Journal of Pharmaceutical and Biomedical Analysis 89 (2014) 150–157
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Pharmacokinetic study of cinnamaldehyde in rats by GC–MS after oral and intravenous administration Hang Zhao a,d,1 , Yanhua Xie a,d,1 , Qian Yang a,d,1 , Yu Cao b,d , Honghai Tu c , Wei Cao a,d,∗ , Siwang Wang a,d,∗ a
Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China Team NO. 1, Cadet Brigade, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China c Institute for Drug and Instrument Control of Xinjiang Military Region, Urumqi 830063, China d The Cultivation Project of Collaborative Innovation Center for Chinese Medicine in QinBa Mountains, Xi’an 710032, China b
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 17 October 2013 Accepted 27 October 2013 Available online 9 November 2013 Keywords: Cinnamaldehyde Methyl cinnamate Pharmacokinetic GC–MS Bioavailability
a b s t r a c t A selective and sensitive method utilizing gas chromatography–mass spectrometry was developed for simultaneous determination of cinnamaldehyde, cinnamyl alcohol, and methyl cinnamate in rat plasma. Cinnamaldehyde and cinnamyl alcohol can inter-convert to one another in rats, thus simultaneous quantifying both analytes provided a reliable and accurate method of assessment. Three qualifying ions (131 m/z, 105 m/z and 92 m/z) were chosen for simultaneous quantification of cinnamaldehyde and its metabolites. In this study, the calibration curves demonstrated a good linearity and reproducibility over the range of 20–2000 ng/ml (r2 ≥ 0.999) for all analytes. Furthermore, the sensitivity of gas chromatography–mass spectrometry revealed sufficient lower limit of quantitation and detection of 20 ng/ml and 5 ng/ml, respectively, in the pharmacokinetic analysis. The intra- and inter-day precision variations were less than 10.4% and 12.2%, respectively, whilst accuracy values ranged from −8.6% to 14.8%. All analytes were stable in plasma and in processed samples at room temperature for 24 h with no significant degradation after three freeze/thaw cycles. A small amount of the administered cinnamaldehyde had long half-life of 6.7 ± 1.5 h. In this study, gas chromatography–mass spectrometry was demonstrated to be a powerful tool for the pharmacokinetic studies of rats after intravenous and oral administration of cinnamaldehyde. © 2013 Elsevier B.V. All rights reserved.
1. Introduction For centuries, Cortex Cinnamomi has been widely used as a herbal medicine and spice in numerous countries [1,2]. Its major active component is cinnamaldehyde (CA) (Fig. 1). CA was isolated from cinnamon essential oils in the nineteenth century, and was mainly used to impart a cinnamon fragrance to medical products, cosmetics, and perfumes [3]. In recent years, CA has been widely used as a flavoring agent with maximum permitted concentrations from 2000 ppm in baby food and desserts to 6400 ppm in fruits and juices [4]. The consumption of CA as a flavoring agent in the United States is estimated to be 500,000 kg/year [5]. Due to its massive use and potential toxic effects, much attention has been placed on CA. Numerous studies indicated that CA exerted a variety of bioactive and pharmacological effects such as anti-viral
∗ Corresponding authors. Tel.: +86 29 84774748; fax: +86 29 84773752. E-mail addresses: [email protected] (W. Cao), [email protected] (S. Wang). 1 These authors contributed equally to this work. 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.10.044
[6], anti-inflammatory [7,8], anti-thrombic [9,10], anti-diabetic [11,12], and anti-cancer effects [13,14]. Recently, we also found that CA had obvious therapeutic effects on viral myocarditis though suppressing the expression of Toll like receptor 4 [15]. The metabolites of CA have also demonstrated similar pharmacological effects [16,17]; therefore, determining the pharmacokinetic data of major active substance and its metabolites is a next logical step. Several researchers have attempted to analyze CA by liquid chromatography (LC) in plasma samples. They found that CA was quickly absorbed and rapidly oxidized to cinnamic acid after oral administration, only a small amount of CA remained in the blood for 24 h [18,19]. The previous methods for assessing CA levels (generally around 1000 ng/ml) are insufficient and unreliable [18]. The concentration of main metabolites such as cinnamic acid in blood or hippuric acid in urine was quantified to predict the concentration of CA [18,20,21]. Providing precise concentration data of CA with the highly sensitive method of gas chromatography–mass spectrometry (GC–MS) is more essential than describing the known data of cinnamic acid with LC. Our previous study indicated that CA had higher pharmacological activity than cinnamic acid [6,15]. Therefore, a sensitive and accurate method is important to be developed
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(SIM) mode. Three qualifying ions (131 m/z, 105 m/z and 92 m/z) were selected for simultaneous investigating CA, methyl cinnamate, and cinnamyl alcohol. The obtained spectra were analyzed by the NIST library (Shimadzu, Kyoto, Japan) and are shown in Fig. 2. 2.3. Preparation of stock solutions
Fig. 1. The molecular structures of propiophenone (I), cinnamaldehyde (II), methyl cinamate (III), and cinnamyl alcohol (IV).
Stock solutions of CA, methyl cinnamate, cinnamyl alcohol, and propiophenone (IS) were prepared in acetonitrile at a concentration of 100 g/ml; these solutions were diluted at a concentration range of 0.3–30 g/ml which was used as the working solutions in the linearity experiment. The internal standard solution was obtained by diluting the IS stock solution to a concentration of 2500 ng/ml. The stock solutions were kept away from light at −20 ◦ C and used within two months. 2.4. Sample preparation
to investigate the accurate pharmacokinetic data of low concentrations of CA. LC was firstly used to analyze CA in traditional Chinese preparations [22,23]. Later, gas chromatography (GC) coupled with flame ionization (FID) was applied to determine CA in similar traditional preparations [24,25]. However, the pharmacokinetic data of CA were limited, due to the fact that the limit of detection of analytes using LC and GC methods was not sensitive enough for pharmacokinetic studies. Recently, the use of LC [26,27] and GC [28–30] combined with an MS detector have become new methods for identification and quantitation of CA at relatively low concentrations. Unfortunately, these data focused on the CA in the essential oil, but provided no information on the pharmacokinetic study of CA and its metabolites in rat plasma. In addition, CA is a reactive aldehyde and can be easily converted to cinnamyl alcohol [3]; thus quantifying both CA and cinnamyl alcohol is essential. GC–MS has been developed and demonstrated to be a sensitive method for investigating the pharmacokinetics of CA. The pharmacokinetic parameters after oral and intravenous (IV) administration were investigated in order to provide comparative analysis of two kinds of metabolic pathways and the data of bioavailability. 2. Materials and methods 2.1. Chemicals and materials CA (99%, purity) was supplied by Yuan Cheng Pharmaceutical Co., Ltd. Propiophenone (98%, purity), methyl cinnamate (98%, purity), and cinnamyl alcohol (98%, purity) were purchased from the Adamas Reagent Co., Ltd. (Shanghai, China). Their chemical structures are shown in Fig. 1. Propiophenone was used as the internal standard (IS). Acetonitrile (HPLC grade) was obtained from Honeywell (Muskegon, MI, USA). All other reagents were of analytical grade. 2.2. GC–MS conditions and instrumentation The analysis was tested on an ISQ Trace Ultra (Thermo Fisher Scientific, Waltham, MA) system combined with a Triplus atuosampler and injector. A DB-5ms capillary column (30 m × 0.25 mm, 0.25 m thickness; Agilent Technologies, USA) was used. The initial oven temperature was 50 ◦ C for 1 min, and the temperature was subsequently increased to 160 ◦ C at 10 ◦ C/min for 1 min and then increased to 280 ◦ C at 20 ◦ C/min for 1 min. Helium was used as carrier gas at a constant flow rate of 1.0 ml/min. The transfer line and ion source temperatures were both 250 ◦ C. Ionization was carried out in electron impact ionization (EI) mode at 70 eV. Detection was operated under the selected ion monitoring
A selective and sensitive sample preparation method was utilized to eliminate the interference of endogenous proteins and to optimize extraction recovery. From among several chemical reagents (methanol, acetonitrile, and formaldehyde) tested in the process acetonitrile showed highest sensitivity and convenience, especially in minimizing endogenous interference and enhancing extraction recovery. Consequently, we choose acetonitrile as the optimal solvent for sample preparation. To each 200 l plasma sample, 50 l internal standard solution (2500 ng/ml) and 500 l acetonitrile were added. Then, the solution was thoroughly vortex-mixed for 120 s. After centrifugation at 12,000g for 10 min, 1 l of the supernatant was injected into the GC–MS for analysis. Feces samples were dried and then powdered using a mortar and pestle. Physiological saline solution (1:2, w/v) was added and homogenized with the pulverized feces. Urine samples were diluted using the physiological saline solution (1:5, w/v) and then processed similarly to the plasma samples. 2.5. Pharmacokinetic and excretion study All animal protocols were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. Male Sprague-Dawley rats, weighing 225–275 g, were supplied from the Experimental Animal Research Center, the Fourth Military Medical University (Xi‘an, China). Three groups of rats (n = 5, each group) received a single oral dose of 500 mg/kg, 250 mg/kg, or 125 mg/kg CA (diluted in corn oil). Blood was collected at 10, 30, 60, 120, 180, 240, 360, 480, 720, 1080, and 1440 min post-administration. In one group of rats (n = 5, each group), blood was collected at 2, 5, 10, 15, 30, 60, 90, 120, and 180 min after IV administration of 20 mg/kg CA. These time plots were selected on the basis of previous studies of CA. The blood samples were processed similarly to the blank sample. The group of rats (n = 5, each group) used for the urinary and fecal excretion study received a single oral dose of 500 mg/kg CA. The rats were kept in stainless-steel metabolic cages with free access to water and food. Urine and feces were collected at 0–4, 4–8, 8–12, 12-18, and 18–24 h post-dosing. The feces were dried at room temperature. All specimens were processed as described previously. 2.6. Method validation 2.6.1. Specificity and sensitivity Five blank plasma samples obtained from individual rats were analyzed to exclude the possibility of interfering peaks at the
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Fig. 2. Full scan of mass spectra of propiophenone (A), cinnamaldehyde (B), methyl cinamate (C), and cinnamyl alcohol (D).
retention times of CA, its metabolites, and IS. The lower limit of detection (LLOD) and the lower limit of quantitation (LLOQ) were determined as the concentration of target compounds which provided a signal to noise (S/N) ratio with 3:1 and 10:1, respectively. 2.6.2. Linearity Calibration samples were prepared by spiking 200 l of blank plasma with 50 l of the working solution, 50 l of the internal standard solution (2500 ng/ml), and 450 l of acetonitrile for
a final plasma concentration of 20 to 2000 ng/ml for CA, methyl cinnamate, and cinnamyl alcohol. The calibration samples were vortex-mixed for 120 s then centrifuged at 12,000g for 10 min. The supernatant was collected for GC–MS quantification, and linearity was assessed by a weighed (1/x2 ) least squares regression analysis. 2.6.3. Precision, accuracy, and recovery High, medium, and low concentrations were prepared at 40, 200, 1000 ng/ml, respectively, for CA, methyl cinnamate, and cinnamyl
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alcohol. These calibration samples were chosen as quality control (QC) samples to evaluate accuracy and precision. Precision was evaluated by intra-day and inter-day relative standard deviation (RSD), which should not have exceeded 15%. Accuracy was evaluated by comparing the observed concentration with the actual concentration. The relative errors of accuracy should were within 15%. The recovery of each analyte was measured by comparing the ratio of the analyte to the IS in the QC samples with the standard solution. QC samples were prepared in the same way using water substituted for plasma. 2.6.4. Stability Assessment of the stability of each analyte was designed to cover all situations facing the analyte in the experiment. A period of 24 h stability was tested using vials at room temperature and then exposing them to three freeze/thaw cycles after processing. Blank plasma spiked at the high, medium, and low level concentrations were assessed after standing at room temperature for 24 h. These samples were compared to the freshly prepared samples, and the comparison ratio was obtained. Freeze/thaw cycles were evaluated as follows. Blank plasma spiked with the above-mentioned concentrations was stocked at −80 ◦ C for 24 h. These samples were then placed at room temperature to thaw. After three freeze/thaw cycles, samples were injected to the GC–MS to compare with the freshly prepared samples. 3. Results and discussion 3.1. Selectivity Six plasma samples from different rats were analyzed to confirm any inference at the retention time of the analytes. Fig. 2 shows the full-scan mass spectra of the analytes obtained by electron impact (EI). The candidates for SIM ions were carefully selected based on the selectivity and sensitivity. The SIM ions of CA, methyl
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cinnamate, cinnamyl alcohol, and the internal standard were chosen as the fragmentation ions at m/z 131, 105 and 92, respectively. Following the above methods, representative chromatograms of blank plasma, blank plasma spiked with IS, CA, methyl cinnamate and cinnamyl alcohol, 10 min after IV injection and 120 min after oral administration are collected in Fig. 3 showing the clear specificity of all analytes.
3.2. Calibration The calibration curves were successfully established with high linearity over the range used in this study. Calibration standards were prepared from 20 to 2000 ng/ml and assayed on different days to demonstrate the linearity of this method. This simple internal standard method described the concentration–response relationship analyzed by a non-weighted least squares linear regression. As shown in Table 1, calibration curves for CA, methyl cinnamate, and cinnamyl alcohol had good linearity, with a correlation coefficient of over 0.999 (r2 ≥ 0.999). The LLOQ of CA, methyl cinnamate, and cinnamyl alcohol was 20 ng/ml (S/N = 10).
3.3. Precision, accuracy and recovery The intra- and inter-day precision and accuracy of the entire method were assessed by analyzing the QC samples at three levels (40, 200, and 1000 ng/ml) in five replicates on the same day. RSD was typically below 12.2% for all analytes at all concentrations, while relative errors ranged from −8.6% to 14.8%. These data are presented in Table 2. The recovery of CA and its metabolites was measured at the same concentration as the QC samples. The peak area ratios of IS to analytes added after the extraction procedure represented 100%. The peak area ratios obtained from the analyte-free plasma spiked
Table 1 The linear range, correlation coefficient (r2 ), and calibration curve parameters with 95% confidence intervals of cinnamaldehyde, methyl cinamate and cinnamyl alcohol. Biological samples
Compound
Linear range (ng/ml)
Regression equation
r2
Plasma
Cinnamaldehyde Methyl cinamate Cinnamyl alcohol Cinnamaldehyde Methyl cinamate Cinnamyl alcohol Cinnamaldehyde Methyl cinamate Cinnamyl alcohol
20–2000 20–2000 20–2000 50–5000 50–5000 50–5000 50–5000 50–5000 50–5000
y = 1577x − 1.2 y = 1319x + 19.7 y = 5470x + 52.1 y = 1594x−3.0 y = 1335x + 20.5 y = 5552x + 42.8 y = 1625x − 2.2 y = 1342x + 34.1 y = 5485x + 63.0
0.9991 0.9991 0.9995 0.9992 0.9994 0.9997 0.9991 0.9992 0.9996
Feces
Urine
Table 2 Intra-day and inter-day precision, accuracy, and recovery for cinnamaldehyde, methyl cinnamate and cinnamyl alcohol. (intraday: n = 6; interday: n = 6 for one day, on three consecutive days; recovery: n = 5). Compound
Cinnamaldehyde
Methyl cinnamate Cinnamyl alcohol
Added conc. (ng/ml)
40 200 1000 40 200 1000 40 200 1000
Inter-day
Intra-day Mean conc. (ng/ml) (mean ± SD)
Precision (RSD%)
Accuracy (RE%)
Mean conc. (ng/ml) (mean ± SD)
Precision (RSD%)
Accuracy (RE%)
44.2 ± 3.5 192.7 ± 4.7 943.1 ± 16.5 43.2 ± 1.4 204.8 ± 6.3 913.6 ± 17.3 45.3 ± 4.7 200.4 ± 6.3 982.2 ± 27.4
7.9 2.4 1.7 3.2 3.1 1.9 10.4 3.1 2.8
10.5 −3.7 −5.7 8.0 2.4 −8.6 13.3 0.2 −1.8
45.3 ± 1.7 212.0 ± 8.0 973.7 ± 25.0 42.8 ± 2.2 193.5 ± 4.7 1012.6 ± 26.1 45.9 ± 5.6 199.7 ± 7.0 987.4 ± 28.6
3.8 3.8 2.6 5.1 2.4 2.6 12.2 3.5 2.9
13.3 6 −2.6 7 −3.3 1.3 14.8 −0.2 −1.3
SD: standard deviation; RSD: relative standard deviation; RE: relative error.
Recovery (%) (RSD%)
110.5 (11.2) 97.3 (5.3) 104.2 (7.3) 98.4 (7.1) 103.1 (6.5) 94.3 (4.2) 98.3 (12.8) 102.4 (6.1) 104.0 (8.3)
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Fig. 3. Chromatograms of blank plasma (A); blank plasma spiked with propiophenone, cinnamaldehyde, methyl cinamate, and cinnamyl alcohol (B); plasma 10 min after IV administration of cinnamaldehyde (20 mg/kg) (C); plasma 120 min after oral administration of cinnamaldehyde (500 mg/kg) (D). Peak 1, propiophenone; Peak 2, cinnamaldehyde. Peak 3, cinnamyl alcohol, Peak 4, methyl cinamate (mean ± S.D., n = 5).
with IS and analytes post-extraction compared to the 100% ratio confirmed the recovery values. The average recovery of the QC samples at three levels (low, medium, high), respectively, were 110.5 ± 11.2%, 97.3 ± 5.3%, 104.2 ± 7.3% for CA, 98.4 ± 7.1% 103.1 ± 6.5%, 94.3 ± 4.2% for methyl cinnamate, and 98.3 ± 12.8%, 102.4 ± 6.1%, 104.0 ± 8.3% for cinnamyl alcohol (Table 2). 3.4. Stability Stability was assessed by reinjection of the above-mentioned QC standards at low, medium, and high concentrations. All
analytes were found to be very stable for 24 h at room temperature; they also underwent freeze–thawing for three cycles without significant degradation; the obtained coefficient variations (CVs) were less than 8% for all analytes. 3.5. Application to pharmacokinetic analyses This selective and sensitive GC–MS method was successfully applied in the pharmacokinetic study of CA in rats. All detected analyte concentrations were >20 ng/ml, and clearly above the LLOQ of the methods. The mean plasma concentration-time profile of CA and its metabolites are shown in Fig. 4, with pharmacokinetic
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Fig. 4. Mean plasma concentration-time profile of cinnamaldehyde (A), methyl cinamate (B) and cinnamyl alcohol (C) in rats after a single oral dose of 500 mg/kg, 250 mg/kg, 125 mg/kg. Mean plasma concentration-time profile of cinnamaldehyde, methyl cinamate and cinnamyl alcohol after intravenous injection of 20 mg/kg (D) (mean ± S.D., n = 5). Table 3 The main pharmacokinetic parameters of cinnamaldehyde, methyl cinnamate, cinnamyl alcohol after oral administration by non-compartmental analysis (500 mg/kg, 250 mg/kg, and 125 mg/kg). CA Parameter
500 mg/kg
250 mg/kg
125 mg/kg
AUC0–t (ng h/ml) AUC0–∞ (ng h/ml) MRT (h) t1/2 (h) Tmax (h) CLz /F (l/h/kg) Vz /F (l/kg) Cmax (ng/ml) F (%) Methyl cinnamate AUC0–t (ng h/ml) AUC0–∞ (ng h/ml) MRT (h) t1/2 (h) Tmax (h) CLz /F (l/h/kg) Vz /F (l/kg) Cmax (ng/ml) Cinnamyl alcohol AUC0–t (ng h/ml) AUC0–∞ (ng h/ml) MRT (h) t1/2 (h) Tmax (h) CLz /F (l/h/kg) Vz /F (l/kg) Cmax (ng/ml)
1984 ± 531 2187 ± 517 7.6 ± 0.6 6.7 ± 1.5 1.6 ± 0.5 239 ± 55 2392 ± 526 249 ± 36 22.3 ± 9.0
1141 ± 265 1299 ± 259 7.1 ± 1.6 6.2 ± 1.5 2 ± 0.7 199 ± 44 1739 ± 367 121 ± 14
677 ± 127 1010 ± 314 5.8 ± 1.3 6.8 ± 2.6 1.8 ± 0.4 133 ± 38 1239 ± 358 82 ± 15
762 ± 262 1866 ± 418 3.7 ± 1.1 3.1 ± 2.1 1.6 ± 0.8 499 ± 293 2384 ± 1460 189 ± 37
568 ± 168 783 ± 165 4.4 ± 0.7 6.1 ± 2.8 2 ± 0.7 331 ± 73 2843 ± 1284 98 ± 17
425 ± 67 981 ± 218 5.4 ± 1.6 15.5 ± 6.5 1.5 ± 0.5 159 ± 36 3329 ± 889 64 ± 22
1105 ± 337 1915 ± 735 4.0 ± 1.4 6.7 ± 2.8 1.5 ± 0.7 294 ± 112 2502 ± 327 221 ± 66
AUC0–t : area under the curve to termination time; AUC0–∞ : area under the curve extrapolated to infinity; MRT: mean residence time; t1/2 : half-life; Tmax : time at maximum plasma concentration; CLz /F: plasma clearance; Vz /F: volume of distribution; Cmax : maximum plasma concentration; F (%): oral bioavailability.
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Table 4 The main pharmacokinetic parameters of cinnamaldehyde and its metabolites after intravenous administration by non-compartmental analysis (20 mg/kg). Parameter
Cinnamaldehyde
Methyl cinamate
Cinnamyl alcohol
AUC0–t (ng h/ml) AUC0–∞ (ng h/ml) MRT (h) t1/2 (h) Tmax (h) CLz /F (l/h/kg) Vz /F (l/kg) Cmax (ng/ml)
355 ± 53 511 ± 156 0.8 ± 0.2 1.7 ± 0.32 0.033 0.042 ± 0.011 0.103 ± 0.021 547 ± 142
227 ± 27 254 ± 29 0.518 ± 0.06 0.634 ± 0.25 0.033 0.08 ± 0.009 0.073 ± 0.027 596 ± 156
534 ± 166 585 ± 185 0.4 ± 0.17 0.58 ± 0.37 0.033 0.037 ± 0.014 0.029 ± 0.016 1899 ± 878
AUC0–t : area under the curve to termination time; AUC0–∞ : area under the curve extrapolated to infinity; MRT: mean residence time; t1/2 : half-life; Tmax : time at maximum plasma concentration; CLz /F: plasma clearance; Vz /F: volume of distribution; Cmax : maximum plasma concentration.
parameters listed in Table 3 and 4. Fig. 4(A)–(C) elaborated the oral administration in three dosages, while Fig. 4(D) described the 20 mg/kg IV administration. Simultaneous determining the levels of CA and its metabolites allowed us to understand the pharmacological interactions of CA and its metabolites. In Tables 3 and 4, the areas under the plasma concentration–time curve (AUC) from 0 min to terminal time of CA were 1984 ± 531 and 355 ± 53 ng h/ml for oral (500 mg/kg) and IV (20 mg/kg) administration, respectively. The bioavailability of CA was approximately 20% after oral administration. The elimination half-lives of CA were 6.7 ± 1.5 and 1.7 ± 0.3 h for oral and IV administration, respectively. From dosage 125 to 500 mg, maximum plasma concentration (Cmax ) and area under the curve to termination time (AUC0–t ) were proportional to the dose; time at maximum plasma concentration (Tmax ) and mean residence time (MRT) did not change following dose escalation (Table 3). In addition, excretion experiment was also performed which is shown in Fig. 5. Lower accumulative ratio of CA was found after 24 h, with the numbers reaching at 0.3% and 0.8% in feces and urine.
As shown in Fig. 4, a double peak was observed in the concentration-time profile of 500 mg/kg oral administration; the Cmax was 249 ± 36 ng/ml and the other peak was 130 ± 56 ng/ml. A similar report which illustrated CA maintained a concentration at 1 g/ml for 24 h also supported this phenomenon [19]. Several mechanisms might account for this phenomenon, such as enterohepatic circulation, fractionated gastric emptying, and separated “absorption windows” [31–33]. Enterohepatic circulation may be an explanation for this because the double-peak was not observed in the IV concentration-time profile; furthermore, the metabolites of CA presented the same phenomenon. Complex drug absorption cannot be clarified by a single experiment, and further studies should be carried out to ascertain whether this phenomenon is related to enterohepatic circulation or other mechanisms. This report revealed new information of the metabolism of CA which might provide guidance for further pharmaceutical and pharmacological studies. 4. Conclusions A selective and sensitive method has been established for the determination of CA, methyl cinnamate and cinnamyl alcohol and was successfully applied in this pharmacokinetic study. As previous studies have demonstrated, after CA enters the body, approximately 60% CA is oxidized to cinnamic acid rapidly [19]; however, a small portion of CA remains unoxidized. Our research clearly defined the time-concentration curve of CA, which make up the deficiency of previous study. Even though GC–MS cannot quantify the concentration of cinnamic acid or hippuric acid, it still provided the concentrations of CA itself and two other metabolites. More importantly, one metabolite, methyl cinnamate was firstly reported. To date, this is the first report using the GC–MS technique to separate and determine CA and its metabolites in rat plasma. This report fills the gap of the pharmacokinetic study of a widely used plant extract and provides a new method for the further investigation of CA. Acknowledgments This study was supported by the National Science Foundation of China (No. 20872180). The authors would like to thank Mr. Hua Li and Ms. Ying Li for their experimental support. References
Fig. 5. Urinary and fecal cumulative excretion of cinnamaldehyde in rats (mean ± S.D, n = 5).
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Pharmacokinetic study of cinnamaldehyde in rats by GC–MS after oral and intravenous administration Hang Zhao a,d,1 , Yanhua Xie a,d,1 , Qian Yang a,d,1 , Yu Cao b,d , Honghai Tu c , Wei Cao a,d,∗ , Siwang Wang a,d,∗ a
Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China Team NO. 1, Cadet Brigade, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China c Institute for Drug and Instrument Control of Xinjiang Military Region, Urumqi 830063, China d The Cultivation Project of Collaborative Innovation Center for Chinese Medicine in QinBa Mountains, Xi’an 710032, China b
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 17 October 2013 Accepted 27 October 2013 Available online 9 November 2013 Keywords: Cinnamaldehyde Methyl cinnamate Pharmacokinetic GC–MS Bioavailability
a b s t r a c t A selective and sensitive method utilizing gas chromatography–mass spectrometry was developed for simultaneous determination of cinnamaldehyde, cinnamyl alcohol, and methyl cinnamate in rat plasma. Cinnamaldehyde and cinnamyl alcohol can inter-convert to one another in rats, thus simultaneous quantifying both analytes provided a reliable and accurate method of assessment. Three qualifying ions (131 m/z, 105 m/z and 92 m/z) were chosen for simultaneous quantification of cinnamaldehyde and its metabolites. In this study, the calibration curves demonstrated a good linearity and reproducibility over the range of 20–2000 ng/ml (r2 ≥ 0.999) for all analytes. Furthermore, the sensitivity of gas chromatography–mass spectrometry revealed sufficient lower limit of quantitation and detection of 20 ng/ml and 5 ng/ml, respectively, in the pharmacokinetic analysis. The intra- and inter-day precision variations were less than 10.4% and 12.2%, respectively, whilst accuracy values ranged from −8.6% to 14.8%. All analytes were stable in plasma and in processed samples at room temperature for 24 h with no significant degradation after three freeze/thaw cycles. A small amount of the administered cinnamaldehyde had long half-life of 6.7 ± 1.5 h. In this study, gas chromatography–mass spectrometry was demonstrated to be a powerful tool for the pharmacokinetic studies of rats after intravenous and oral administration of cinnamaldehyde. © 2013 Elsevier B.V. All rights reserved.
1. Introduction For centuries, Cortex Cinnamomi has been widely used as a herbal medicine and spice in numerous countries [1,2]. Its major active component is cinnamaldehyde (CA) (Fig. 1). CA was isolated from cinnamon essential oils in the nineteenth century, and was mainly used to impart a cinnamon fragrance to medical products, cosmetics, and perfumes [3]. In recent years, CA has been widely used as a flavoring agent with maximum permitted concentrations from 2000 ppm in baby food and desserts to 6400 ppm in fruits and juices [4]. The consumption of CA as a flavoring agent in the United States is estimated to be 500,000 kg/year [5]. Due to its massive use and potential toxic effects, much attention has been placed on CA. Numerous studies indicated that CA exerted a variety of bioactive and pharmacological effects such as anti-viral
∗ Corresponding authors. Tel.: +86 29 84774748; fax: +86 29 84773752. E-mail addresses: [email protected] (W. Cao), [email protected] (S. Wang). 1 These authors contributed equally to this work. 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.10.044
[6], anti-inflammatory [7,8], anti-thrombic [9,10], anti-diabetic [11,12], and anti-cancer effects [13,14]. Recently, we also found that CA had obvious therapeutic effects on viral myocarditis though suppressing the expression of Toll like receptor 4 [15]. The metabolites of CA have also demonstrated similar pharmacological effects [16,17]; therefore, determining the pharmacokinetic data of major active substance and its metabolites is a next logical step. Several researchers have attempted to analyze CA by liquid chromatography (LC) in plasma samples. They found that CA was quickly absorbed and rapidly oxidized to cinnamic acid after oral administration, only a small amount of CA remained in the blood for 24 h [18,19]. The previous methods for assessing CA levels (generally around 1000 ng/ml) are insufficient and unreliable [18]. The concentration of main metabolites such as cinnamic acid in blood or hippuric acid in urine was quantified to predict the concentration of CA [18,20,21]. Providing precise concentration data of CA with the highly sensitive method of gas chromatography–mass spectrometry (GC–MS) is more essential than describing the known data of cinnamic acid with LC. Our previous study indicated that CA had higher pharmacological activity than cinnamic acid [6,15]. Therefore, a sensitive and accurate method is important to be developed
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(SIM) mode. Three qualifying ions (131 m/z, 105 m/z and 92 m/z) were selected for simultaneous investigating CA, methyl cinnamate, and cinnamyl alcohol. The obtained spectra were analyzed by the NIST library (Shimadzu, Kyoto, Japan) and are shown in Fig. 2. 2.3. Preparation of stock solutions
Fig. 1. The molecular structures of propiophenone (I), cinnamaldehyde (II), methyl cinamate (III), and cinnamyl alcohol (IV).
Stock solutions of CA, methyl cinnamate, cinnamyl alcohol, and propiophenone (IS) were prepared in acetonitrile at a concentration of 100 g/ml; these solutions were diluted at a concentration range of 0.3–30 g/ml which was used as the working solutions in the linearity experiment. The internal standard solution was obtained by diluting the IS stock solution to a concentration of 2500 ng/ml. The stock solutions were kept away from light at −20 ◦ C and used within two months. 2.4. Sample preparation
to investigate the accurate pharmacokinetic data of low concentrations of CA. LC was firstly used to analyze CA in traditional Chinese preparations [22,23]. Later, gas chromatography (GC) coupled with flame ionization (FID) was applied to determine CA in similar traditional preparations [24,25]. However, the pharmacokinetic data of CA were limited, due to the fact that the limit of detection of analytes using LC and GC methods was not sensitive enough for pharmacokinetic studies. Recently, the use of LC [26,27] and GC [28–30] combined with an MS detector have become new methods for identification and quantitation of CA at relatively low concentrations. Unfortunately, these data focused on the CA in the essential oil, but provided no information on the pharmacokinetic study of CA and its metabolites in rat plasma. In addition, CA is a reactive aldehyde and can be easily converted to cinnamyl alcohol [3]; thus quantifying both CA and cinnamyl alcohol is essential. GC–MS has been developed and demonstrated to be a sensitive method for investigating the pharmacokinetics of CA. The pharmacokinetic parameters after oral and intravenous (IV) administration were investigated in order to provide comparative analysis of two kinds of metabolic pathways and the data of bioavailability. 2. Materials and methods 2.1. Chemicals and materials CA (99%, purity) was supplied by Yuan Cheng Pharmaceutical Co., Ltd. Propiophenone (98%, purity), methyl cinnamate (98%, purity), and cinnamyl alcohol (98%, purity) were purchased from the Adamas Reagent Co., Ltd. (Shanghai, China). Their chemical structures are shown in Fig. 1. Propiophenone was used as the internal standard (IS). Acetonitrile (HPLC grade) was obtained from Honeywell (Muskegon, MI, USA). All other reagents were of analytical grade. 2.2. GC–MS conditions and instrumentation The analysis was tested on an ISQ Trace Ultra (Thermo Fisher Scientific, Waltham, MA) system combined with a Triplus atuosampler and injector. A DB-5ms capillary column (30 m × 0.25 mm, 0.25 m thickness; Agilent Technologies, USA) was used. The initial oven temperature was 50 ◦ C for 1 min, and the temperature was subsequently increased to 160 ◦ C at 10 ◦ C/min for 1 min and then increased to 280 ◦ C at 20 ◦ C/min for 1 min. Helium was used as carrier gas at a constant flow rate of 1.0 ml/min. The transfer line and ion source temperatures were both 250 ◦ C. Ionization was carried out in electron impact ionization (EI) mode at 70 eV. Detection was operated under the selected ion monitoring
A selective and sensitive sample preparation method was utilized to eliminate the interference of endogenous proteins and to optimize extraction recovery. From among several chemical reagents (methanol, acetonitrile, and formaldehyde) tested in the process acetonitrile showed highest sensitivity and convenience, especially in minimizing endogenous interference and enhancing extraction recovery. Consequently, we choose acetonitrile as the optimal solvent for sample preparation. To each 200 l plasma sample, 50 l internal standard solution (2500 ng/ml) and 500 l acetonitrile were added. Then, the solution was thoroughly vortex-mixed for 120 s. After centrifugation at 12,000g for 10 min, 1 l of the supernatant was injected into the GC–MS for analysis. Feces samples were dried and then powdered using a mortar and pestle. Physiological saline solution (1:2, w/v) was added and homogenized with the pulverized feces. Urine samples were diluted using the physiological saline solution (1:5, w/v) and then processed similarly to the plasma samples. 2.5. Pharmacokinetic and excretion study All animal protocols were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. Male Sprague-Dawley rats, weighing 225–275 g, were supplied from the Experimental Animal Research Center, the Fourth Military Medical University (Xi‘an, China). Three groups of rats (n = 5, each group) received a single oral dose of 500 mg/kg, 250 mg/kg, or 125 mg/kg CA (diluted in corn oil). Blood was collected at 10, 30, 60, 120, 180, 240, 360, 480, 720, 1080, and 1440 min post-administration. In one group of rats (n = 5, each group), blood was collected at 2, 5, 10, 15, 30, 60, 90, 120, and 180 min after IV administration of 20 mg/kg CA. These time plots were selected on the basis of previous studies of CA. The blood samples were processed similarly to the blank sample. The group of rats (n = 5, each group) used for the urinary and fecal excretion study received a single oral dose of 500 mg/kg CA. The rats were kept in stainless-steel metabolic cages with free access to water and food. Urine and feces were collected at 0–4, 4–8, 8–12, 12-18, and 18–24 h post-dosing. The feces were dried at room temperature. All specimens were processed as described previously. 2.6. Method validation 2.6.1. Specificity and sensitivity Five blank plasma samples obtained from individual rats were analyzed to exclude the possibility of interfering peaks at the
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Fig. 2. Full scan of mass spectra of propiophenone (A), cinnamaldehyde (B), methyl cinamate (C), and cinnamyl alcohol (D).
retention times of CA, its metabolites, and IS. The lower limit of detection (LLOD) and the lower limit of quantitation (LLOQ) were determined as the concentration of target compounds which provided a signal to noise (S/N) ratio with 3:1 and 10:1, respectively. 2.6.2. Linearity Calibration samples were prepared by spiking 200 l of blank plasma with 50 l of the working solution, 50 l of the internal standard solution (2500 ng/ml), and 450 l of acetonitrile for
a final plasma concentration of 20 to 2000 ng/ml for CA, methyl cinnamate, and cinnamyl alcohol. The calibration samples were vortex-mixed for 120 s then centrifuged at 12,000g for 10 min. The supernatant was collected for GC–MS quantification, and linearity was assessed by a weighed (1/x2 ) least squares regression analysis. 2.6.3. Precision, accuracy, and recovery High, medium, and low concentrations were prepared at 40, 200, 1000 ng/ml, respectively, for CA, methyl cinnamate, and cinnamyl
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alcohol. These calibration samples were chosen as quality control (QC) samples to evaluate accuracy and precision. Precision was evaluated by intra-day and inter-day relative standard deviation (RSD), which should not have exceeded 15%. Accuracy was evaluated by comparing the observed concentration with the actual concentration. The relative errors of accuracy should were within 15%. The recovery of each analyte was measured by comparing the ratio of the analyte to the IS in the QC samples with the standard solution. QC samples were prepared in the same way using water substituted for plasma. 2.6.4. Stability Assessment of the stability of each analyte was designed to cover all situations facing the analyte in the experiment. A period of 24 h stability was tested using vials at room temperature and then exposing them to three freeze/thaw cycles after processing. Blank plasma spiked at the high, medium, and low level concentrations were assessed after standing at room temperature for 24 h. These samples were compared to the freshly prepared samples, and the comparison ratio was obtained. Freeze/thaw cycles were evaluated as follows. Blank plasma spiked with the above-mentioned concentrations was stocked at −80 ◦ C for 24 h. These samples were then placed at room temperature to thaw. After three freeze/thaw cycles, samples were injected to the GC–MS to compare with the freshly prepared samples. 3. Results and discussion 3.1. Selectivity Six plasma samples from different rats were analyzed to confirm any inference at the retention time of the analytes. Fig. 2 shows the full-scan mass spectra of the analytes obtained by electron impact (EI). The candidates for SIM ions were carefully selected based on the selectivity and sensitivity. The SIM ions of CA, methyl
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cinnamate, cinnamyl alcohol, and the internal standard were chosen as the fragmentation ions at m/z 131, 105 and 92, respectively. Following the above methods, representative chromatograms of blank plasma, blank plasma spiked with IS, CA, methyl cinnamate and cinnamyl alcohol, 10 min after IV injection and 120 min after oral administration are collected in Fig. 3 showing the clear specificity of all analytes.
3.2. Calibration The calibration curves were successfully established with high linearity over the range used in this study. Calibration standards were prepared from 20 to 2000 ng/ml and assayed on different days to demonstrate the linearity of this method. This simple internal standard method described the concentration–response relationship analyzed by a non-weighted least squares linear regression. As shown in Table 1, calibration curves for CA, methyl cinnamate, and cinnamyl alcohol had good linearity, with a correlation coefficient of over 0.999 (r2 ≥ 0.999). The LLOQ of CA, methyl cinnamate, and cinnamyl alcohol was 20 ng/ml (S/N = 10).
3.3. Precision, accuracy and recovery The intra- and inter-day precision and accuracy of the entire method were assessed by analyzing the QC samples at three levels (40, 200, and 1000 ng/ml) in five replicates on the same day. RSD was typically below 12.2% for all analytes at all concentrations, while relative errors ranged from −8.6% to 14.8%. These data are presented in Table 2. The recovery of CA and its metabolites was measured at the same concentration as the QC samples. The peak area ratios of IS to analytes added after the extraction procedure represented 100%. The peak area ratios obtained from the analyte-free plasma spiked
Table 1 The linear range, correlation coefficient (r2 ), and calibration curve parameters with 95% confidence intervals of cinnamaldehyde, methyl cinamate and cinnamyl alcohol. Biological samples
Compound
Linear range (ng/ml)
Regression equation
r2
Plasma
Cinnamaldehyde Methyl cinamate Cinnamyl alcohol Cinnamaldehyde Methyl cinamate Cinnamyl alcohol Cinnamaldehyde Methyl cinamate Cinnamyl alcohol
20–2000 20–2000 20–2000 50–5000 50–5000 50–5000 50–5000 50–5000 50–5000
y = 1577x − 1.2 y = 1319x + 19.7 y = 5470x + 52.1 y = 1594x−3.0 y = 1335x + 20.5 y = 5552x + 42.8 y = 1625x − 2.2 y = 1342x + 34.1 y = 5485x + 63.0
0.9991 0.9991 0.9995 0.9992 0.9994 0.9997 0.9991 0.9992 0.9996
Feces
Urine
Table 2 Intra-day and inter-day precision, accuracy, and recovery for cinnamaldehyde, methyl cinnamate and cinnamyl alcohol. (intraday: n = 6; interday: n = 6 for one day, on three consecutive days; recovery: n = 5). Compound
Cinnamaldehyde
Methyl cinnamate Cinnamyl alcohol
Added conc. (ng/ml)
40 200 1000 40 200 1000 40 200 1000
Inter-day
Intra-day Mean conc. (ng/ml) (mean ± SD)
Precision (RSD%)
Accuracy (RE%)
Mean conc. (ng/ml) (mean ± SD)
Precision (RSD%)
Accuracy (RE%)
44.2 ± 3.5 192.7 ± 4.7 943.1 ± 16.5 43.2 ± 1.4 204.8 ± 6.3 913.6 ± 17.3 45.3 ± 4.7 200.4 ± 6.3 982.2 ± 27.4
7.9 2.4 1.7 3.2 3.1 1.9 10.4 3.1 2.8
10.5 −3.7 −5.7 8.0 2.4 −8.6 13.3 0.2 −1.8
45.3 ± 1.7 212.0 ± 8.0 973.7 ± 25.0 42.8 ± 2.2 193.5 ± 4.7 1012.6 ± 26.1 45.9 ± 5.6 199.7 ± 7.0 987.4 ± 28.6
3.8 3.8 2.6 5.1 2.4 2.6 12.2 3.5 2.9
13.3 6 −2.6 7 −3.3 1.3 14.8 −0.2 −1.3
SD: standard deviation; RSD: relative standard deviation; RE: relative error.
Recovery (%) (RSD%)
110.5 (11.2) 97.3 (5.3) 104.2 (7.3) 98.4 (7.1) 103.1 (6.5) 94.3 (4.2) 98.3 (12.8) 102.4 (6.1) 104.0 (8.3)
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Fig. 3. Chromatograms of blank plasma (A); blank plasma spiked with propiophenone, cinnamaldehyde, methyl cinamate, and cinnamyl alcohol (B); plasma 10 min after IV administration of cinnamaldehyde (20 mg/kg) (C); plasma 120 min after oral administration of cinnamaldehyde (500 mg/kg) (D). Peak 1, propiophenone; Peak 2, cinnamaldehyde. Peak 3, cinnamyl alcohol, Peak 4, methyl cinamate (mean ± S.D., n = 5).
with IS and analytes post-extraction compared to the 100% ratio confirmed the recovery values. The average recovery of the QC samples at three levels (low, medium, high), respectively, were 110.5 ± 11.2%, 97.3 ± 5.3%, 104.2 ± 7.3% for CA, 98.4 ± 7.1% 103.1 ± 6.5%, 94.3 ± 4.2% for methyl cinnamate, and 98.3 ± 12.8%, 102.4 ± 6.1%, 104.0 ± 8.3% for cinnamyl alcohol (Table 2). 3.4. Stability Stability was assessed by reinjection of the above-mentioned QC standards at low, medium, and high concentrations. All
analytes were found to be very stable for 24 h at room temperature; they also underwent freeze–thawing for three cycles without significant degradation; the obtained coefficient variations (CVs) were less than 8% for all analytes. 3.5. Application to pharmacokinetic analyses This selective and sensitive GC–MS method was successfully applied in the pharmacokinetic study of CA in rats. All detected analyte concentrations were >20 ng/ml, and clearly above the LLOQ of the methods. The mean plasma concentration-time profile of CA and its metabolites are shown in Fig. 4, with pharmacokinetic
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Fig. 4. Mean plasma concentration-time profile of cinnamaldehyde (A), methyl cinamate (B) and cinnamyl alcohol (C) in rats after a single oral dose of 500 mg/kg, 250 mg/kg, 125 mg/kg. Mean plasma concentration-time profile of cinnamaldehyde, methyl cinamate and cinnamyl alcohol after intravenous injection of 20 mg/kg (D) (mean ± S.D., n = 5). Table 3 The main pharmacokinetic parameters of cinnamaldehyde, methyl cinnamate, cinnamyl alcohol after oral administration by non-compartmental analysis (500 mg/kg, 250 mg/kg, and 125 mg/kg). CA Parameter
500 mg/kg
250 mg/kg
125 mg/kg
AUC0–t (ng h/ml) AUC0–∞ (ng h/ml) MRT (h) t1/2 (h) Tmax (h) CLz /F (l/h/kg) Vz /F (l/kg) Cmax (ng/ml) F (%) Methyl cinnamate AUC0–t (ng h/ml) AUC0–∞ (ng h/ml) MRT (h) t1/2 (h) Tmax (h) CLz /F (l/h/kg) Vz /F (l/kg) Cmax (ng/ml) Cinnamyl alcohol AUC0–t (ng h/ml) AUC0–∞ (ng h/ml) MRT (h) t1/2 (h) Tmax (h) CLz /F (l/h/kg) Vz /F (l/kg) Cmax (ng/ml)
1984 ± 531 2187 ± 517 7.6 ± 0.6 6.7 ± 1.5 1.6 ± 0.5 239 ± 55 2392 ± 526 249 ± 36 22.3 ± 9.0
1141 ± 265 1299 ± 259 7.1 ± 1.6 6.2 ± 1.5 2 ± 0.7 199 ± 44 1739 ± 367 121 ± 14
677 ± 127 1010 ± 314 5.8 ± 1.3 6.8 ± 2.6 1.8 ± 0.4 133 ± 38 1239 ± 358 82 ± 15
762 ± 262 1866 ± 418 3.7 ± 1.1 3.1 ± 2.1 1.6 ± 0.8 499 ± 293 2384 ± 1460 189 ± 37
568 ± 168 783 ± 165 4.4 ± 0.7 6.1 ± 2.8 2 ± 0.7 331 ± 73 2843 ± 1284 98 ± 17
425 ± 67 981 ± 218 5.4 ± 1.6 15.5 ± 6.5 1.5 ± 0.5 159 ± 36 3329 ± 889 64 ± 22
1105 ± 337 1915 ± 735 4.0 ± 1.4 6.7 ± 2.8 1.5 ± 0.7 294 ± 112 2502 ± 327 221 ± 66
AUC0–t : area under the curve to termination time; AUC0–∞ : area under the curve extrapolated to infinity; MRT: mean residence time; t1/2 : half-life; Tmax : time at maximum plasma concentration; CLz /F: plasma clearance; Vz /F: volume of distribution; Cmax : maximum plasma concentration; F (%): oral bioavailability.
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Table 4 The main pharmacokinetic parameters of cinnamaldehyde and its metabolites after intravenous administration by non-compartmental analysis (20 mg/kg). Parameter
Cinnamaldehyde
Methyl cinamate
Cinnamyl alcohol
AUC0–t (ng h/ml) AUC0–∞ (ng h/ml) MRT (h) t1/2 (h) Tmax (h) CLz /F (l/h/kg) Vz /F (l/kg) Cmax (ng/ml)
355 ± 53 511 ± 156 0.8 ± 0.2 1.7 ± 0.32 0.033 0.042 ± 0.011 0.103 ± 0.021 547 ± 142
227 ± 27 254 ± 29 0.518 ± 0.06 0.634 ± 0.25 0.033 0.08 ± 0.009 0.073 ± 0.027 596 ± 156
534 ± 166 585 ± 185 0.4 ± 0.17 0.58 ± 0.37 0.033 0.037 ± 0.014 0.029 ± 0.016 1899 ± 878
AUC0–t : area under the curve to termination time; AUC0–∞ : area under the curve extrapolated to infinity; MRT: mean residence time; t1/2 : half-life; Tmax : time at maximum plasma concentration; CLz /F: plasma clearance; Vz /F: volume of distribution; Cmax : maximum plasma concentration.
parameters listed in Table 3 and 4. Fig. 4(A)–(C) elaborated the oral administration in three dosages, while Fig. 4(D) described the 20 mg/kg IV administration. Simultaneous determining the levels of CA and its metabolites allowed us to understand the pharmacological interactions of CA and its metabolites. In Tables 3 and 4, the areas under the plasma concentration–time curve (AUC) from 0 min to terminal time of CA were 1984 ± 531 and 355 ± 53 ng h/ml for oral (500 mg/kg) and IV (20 mg/kg) administration, respectively. The bioavailability of CA was approximately 20% after oral administration. The elimination half-lives of CA were 6.7 ± 1.5 and 1.7 ± 0.3 h for oral and IV administration, respectively. From dosage 125 to 500 mg, maximum plasma concentration (Cmax ) and area under the curve to termination time (AUC0–t ) were proportional to the dose; time at maximum plasma concentration (Tmax ) and mean residence time (MRT) did not change following dose escalation (Table 3). In addition, excretion experiment was also performed which is shown in Fig. 5. Lower accumulative ratio of CA was found after 24 h, with the numbers reaching at 0.3% and 0.8% in feces and urine.
As shown in Fig. 4, a double peak was observed in the concentration-time profile of 500 mg/kg oral administration; the Cmax was 249 ± 36 ng/ml and the other peak was 130 ± 56 ng/ml. A similar report which illustrated CA maintained a concentration at 1 g/ml for 24 h also supported this phenomenon [19]. Several mechanisms might account for this phenomenon, such as enterohepatic circulation, fractionated gastric emptying, and separated “absorption windows” [31–33]. Enterohepatic circulation may be an explanation for this because the double-peak was not observed in the IV concentration-time profile; furthermore, the metabolites of CA presented the same phenomenon. Complex drug absorption cannot be clarified by a single experiment, and further studies should be carried out to ascertain whether this phenomenon is related to enterohepatic circulation or other mechanisms. This report revealed new information of the metabolism of CA which might provide guidance for further pharmaceutical and pharmacological studies. 4. Conclusions A selective and sensitive method has been established for the determination of CA, methyl cinnamate and cinnamyl alcohol and was successfully applied in this pharmacokinetic study. As previous studies have demonstrated, after CA enters the body, approximately 60% CA is oxidized to cinnamic acid rapidly [19]; however, a small portion of CA remains unoxidized. Our research clearly defined the time-concentration curve of CA, which make up the deficiency of previous study. Even though GC–MS cannot quantify the concentration of cinnamic acid or hippuric acid, it still provided the concentrations of CA itself and two other metabolites. More importantly, one metabolite, methyl cinnamate was firstly reported. To date, this is the first report using the GC–MS technique to separate and determine CA and its metabolites in rat plasma. This report fills the gap of the pharmacokinetic study of a widely used plant extract and provides a new method for the further investigation of CA. Acknowledgments This study was supported by the National Science Foundation of China (No. 20872180). The authors would like to thank Mr. Hua Li and Ms. Ying Li for their experimental support. References
Fig. 5. Urinary and fecal cumulative excretion of cinnamaldehyde in rats (mean ± S.D, n = 5).
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