaw, P.C., 2007. Authentication of Radix Aucklandiae and its substitutes by GC–MS and hierarchical clustering analysis. J. Sep. Sci. 30, 3233–3239. Taniguchi, M., Kataoka, T., Suzuki, H., Uramoto, M., Ando, M., Arao, K., Magae, J., Nishimura, T., Otake, N., Nagai, K., 1995. Costunolide and dehydrocostus lactone as inhibitors of killing function of cytotoxic T lymphocytes. Biosci. Biotechnol. Biochem. 59, 2064–2067. Wu, H., Zhu, Z.Y., Zhang, G.Q., Zhao, L., Zhang, H, Zhu, D.L., Chai, Y.F., 2009. Comparative pharmacokinetic study of paeoniflorin after oral administration of pure paeoniflorin, extract of Cortex Moutan and Shuang-Dan prescription to rats. J. Ethnopharmacol. 125, 444–449. Wang, Y.B., Xu, H., Zhang, Y.F., 2000. Study on quality evaluation of muxiang: HPLC determination of two kinds of sesquiterpene lactone content. Chin. J. Pharm. Anal. 6, 316–317. Wang, L., Zhao, F., He, E.Q., Wang, S., Xu, H., Liu, K., 2008. Effects of eighteen sesquiterpenes from Saussurea lappa on the proliferation of six human cancer cell lines. Nat. Prod. Res. Dev. 20, 8082–8812. Xu, M.J., Wang, G.J., Xie, H.T., Huang, Q., Wang, W., Jia, Y.W., 2008. Pharmacokinetic comparisons of schizandrin after oral administration of schizandrin monomer, fructus Schisandrae aqueous extract and Sheng-Mai-San to rats. J. Ethnopharmacol. 115, 483–488. Yamahara, J., Kobayashi, M., Miki, K., Kozuka, M., Sawada, T., Fujimura, H., 1985. Cholagogic and antiulcer effect of Saussurea radix and its components. Chem. Pharm. Bull. 33, 1285–1288. Yang, Li, Chen, Xu, Qiang, Zhang, Jun, Yan, Ren, Liu S, Xiang, Tan, 2005. In vitro antiHelicobacter pylori action of 30 Chinese herbal medicines used to treat ulcer diseases. J. Ethnopharmacol. 98, 329–333. Yu, H.H., Lee, J.S., Lee, K.H., Kim, K.Y., You, Y.O., 2007. Saussurea lappa inhibits the growth, acid production, adhesion, and water-insoluble glucan synthesis of streptococcus mutans. J. Ethnopharmacol. 111, 413–417.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Pharmacokinetic study on costunolide and dehydrocostuslactone after oral administration of traditional medicine Aucklandia lappa Decne. by LC/MS/MS Jingze Zhang a,1, Xiao Hu b,1, Wenyuan Gao c,n, Zhuo Qu c, Huimin Guo c, Zhen Liu c, Changxiao Liu d a
Department of Pharmacy, Logistics College of Chinese People's Armed Police Forces, Tianjin 300162, China Tianjin Key Laboratory for Prevention and Control of Occupational and Environmental Hazard. Logistics College of Chinese People’s Armed Police Forces, Tianjin 300162, China c School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China d The State Key Laboratories of Pharmacodynamics and Pharmacokinetics, Tianjin 300193, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 30 May 2013 Received in revised form 30 July 2013 Accepted 7 October 2013 Available online 8 November 2013
Ethnopharmacological relevance: Radix Aucklandiae (RA), a well known traditional Chinese medicine, is widely used for treating various problems in digestive system. A selective and sensitive highperformance liquid chromatography coupled with mass spectrometry method was first developed and validated for simultaneous quantification of costunolide and dehydrocostuslactone in rat plasma with diazepam as internal standard after oral administration of RA extraction. Materials and methods: Plasma samples were extracted via solid-phase extraction and detected by multiple-reaction monitoring mode under positive electrospray. Chromatographic separation was accomplished on an Agilent C18 column (2.1 mm 150 mm, 5 mm), with 0.1% formic acid and acetonitrile (1:1) as the mobile phase at a flow rate of 0.5 mL/min. Results: The quantification was performed using the transitions of m/z 233/187 for costunolide, m/z 231/ 185 for dehydrocostuslactone and m/z 285/193 for diazepam, respectively. Calibration curves were linear over the concentration range of 0.7–769.7 ng/mL for costunolide and 0.9–956.0 ng/mL for dehydrocostuslactone. The intra-day and inter-day precisions (RSD%) for two compounds was less than 8.76% and 9.70% and the accuracy (RE%) range from 6.14% to 5.35%. The time to reach the maximum plasma concentration (Tmax) was 10.46 h for costunolide, 12.39 h dehydrocostuslactone. The elimination halftime (t1/2) of costunolide and dehydrocostuslactone was 5.547 0.81 and 4.32 70.71 (h). The AUC of costunolide and dehydrocostuslactone was 308.83 and 7884.51 respectively (ng h/mL). Conclusions: It was the first report for the study of pharmacokinetic profile of costunolide and dehydrocostuslactone in rat plasma after oral administration of RA extract. These results provided a meaningful basis for better understanding the absorption of traditional medicine, RA, and provide useful scientific data for clinical application. & 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sesquiterpene lactones Radix Aucklandiae Pharmacokinetic HPLC–MS
1. Introduction Radix Aucklandiae (RA), known as Muxiang in Chinese, is derived from the dried root of Aucklandia lappa Dence. belonging to the family Asteraceae. It is one of the commonly used traditional Chinese medicines which listed in Chinese Phaemacopoeia (Committee of National Pharmacopoeia, 2010). Costunolide (1) and dehydrocostuslactone (2) with a guaiane skeleton belonging to sesquiterpene lactones are the major active constituents in n
Corresponding author. Tel./fax: þ 86 22 87401895. E-mail addresses: [email protected], [email protected] (W. Gao). 1 These two authors contributed equally to this work.
0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.10.024
RA (Li et al., 2005). In Chinese Phaemacopoeia, as the makers to control herb quality, the total content of costunolide and dehydrocostuslactone was no less than 1.8%. In recent years, researches on RA have been mainly focused on the chemical constituent (Chhabra et al., 1998; Hou et al., 1998) and pharmacology (Pandey et al., 2007; Parekh and Chanda, 2007; Yu et al., 2007). However, there is no literature available for the pharmacokinetic of main constituents in RA. In previous study of the chemical constituents, Shum et al. (2007) reported that ninety chemicals can be detected in the essential oil of Aucklandia lappa and among them 28 were unique to RA. Isodihydrocostunolide, a new sesquiterpene lactone from the roots of Saussurea lappa, exhibited good activity on cancer cell lines and IC50 values (Robinson et al., 2008). The
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effects of eighteen sesquiterpenes isolated from Saussurea lappa on the proliferation of six human cancer cell lines were evaluated by MTT assay (Wang et al., 2008). RA has been widely used for the treatment of various problems in digestive system, including loss of appetite, indigestion, diarrhea, and abdominal pain (Chang and But, 1986). In addition, it possesses activities of antispasmodic (Shoji et al., 1986) antiinflammatory (Damre et al., 2003; Choi et al., 2012), anticancer (Yang et al., 2005), anti-ulcer (Taniguchi, et al., 1995; Yamahara, et al., 1985) and cholagogic (Mitra et al., 1996). Several analytical techniques have been published for the analysis of costunolide and dehydrocostuslactone in the herb by high performance liquid chromatography (HPLC) (Wang et al., 2000), gas chromatography with mass detection (GC–MS) (Qiu. et al., 2001). Hu et al. established a sensitive UPLC-MS/MS for the quantification of costunolide and dehydrocostuslactone in rat plasma after oral administration of the mixture of two monomers (Hu et al., 2011). However, no study focused on the pharmacokinetic profile of costunolide and dehydrocostuslactone after administration of the herb extract of RA. Prior published reports showed the pharmacokinetic parameters were significant differently after oral administration of one active component, herb or its prescription. The different pharmacokinetic parameters of paeoniflorin were obtained after oral administration of Cortex Moutan or Shuang-Dan decoction (Wu et al., 2009), while the significant changes were obviously observed following administration of pure baicalin, Radix scutellariae extract and Huang-Lian-Jie-Du-Tang (Lu et al., 2007). Therefore comparing with the pharmacokinetic parameters of the pure monomer, those may change after oral administration of the herb. In present study, the pharmacokinetic parameters were studied by a simple, rapid and sensitive HPLC–MS/MS method for quantification of costunolide and dehydrocostuslactone in the rat plasma after oral administration of the herb extract of RA. The preliminary pharmacokinetic behavior of two sesquiterpene lactones in rat plasma after oral administration of the dried root of Aucklandia lappa Dence. was firstly elucidated.
2. Materials and methods 2.1. Chemicals HPLC grade acetonitrile was purchased from Fisher (USA). Water was purified by a Milli-Q water purification system (Millipore, USA). Methanol of analytical grade was purchased from Guangfu Technology Limited Company (Tianjin, China).
Standards including costunolide, dehydrocostuslactone and diazepam (internal standard, IS) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All the reference compounds have over 98% purity. Aucklandia lappa Decne. was provided by Tianjin Zhongxin Pharmaceutical Group Co., Ltd. (Tianjin, China) and identified by Professor Wenyuan Gao from School of Pharmaceutical Science and Technology, Tianjin University, China. The voucher specimens (Voucher no. MX100108) are available in the herbarium of Research Center of Tianjin Zhongxin Pharmaceuticals. 2.2. Instrumentation The LC-MS/MS system consisted of an Agilent 1200 mod. liquid chromatography system (Agilent, MA, USA) equipped with a binary solvent delivery system, an autosampler, a column compartment and a 6410 triple quadrupole mass spectrometer with electrospray ionization (ESI) source. Data were analyzed by MassHunter software (Agilent Corporation, MA, USA). The analytical column was a Agilent C18 (150 mm 4.6 mm, 5 μm). A bionary mobile phase system consisted of (A) acetonitrile and (B) 0.1% formic acid (1:1, v/v) at a flow rate of 0.5 mL/min. 2.3. Preparation of the extracts In the present study, twenty grams of Aucklandia lappa Decne. were powdered and extracted with 200 mL methanol for 2 h in a reflux condenser. The filtrate was collected and the residue was reextracted with 200 mL methanol. Then the solvent was removed under reduced pressure in a rotary evaporator. Costunolide and dehydrocostuslactone (the structures given in Fig. 1) were main components in Aucklandia lappa Decne. and the contents of two constituents in the extract checked by HPLC methods (Wu et al., 2009) were 0.79% and 3.59% respectively. 2.4. Animals Healthy male Wistar rats, weighing about 250–280 g were used in the experiments. The animals were purchased from the Experimental Animal Center, Chinese Academy of Medical Sciences, Peking (SCXK-2007-004). The animals were housed in an environmentally (t¼ 25 1C) and air humidity (60%) controlled room with a 12 h light-dark (07:00–19:00 h and 19:00–07:00 h) cycle, kept on a standard laboratory diet and drinking water ad libitum. This study was carried out in accordance with the Regulation for the Administration of Affairs Concerning Experimental Animals (State Council of China, 1988).
Fig. 1. Chemial structures of costunolide, dehydrodehydrocostus and diazepam (IS).
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2.5. Rat plasma sample preparation A 100 μL aliquot of rat plasma sample and 10 μL of diazepam (internal standard, 100 ng/mL) were added to a 1.5 mL heparinized eppendorf tube. After vortexing for 3 min, the sample was centrifuged at 10,000 rpm for 5 min and then the supernatant was loaded onto the preconditioned SPE (i.e., preconditioned with 1 mL methanol followed by 2 mL purified water). The cartridge was rinsed twice with 1 mL of water and the constituents were eluted with 1 mL methanol. The methanol fraction was dried with nitrogen at 45 1C and the dry residue was reconstituted with 50 μL of methanol. After centrifugation at 10,000 rpm for 5 min, 10 μL of the sample was directly injected into the HPLC–MS system for analysis. 2.6. Preparation of standard solution and quality control samples The linearity was determined by analysis of standard solutions at nine different concentrations. Known amounts of costunolide, dehydrocostuslactone and IS were added into 100 mL of blank plasma to prepare the following series of standards. The standard sets of costunolide had good linearity with its own linearity range at 0.70, 6.01, 12.03, 24.05, 48.11, 96.21, 192.42, 384.85 and 769.70 ng/mL. And the standard sets of dehydrocostuslactone ranged at 0.96, 7.47, 14.94, 29.88, 59.75, 119.50, 239.00, 478.00 and 956.00 ng/mL. The quality control (QC) samples were prepared at three different concentrations 6.01, 48.11, 384.85 ng/mL for costunolide and 7.47, 59.75, 478.00 ng/mL for dehydrocostuslactone. 2.7. Method validation Validation was performed by establishing the within-batch and between-batch accuracy, precision, recovery and stability of the method on quality control (QC) samples. 2.7.1. Selectivity Spiked blank rat plasma with costunolide, dehydrocostuslactone and IS, blank plasma sample and the mixed standard (costunolide, dehydrocostuslactone and IS) were tested. Chromatographic peaks of the analytes were identified based on their retention times and MRM responses. 2.7.2. Linearity, limit of detection (LOD) and limit of quantification (LLOQ) Calibration curves were constructed by the analysis of plasma samples spiked with nine different concentrations of standards. The LOD was determined using the signal-to-noise ratio (S/N) of 3:1 by comparing test results from samples with known concentrations of analyte with blank samples. The LLOQ was defined as the lowest concentration point of the calibration curve at which can be detected with acceptable precision and accuracy. 2.7.3. Precision and accuracy The intra-day and inter-day precisions were determined by the assay of standard solutions at three concentrations on a single day and three continuous days. Five determinations of each concentration level of costunolide and dehydrocostuslactone were analyzed and precision and accuracy were expressed as relative standard deviation (RSD%) and deviation from theoretical value. 2.7.4. Stability The stability of costunolide and dehydrocostuslactone in rat plasma was tested by analysis of freeze-thawing three times and stored in room temperature for 24 h. Spiked plasma samples were
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repeated in triplicate (n ¼3) at three concentration levels of each standard. 2.7.5. Recovery and matrix effect The extraction efficiency of costunolide, dehydrocostuslactone and IS was analyzed by the determination of QC samples of three concentration levels. Five replicates of each sample were spiked with the analytes prior to and after extraction and injected into the analytical column. Recovery of costunolide and dehydrocostuslacton was calculated using the following equation: recovery ¼peak area after extraction/peak area direct injectionn100%. The matrix effect was measured by comparing the peak area of samples spiked post-extraction with those of standard solution evaporated and reconstituted in mobile phase. The recovery and matrix effect of two components were detected at three levels of QC samples in five replicates. 2.8. Applications in pharmacokinetics studies The extract was suspended in 0.5% CMC and six rats were orally administrated the herb at the dose of 2 g/kg body weight, equal to the content of costunolide 15.7 mg/kg and dehydrocostuslactone 71.9 mg/kg. Blood samples of 100 μL were collected in heparinized eppendorf tubes via angular vein before dosing and subsequently at 0.083, 0.33, 0.67, 1.33, 2, 3, 4, 6, 8, 10, 12, 24, 36 and 48 h after oral administration. The rats were given free access to water during the experiment. Blood samples were immediately heparinized and centrifuged at 10,000 rpm for 5 min and the supernatants were placed into 1.5 mL polypropylene tubes and stored at 20 1C for the further analysis. The plasma concentrations of costunolide and dehydrocostuslactone were evaluated using the equation from the standard curves that were run with each batch of samples. The plasma concentration-time data were analyzed with the 3P97 software. The following pharmacokinetic parameters were calculated: maximum plasma concentration (Cmax) and the time to reach maximum (Tmax) were determined directly from the maximum measured data; the terminal elimination rate constant (ke) was determined by linear least-squares regression of the terminal portion of the plasma concentration–time curve, and the elimination half-life (t1/2) was calculated as 0.693/ke; the area under the plasma concentration–time curve from time 0 to the time of the last measurable concentration (AUC0-t) was calculated by the trapezoidal rule.
3. Result and discussion 3.1. Optimization of analysis conditions Chromatographic analysis of the analytes and IS was initiated under isocratic conditions aiming to develop a simple separation process with good resolution of adjacent peaks in a short run time. HPLC analytical parameters including separation column, mobile phase and its elution mode were all investigated. A simple and accurate analysis method for the determination of the two sesquiterpenes in rat serum was performed with a C18 column (Agilent, 2.1 mm 150 mm, i.d., 5 mm) and a C18 guard column (4.6 mm 7.5 mm, i.d., 5 mm). And the flow rate was 0.5 mL/min with the mobile phase composed of 0.1% formic acid and acetonitrile (1:1) and the column temperature was maintained at 351 C. No endogenous interference was observed at retention times of costunolide (10.6 min), dehydrocostuslactone (11.7 min) and IS (3.6 min) because of the high selectivity of MRM mode. Typical
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Fig. 2. Representative of A, B and C stand for the blank plasma, blank plasma spiked with the standards and the plasma sample after 1 h oral administration of RA. a: MRM spectrums of costunolide; b: MRM spectrums of dehydrocostuslactone; c: MRM spectrums of IS.
chromatograms of blank plasma, spiked plasma and subject samples were shown in Fig. 2. The analytes and IS standard solutions by direct full scan method indicated that the sensitive signals were got from electrospray ionization source in positive mode. The electrospray ionization of costunolide and dehydrocostuslactone produced the abundant protonated molecular ions [Mþ H] þ at m/z 231 and 233 which using different collision energies of 40 ev and 45 ev. 3.2. Sample preparation In order to obtain the optimism method of rat sample preparation, three methods of extraction including liquid–liquid extraction with ethyl acetate, protein precipitation with methanol or acetonitrile, and C18 SPE cartridges were used for the preparation of plasma samples. According to the results of high recovery and low endogenous matrix inference, deproteinization with C18 SPE cartridges was used in the present experiment. In order to reduce the matrix effect of the sample and improve sensitivity to the constituents, interfering compounds can be removed by deionized water. Diazepam was selected as IS because it behaves very reproducibly under the optimized extraction procedure and it completely resolved from two sesquiterpene lactones and endogenous compounds. 3.3. Method validation 3.3.1. Selectivity Typical chromatograms of drug-free rat plasma, blank plasma spiked with costunolide, dehydrocostuslactone and IS, and plasma samples obtained from 10 h after oral administration of the herb extract were displayed in Fig. 2. As shown that costunolide and dehydrocostuslactone gave retention times of 10.60 and 11.74 min, respectively, and the retention of time of IS was at 3.22 min. In the run time of 15 min, two sesquiterpene lactones and IS in biological samples got a good separation and no interference from endogenous substances were observed at the time of the analytes and IS. In order to obtain a sensitive mass response of the analytes, the transitions of the targets and the main parameters, such as cone, capillary and collision voltages, desolvation and source temperatures were investigated carefully. All MS parameters were optimized to find the most abundant and characteristic molecular and product ions. Full scan product ion spectra of sesquiterpene lactones and IS were investigated with MRM mode. Compared with the previous method for the determination of costunolide and dehydrocostuslactone in rat plasma, the present HPLC–MS/MS method added diazepam into the plasma sample as the internal standard. For costunolide, typical fragment ion of m/z 187.1 was observed and for dehydrocostuslactone, the fragment was m/z 185. The characteristic fragment ion of m/z 193.1 for IS was
selected. Their fragmentation pathways are shown in Fig. 3. No significant interference in the MRM channels at the relevant retention times was observed during the study. 3.3.2. Calibration curves and LLOQ Two sesquiterpene lactones calibration curves were constructed by plotting peak area ratio (y) of costunolide or dehydrocostuslactone to the internal standard, versus their concentration (x). Linearity was assessed by weighted (1/x) linear regression of calibration curves generated in triplicate on three consecutive days using analyte-internal standard peak area ratios. Typical equations of the calibration curves were y¼6.754x 4.568 for costunolide, and y¼0.792x 0.109 for dehydrocostuslactone. Good linearity was obtained over the concentration range from 0.7 to 769.7 ng/mL for costunolide with correlation coefficients (r2) ¼ 0.9978, and from 0.9 to 956.0 ng/mL for dehydrocostuslactone with r2 ¼0.9979. The results of LLOQ of two sesquiterpene lactones were established at 0.7 ng/ml for costunolide and 0.9 ng/ml for dehydrocostuslactone, which were sensitive enough for pharmacokinetic study after oral administration of herb extract. The results of limit of detection (LOD), defined as a signal to noise ratio of 3:1, were 0.2 and 0.3 ng/mL respectively. 3.3.3. Precision and accuracy The intra- and inter-batch precision and accuracy of the assay were assessed by analyzing QC samples which were prepared at three different concentrations 6.01, 48.11, 384.85 ng/mL for costunolide and 7.47, 59.75, 478.00 for dehydrocostuslactone. In this assay, the intra- and inter-batch precisions were less than 8.76% and 9.70%, and the accuracy was within 6.14% and 5.35% for two sesquiterpene lactones. The results, summarized in Table 1, demonstrated that the values of precision and accuracy were within the acceptable range and indicated that the method was accurate and precise. 3.3.4. Recovery and matrix effect The extraction recovery was calculated by comparing the peak areas of the plasma sample spiked with standards with those of the standard solutions. Average recovery of costunolide from rat plasma was 76.23%, 77.74% and 77.39% for low, middle and high level of QC samples, while 82.85%, 82.57% and 80.04% for dehydrocostuslactone respectively. The results of recovery and matrix detection of costunolide, dehydrocostuslactone and IS were displayed in Table 2. 3.3.5. Stability The stability of costunolide and dehydrocostuslactone in rat plasma was investigated using three concentration levels of QC samples. The stability results showed that all analytes were stable
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Fig. 3. MS spectrum of costunolide, dehydrocostuslactone and IS, the quantifier ions m/z transitions for costunolide, dehydrocostuslactone and IS were 233-187, 231-185 and 285-193, respectively.
in plasma samples after three freeze-thaw cycles, 24 h at room temperature and 30 days kept frozen at 20 C. Stability data are summarized in Table 3.
3.3.6. Pharmacokinetic studies Mean plasma concentration–time profiles of costunolide and dehydrocostuslactone after oral administration of the extract of
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Table 1 Precision and accuracy of the determination of costunolide and dehydrocostuslactone. Components
Spiked concentration (lg/mL)
Intra-day (n¼6)
Inter-day (n¼ 6)
Measured concentration (lg/mL, mean 7S.D.)
RSD (%)
RE (%)
Measured concentration (lg/mL, mean 7S.D.)
RSD (%)
RE (%)
Costunolide
6.01 48.11 384.85
5.96 47.41 375.72
7.15 5.50 3.52
0.92 1.45 2.37
5.80 47.15 365.55
8.86 6.82 7.28
3.44 2.00 5.01
Dehydrocostuslactone
7.47 59.75 478.00
7.93 59.98 470.78
5.28 8.76 6.93
6.14 0.38 1.51
7.87 60.37 467.68
4.46 9.70 8.92
5.35 1.04 2.16
Table 2 Extract recovery and matrix effect of costunolide and dehydrocostuslactone in rat plasma. Components
Spiked concentration (lg/mL)
Extract recovery (n¼ 6) RSD(%)
Matrix effect (n¼ 6) RSD(%)
Costunolide
6.01 48.11 384.85
76.23 7 3.07 77.74 7 6.79 77.39 7 3.98
92.22 7 3.78 93.20 7 2.42 92.677 4.59
Dehydrocostuslactone
7.47 59.75 478.00
82.85 7 2.79 82.57 7 2.95 80.04 7 3.13
93.147 2.15 91.34 7 3.60 91.23 7 3.60
Table 3 Stability of costunolide and dehydrocostuslactone in rat plasma. Components
Concentration added (lg/mL)
Accuracy (%, mean7 S.D.) 24 h at room temperature
Freeze-thaw cycles 3 times
30 days storage at 20 1C
Costunolide
6.01 48.11 384.85
97.84 7 6.48 97.93 7 3.49 99.89 7 5.42
99.89 7 5.00 98.45 7 4.62 100.767 7.13
92.01 7 8.78 104.36 7 9.46 98.18 7 6.99
Dehydrocostuslactone
7.47 59.75 478
101.03 7 5.41 99.85 7 3.64 100.52 7 4.42
100.09 7 5.78 98.80 7 7.32 98.007 5.67
99.06 7 4.74 99.85 7 3.64 100.52 7 4.42
Fig. 4. Time–concentration curve for costunolide (A) and dehydrocostuslactone (B) after a single dose of oral administration of RA.
Aucklandia lappa Decne. were shown in Fig. 4. The main pharmacokinetic parameters were listed in Table 4. The results showed that the concentrations of the anlytes in rat plasma were detected at least 48 h using the validated analytical method in present study. The dose of 2 g/kg herb extract which containing 15 mg costunolide and 72 mg dehydrocostuslactone was orally administrated to rats. After 5 min of oral administration, costunolide and dehydrocostuslactone
were detected in rat plasma, while the plasma concentrations of two sesquiterpene lactones within 48 h were analyzed. The time to reach peak concentration (Tmax) of costunolide and dehydrocostuslactone was the same at 12 h and peak concentration (Cmax) attained to 19.84 and to 493.00 ng/mL respectively. As the mean plasma concentration–time profiles showed that costunolide and dehydrocostuslactone had similar pharmacokinetic
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Table 4 Pharmacokinetic parameters of costunolide (Co) and dehydrocostuslactone (De) after i.g. administration of RA. Parameter
Co
De
Ke(1/h) Ka(1/h) t1/2(ka) (h) t1/2(ke) (h) Tmax(peak) (h) Cmax(ng/ml) AUC((ng/ml)nh) CL/F(s) (mg/h/(ng/ml)) V/F(c) ((mg)/(ng/ml))
0.071 0.13 5.54 9.74 10.46 19.84 308.83 0.0032 0.046
0.13 0.22 4.32 5.33 12.39 493.00 7884.51 0.032 0.019
behaviors and the pharmacokinetic parameters were obvious different to those after oral administration of the monomers in previously report. The pharmacokinetic parameters of dehydrocostuslactone displayed the significant changes that comparison to that following oral administration of the monomers the Tmax was delayed and Cmax was increased. Except that the literature survey indicated that sesquiterpene lactones were rich in costus roots (3% of fresh weight), of which costunolide and dehydrocostuslactone were the chief constituent, several other sesquiterpene lactones were separated from RA, such as β-cyclocostunolide, dihydro costunolide, dehydro costunolide and isodihydrocostunolide et al. Owing to the interaction of these chemical constituent in the herb, it may lead to the change of the pharmacokinetic behavior in vivo. Xu et al. studied on the pharmacokinetic comparisons of schizandrin after oral administration of schizandrin monomer and Fructus Schisandrae aqueous extract (Xu et al., 2008). Their report indicated that some parameters such as T1/2 and AUC displayed significant differences between the groups of the monomer and the herb extract. The similar phenomenon found in the studied of the pharmacokinetic comparisons of baicalin, pure paeoniflorin (Wu et al., 2009; Lu et al., 2007). The results indicated that pharmacokinetic behavior were differences between the monomer and the herb. Except for sesquiterpene lactones, monoterpene and triterpene also detected in the extract of RA. Sesquiterpene lactones were the main activity components and their pharmacokinetic profiles may be influenced by other constituents. 4. Conclusion A sensitive, specific and accurate method with estazolam as IS was the first time to describe for the determination of costunolide and dehydrocostuslactone in rat plasma by LC–MS/MS in positive electrospray ionization mode using MRM and fully validated according to commonly accepted criteria. In terms of high selectivity, low LLOQ and wide linear range, the method exhibited excellent performance by deproteinization with C18 SPE cartridges. The results of validation indicated that the method suited for the studied on the pharmacokinetic of two sesquiterpene lactones after oral administration of the herb extract of RA at a single dose of 2 g/kg. Different from the pharmacokinetic parameters following oral administration of the monomer, it is most important to provide the data for the reaches of the pharmacokinetic of traditional Chinese medicine. Acknowledgement The work was supported by the Natural Science Foundation of Tianjin, China (No.13JCQNJC13000).
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