Eur J Drug Metab Pharmacokinet DOI 10.1007/s13318-015-0272-7
ORIGINAL PAPER
A pharmacokinetic study of patchouli alcohol after a single oral administration of patchouli alcohol or patchouli oil in rats Ruoqi Zhang1 • Peiao Yan1 • Yunxia Li1 • Liang Xiong1 • Xiaohong Gong1 Cheng Peng1
•
Received: 12 November 2014 / Accepted: 3 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract Pogostemonis herba is used in traditional Chinese medicine to remove dampness, relieve sunstroke, stop vomiting and increase appetite. Patchouli alcohol, an ingredient in pogostemonis herba, has the potential to treat inflammation as well as bacterial and fungal infections. The essential oil of pogostemonis herba (patchouli oil) is commonly given orally in clinical settings; however, no pharmacokinetic studies have examined its oral administration. The goal of this study was to investigate the pharmacokinetic behavior of patchouli alcohol following single-dose oral administration in rats; the influence of other patchouli oil components on the pharmacokinetic profile of patchouli alcohol was also examined. In this study, a simple and selective GC/MS method was developed and validated to measure the level of patchouli alcohol in rat plasma. The study revealed that the pharmacokinetics profile was linear in both the patchouli alcohol and patchouli oil groups. The Cmax and AUC0–t of patchouli alcohol were greater in all three doses of patchouli alcohol compared to corresponding patchouli oil doses. Additionally, the Tmax values were significantly greater in the patchouli oil group. These results suggest that the other ingredients in patchouli oil influence the pharmacokinetic behavior of patchouli alcohol during its absorption. The results provide a meaningful basis for evaluating
& Cheng Peng [email protected] Ruoqi Zhang [email protected] 1
Pharmacy College, Chengdu University of Traditional Chinese Medicine, No. 1166, Liutai Avenue, Chengdu 611137, People’s Republic of China
the clinical application of patchouli oil and patchouli alcohol. Keywords Patchouli alcohol Pogostemon cablin (Blanco) Benth GC/MS Oral administration Pharmacokinetics
1 Introduction Pogostemonis herba is the aerial part of dried Pogostemon cablin (Blanco) Benth (Pogostemon, Lamiaceae). It is used in traditional Chinese medicine to remove dampness, relieve sunstroke, stop vomiting and increase appetite [The Pharmacopoeia Commission of People’s Republic of China (PRC) 2010]. Patchouli oil is the essential oil of pogostemonis herba, and it has been widely used to relieve clinical sunstroke (The Pharmacopoeia Commission of PRC 2010). Recent studies have demonstrated that patchouli oil has various pharmacological activities, including antiemetic properties and trypanocidal, anti-bacterial, anti-fungal, and Ca2? antagonist activities; it also repels mosquitoes (Pohlit et al. 2011; Yang et al. 1999, 2000; Kiuchi et al. 2004; Ichikawa et al. 1989). In addition, our previous work has demonstrated that patchouli oil can inhibit the proliferation of and induce apoptosis in PC3 human prostate cancer cells (Cai et al. 2013). Patchouli alcohol (PA, Fig. 1a) comprises 20–26 % of patchouli oil by weight and is the primary active ingredient. PA is the phytochemical marker used to determine the quality of pogostemonis herba and patchouli oil in traditional Chinese medicine (The Pharmacopoeia Commission of PRC 2010). PA has various pharmacological activities, acting as an inhibitor of asexual fungal propagation (Weide et al. 2006), inflammation (Li et al. 2011), the influenza
Eur J Drug Metab Pharmacokinet Fig. 1 Chemical structures and ion spectrum of PA (a) and IS (b)
virus (Lai et al. 2009) and tumorigenesis (Jeong et al. 2013). Thus far, studies have focused on its pharmacology, synthesis (Na¨f et al. 1981), pharmaceutics (You et al. 2010), and clinical use (Wang et al. 2013; Wan et al. 2013; Cui 2013; Fan et al. 2012; Yang et al. 2012). Because patents already cover new PA applications (Lai et al. 2012; Peng et al. a, b) and pogostemonis herbal drugs are generally well accepted, preclinical research has become necessary to promote the development of new PA therapeutic strategies. The low number of pharmacokinetic studies on PA is detrimental to potential new applications. Only one pharmacokinetic study (Yang et al. 2004) on PA, which examined an intravenous injection in rats, has been reported, but all pogostemonis herbal drugs are administered orally in clinical settings. It is therefore essential to study the compound’s pharmacokinetic behavior after oral administration to illustrate its absorption mechanisms and to provide information for clinical use. In addition, patchouli oil is commonly used clinically (Wang et al. 2013; Wan et al. 2013; Cui 2013) as the main form of pogostemonis herba, and thus, its other components (approximately 74–80 %), such as b-patchoulene,
caryophyllene, a-guaiene, seychellene, b-guaiene, and pogostone (Hu et al. 2006), may influence PA’s action. This paper examines the pharmacokinetics of increasing doses of PA and patchouli oil by single-dose oral administration in rats.
2 Experimental 2.1 Chemicals and reagents Pogostemon cablin (Blanco) Benth was collected from the pogostemonis herba Good Agriculture Practice for Chinese Crude Drugs Planting Base in the Guangzhou province of China in August 2012. According to the Pharmacopoeia of P. R. China, pogostemonis herba was identified by Dr. Xiong, a member of our research group (The Pharmacopoeia Commission of PRC 2010). Patchouli oil from pogostemonis herba was obtained by steam distillation (The Pharmacopoeia Commission of PRC 2010). PA was identified by gas chromatography–mass spectrometry (GC/ MS) (Hu et al. 2006) and accounted for 20 % of the oil
Eur J Drug Metab Pharmacokinet
composition based on the area normalization method. PA [98 %purity by GC/MS analysis (Hu et al. 2006)] was isolated from the patchouli oil as previously reported (Zhu et al. 2013). Isoalantolactone, used as an internal standard (IS, Fig. 1b), was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China, purity [98 %). Methanol was purchased from the TEDIA Company (Fairfield, IA, USA), and ethyl acetate was obtained from ARK Chemical (Chengdu, China). The water used was distilled in glass and passed through a Milli-Q water purification system (Millipore, Bedford, MA, USA). Methanol and water were HPLC grade. Ethyl acetate was analytically pure and redistilled. Other reagents were also analytically pure. 2.2 Equipment and GC/MS conditions GC/MS was performed with an Agilent 7890A GC instrument, an Agilent 5975C MS and Agilent ChemStation software (Agilent Technologies, Palo Alto, CA, USA). The injector was operated in the splitless mode at 250 °C (1 lL injection volume). Compounds were separated on an Agilent HP-5 ms capillary column (30 m 9 0.25 mm i.d. 9 0.25 lm coated with 5 % phenyl methyl siloxane film). The column temperature was set to 100 °C for injection and held for 1 min followed by increasing the temperature by 14 °C min-1 to 240 °C, a 1-min hold, and then a 35 °C min-1 increase to 280 °C and another 1-min hold. The spectrometer was operated in electron-impact (EI) mode with an ionization energy of 70 eV and a scan rate of 0.30 s per scan. The inlet ionization source temperature was 280 °C. The parent ion and product ion mass spectra of the base ion peaks at 222.2 and 190.1 m/z for PA and IS, respectively, were acquired with selected ion monitoring. 2.3 Preparation of standards and quality control samples Stock solutions of PA and IS were prepared in methanol at concentrations of approximately 20 and 100 lg/mL, respectively. A 5-, 10- and 20-fold dilution of stock solution of PA were prepared as working solution þ (4000 ng/mL), Þ (2000 ng/mL) and ˜ (1000 ng/mL). A 5- and 10-fold dilution of working solution ˜ were prepared as ˆ (200 ng/mL) and ´ (100 ng/mL). A 5- and 10-fold dilution of working solution ´ were prepared as ` (20 ng/mL) and (10 ng/mL). All the working standard solutions were prepared on the day of analysis by diluting the stock solution with methanol. The working concentration of IS was approximately 10 lg/mL. Each day, before extraction, the plasma calibration curve was prepared by spiking blank plasma (100 lL) with
known amounts of PA. The standard curve in plasma was created with 5, 10, 50, 100, 500, 1000, and 2000 ng/mL. The concentration of IS in plasma was 2 lg/mL. The quality control (QC) samples were prepared fresh in bulk on the day of analysis at four concentrations: 5 (LLQC), 15 (LQC), 500 (MQC), and 1500 (HQC) ng/mL. 2.4 Sample treatment Control (blank) plasma was obtained from untreated rats for calibration, and bulk spiked QC samples were removed from the freezer and allowed to thaw to room temperature before extraction. Control plasma with a volume of 170 lL (100 lL of blank plasma spiked with 50 lL of a working standard PA solution and 20 lL of IS solution) and sample plasma with a volume of 170 lL (100 lL of sample plasma added to 70 lL of methanol) were prepared for extraction. Then, a sample was extracted using 2 mL of ethyl acetate in a vortex shaker (Vortex Genius 3, IKAWerke GmbH & Co., Staufen, Germany). After centrifugation, the upper organic phase was transferred and evaporated to dryness under a stream of nitrogen at 30 °C (N-EVAP 11155, Organomation Associates, Inc., Berlin, Massachusetts, USA). The residue was reconstituted in 100 lL of methanol. The sample was transferred to a glass autosampler vial insert, and 1 lL was injected into the chromatographic system. 2.5 Method validation The assay method was validated for its selectivity, linearity, sensitivity, accuracy, precision, extraction recovery and stability. The selectivity was determined by analyzing six different blank plasma samples. No interference from endogenous or exogenous materials was present at the retention times of either PA or IS. The calibration curves were constructed over a linear range of 5–2000 ng/mL using the analyte/IS peak area ratio (y-axis) vs. the nominal concentration of the analyte (xaxis), and the curves were fitted by linear least-squares regression analysis with a weighting factor of 1/x2. A correlation coefficient (r) of 0.99 was considered to be acceptable for all calibration curves. The sensitivity of the method was evaluated in terms of the lower limit of quantification (LLOQ), at which point the analyte response must be at least five times the blank response. The intra-batch accuracy and precision of the method were evaluated by analyzing five replicates at four different QC levels, and the inter-batch accuracy and precision were evaluated by analyzing the QC samples in three different batches. The accuracy was expressed as a percentage of the nominal concentration (%). The mean accuracy should not
Eur J Drug Metab Pharmacokinet Fig. 2 Chromatograms of the base ion peak at m/z 222.2 of PA and the base ion peak at m/z 190.1 of IS. a Blank plasma; b blank plasma spiked with standard and IS; c plasma sample
Table 1 Precision, accuracy and extraction recovery for the analysis of PA in rat plasma Nominal concentration (ng/mL)
Intra-batch (n = 5)
Inter-batch (n = 3)
Extraction recovery (%)
RSD (%)
Accuracy (%)
RSD (%)
Accuracy (%)
PA
IS
5 15
6.49 5.95
98.23 ± 6.42 97.58 ± 5.79
6.09 6.27
100.58 ± 6.11 100.53 ± 6.31
77.63 ± 6.60
74.26 ± 7.01
500
5.86
99.48 ± 6.03
6.43
98.29 ± 6.47
72.43 ± 7.35
1500
4.72
99.07 ± 4.66
5.26
100.04 ± 5.25
79.25 ± 5.49
deviate by more than 15 %, except for the LLQC (±20 %). The precision was expressed as a relative standard deviation (RSD) of the replicate samples and should not exceed 15 %, except for the LLQC (20 %). The extraction recoveries for PA and IS were evaluated by assaying two groups of samples: (1) neat standard solutions of PA and IS in a solution of methanol, and (2) extracts from plasma spiked with PA and IS. Samples of each group were prepared at three QC levels: LQC, MQC and HQC. By comparing the absolute peak areas of the
analytes obtained in Groups 1 and 2, the extraction recoveries of the analytes were calculated as follows: extraction recovery (%) = 100 9 Group 2/Group 1. The stability of PA was assessed during sample collection and handling and after short-term (at room temperature for 14 h) and long-term (at -20 °C for 21 days) storage, after three freeze and thaw cycles from -20 °C to 25 °C, and after being kept in the autosampler for 12 h at 25 °C. The samples were processed and analyzed with the freshly prepared samples.
Eur J Drug Metab Pharmacokinet Table 2 Stability of PA in rat plasma (n = 3) Analyte concentration (ng/mL)
Bias (%) Short-term stability
Long-term stability
Three freeze–thaw cycle stability
Autosampler stability
15
4.99
10.81
8.23
3.56
500
3.88
4.32
4.23
3.6
1500
4.09
4.82
3.43
2.06
Bias (%) = (the absolute value of mean measured concentration of stability sample minus freshly prepared sample/measured concentration of freshly prepared sample) 9 100
(International Business Machines Corporation, USA). The PA group was administered orally at three doses (10, 30 and 100 mg/kg, dissolved in 0.5 % sodium carboxymethyl cellulose). The oil group was gavaged with equivalent amounts of PA as those used in the PA group (50, 150 and 500 mg/kg, dissolved in 0.5 % sodium carboxymethyl cellulose). Six rats were used for each dose. All rats were fasted for 12 h before dosing and for 4 h after dosing. Water was provided ad libitum. Blood samples (approximately 0.3 mL) were withdrawn from the orbital veins of each rat before dosing and 5, 10, 20 and 40 min and 1, 2, 4, 6, 10, 16, 24, 36 and 48 h after dosing. The heparinized blood was centrifuged at 4500 rpm for 10 min, and the plasma in the upper layer was transferred and stored at -20 °C until analysis. 2.7 Data analysis
Fig. 3 Mean plasma concentration–time profiles in rats administered PA (a) and patchouli oil (b) (n = 6)
All stability experiments were performed at the three QC levels of LQC, MQC and HQC in triplicate. The samples were considered stable if the deviations from the calculated concentrations of freshly prepared QC samples were within 15 %. 2.6 Pharmacokinetic study The animal protocol was approved by the Committee of Scientific Research and the Committee of Animal Care of the Chengdu University of Traditional Chinese Medicine (Chengdu, China). Thirty-six Sprague Dawley rats (SPF, females) weighing 200–220 g were purchased from the Animal Experiment Center of Chengdu University of Traditional Chinese Medicine (Chengdu, China). All rats were randomly allocated to either the PA group (n = 18) or the oil group (n = 18) using PASW Statistics 18.0
All data were processed using Phoenix WinNonlin 6.3 (Pharsight Corporation, USA) to construct pharmacokinetic profiles. The Newman–Keuls test was used to evaluate the differences in AUC0–t/dose, Cmax/dose, and t1/2z among the 3 doses administered. The statistical significance of t1/2z, Cmax, and AUC0–t between the PA and oil groups was evaluated using the t test. The Wilcoxon t rank-sum test was used to analyze the differences in Tmax. A value of P \ 0.05 was considered statistically significant. 2.8 Results and discussion The GC/MS analyses of PA and IS are shown in Fig. 1a, b, respectively. Using proper procedure, an accurate identification of PA and an optimal chromatographic peak were satisfactorily acquired from the rat plasma samples. The selectivity of this method toward the endogenous plasma matrix was assessed using the plasma of all rats. The typical chromatograms are shown in Fig. 2. The retention times of PA and IS were approximately 8.6 and 10.8 min, respectively (Fig. 2a, b). The matrix effect of the analytes was considered, and no signal interfered under these conditions. The peak area ratios of PA to IS vs. the nominal concentrations exhibited acceptable linearity in plasma over the concentration range of 5–2000 ng/mL. The typical
Eur J Drug Metab Pharmacokinet Table 3 Pharmacokinetic parameters [mean (SD)] of PA and oil groups in rat plasma (n = 6) Parameter
PA group (mg/kg) 10
Oil group (mg/kg) 30
100
50
150
500
AUC0–t, ng/mLh
1110.40 (476.74)
4434.36 (1700.03)
15,203.61 (4615.59)
810.38 (257.68)
2576.08 (654.33)
9352.50 (2601.90)
AUC0–?, ng/mLh
1563.84 (529.08)
5005.28 (1441.81)
17,120.06 (5566.02)
960.05 (281.71)
3015.50 (770.86)
12,845.02 (1588.04)
MRT0–t, h
14.02 (3.89)
14.74 (1.72)
13.04 (1.24)
12.78 (2.61)
14.10 (1.82)
14.23 (3.98)
MRT0–?, h
29.58 (11.42)
23.44 (8.39)
19.97 (7.08)
20.29 (4.20)
23.03 (8.21)
39.49 (20.96)
t1/2z, h
21.51 (8.46)
17.81 (7.52)
17.82 (9.29)
15.28 (3.68)
17.83 (7.10)
26.72 (12.26)
Tmax, h
0.83 (0.39)
0.72 (0.25)
1.56 (0.68)
3.33 (1.03)
2.67 (1.03)
3.44 (1.36)
Vz/F, L CLz/F, L/h
41.85 (25.60) 1.49 (0.53)
36.30 (24.13) 1.29 (0.38)
30.77 (13.03) 1.31 (0.53)
47.42 (9.64) 2.25 (0.70)
52.40 (22.38) 2.08 (0.45)
81.00 (34.82) 1.58 (0.19)
Cmax, ng/mL
99.83 (28.33)
367.46 (155.38)
1167.93 (221.34)
48.00 (10.96)
161.91 (35.02)
674.82 (258.40)
Fig. 4 Statistical results of AUC0–t/dose, Cmax/dose, t1/2z and Tmax among the 3 doses administered in PA (10, 30 and 100 mg/kg) and patchouli oil groups (50, 150 and 500 mg/kg)
regression equation was y = 0.2775x - 0.0033 with r = 0.9979. The LLOQ (signal-to-noise ratio, S/N, C5) of PA was 5 ng/mL. Table 1 shows the intra-batch and inter-batch precision and accuracy of PA. The intra-batch and inter-batch precision RSD were 4.72–6.49 % and 5.26–6.43 %, respectively. The intra- and inter-batch accuracy ranged from 97.58 to 99.48 % and from 98.29 to 100.58 %, respectively. The deviations of mean accuracy were within 15 %. The extraction recoveries for PA and IS are shown in Table 1. The mean recovery of PA was 76.44 % (n = 15). The mean recovery of IS was 74.26 % (n = 15). The RSDs of these values were less than 15 %. The stability tests found that the plasma samples at the three QC concentrations were stable (Table 2). The PA in the reconstitution solution was also stable under the autosampler conditions (25 °C) for 12 h.
The results showed that the established method was sensitive enough to detect PA following oral administration to rats at a dose of 10–100 mg/kg. Figure 3 depicts the mean plasma PA concentration vs. time profiles in both groups at each of three doses. The pharmacokinetic parameters of PA are shown in Table 3. The statistical results for the pharmacokinetic parameters among the 3 PA doses administered alone and in patchouli oil are shown in Fig. 4. The mean Cmax and AUC0–t values were both linear in the range of the doses administered. The pharmacokinetic parameters of AUC0–t/dose, Cmax/dose, t1/2z and Tmax were similar between three doses in the PA and patchouli oil groups. The Cmax, Tmax, t1/2z, and AUC0–t values were compared between the PA and oil groups, and the statistical results are shown in Fig. 5. The Cmax values were significantly higher in the PA group than in the oil group (mean [SD] 99.83
Eur J Drug Metab Pharmacokinet
Fig. 5 Statistical results of AUC0–t, t1/2z, Cmax and Tmax between the PA and patchouli oil groups (the contents of PA were equal in PA group, both recorded with doses of 10, 30 and 100 mg/kg). **P \ 0.01 and *P \ 0.05 vs. the oil group
Table 4 A comparative pharmacokinetic results (mean ± SD) of PA and oil groups in rat plasma (n = 6) between oral and intravenous administration Dose(mg/ kg) PA group (ig) Oil group (ig) PA group (iv) Oil group (iv)
AUC0–t, ng/mLh
t1/2, h
10
1110.40 ± 476.74
21.51 ± 8.46
30
4434.36 ± 1700.03
17.81 ± 7.52
100
15,203.61 ± 4615.59
50
810.38 ± 257.68
17.82 ± 9.29 15.28 ± 3.68
150
2576.08 ± 654.33
17.83 ± 7.10
500
9352.50 ± 2601.90
26.72 ± 12.26
283.97 ± 19.55
0.67 ± 0.05
2 4
528.37 ± 15.77
0.60 ± 0.05
8
1014.38 ± 123.88
0.59 ± 0.01
5
435.38 ± 45.35
0.83 ± 0.08
10 20
950.67 ± 96.73 1845.68 ± 185.05
0.83 ± 0.10 0.83 ± 0.10
[28.33] vs. 48.00 [10.96] ng/mL, P = 0.005, for a 10 mg/ kg dose; 367.46 [155.38] vs. 161.91 [35.02] ng/mL, P = 0.022, for a 30 mg/kg dose; and 1167.93 [221.34] vs. 674.82 [258.40] ng/mL, P = 0.005, for a 100 mg/kg dose). The values of the apparent t1/2 were significantly larger after oral administration than those after iv administration (Lai et al. 2009; Yang et al. 2004). The Tmax values were significantly greater after oil administration at all doses compared with PA alone. The AUC0–t values at the 30 and 100 mg/kg doses were significantly greater in the PA group
than in the oil group (mean [SD] 4434.36 [1700.03] vs. 2576.08 [654.33] ng/mLh, P = 0.044; and 15,203.61 [4615.59] vs. 9352.50 [2601.90] ng/mLh, P = 0.027, respectively), which disagrees with the previous pharmacokinetic study (Yang et al. 2004). The comparative pharmacokinetics results between this study and the previous pharmacokinetic study (Yang et al. 2004) are summarized in Table 4. The differences in the rate and degree of absorption may have attributed to the saturation of some enzymes and to transporter competition between PA and some other ingredient in the volatile oil. In this rat model, a simple and selective GC/MS method for the quantification of PA in rat plasma was developed and validated. The desired sensitivity was achieved with an LLOQ of 5 ng/mL. This method was successfully applied to a pharmacokinetic study of single-dose oral PA administration (10–100 mg/kg) in rats. The system’s response to PA appeared to be dose proportional over the range of 10–100 mg/kg. The results indicated that there was a significant difference in the resulting plasma concentration between the oral administration of PA and that of patchouli oil. The ingredients in patchouli oil decreased the rate and amount of PA. These findings will aid the development of new therapeutic strategies using PA. This study highlighted the potential effects that the other components in patchouli oil can have on PA bioactivity. Acknowledgments The authors gratefully acknowledge the assistance of Feng Wan and Doctor Liang Xiong in the extraction of PA
Eur J Drug Metab Pharmacokinet and patchouli oil. We thank Associate Professor of Pharmacy Yun-tao Jia and Dr. Lin Song (Department of Pharmacy, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China) for reviewing this article and advices. This study was supported by science and technology support program (No. 2014SZ0071) by Sichuan Province. Conflict of interest interest.
All the authors have declared no conflict of
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ORIGINAL PAPER
A pharmacokinetic study of patchouli alcohol after a single oral administration of patchouli alcohol or patchouli oil in rats Ruoqi Zhang1 • Peiao Yan1 • Yunxia Li1 • Liang Xiong1 • Xiaohong Gong1 Cheng Peng1
•
Received: 12 November 2014 / Accepted: 3 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract Pogostemonis herba is used in traditional Chinese medicine to remove dampness, relieve sunstroke, stop vomiting and increase appetite. Patchouli alcohol, an ingredient in pogostemonis herba, has the potential to treat inflammation as well as bacterial and fungal infections. The essential oil of pogostemonis herba (patchouli oil) is commonly given orally in clinical settings; however, no pharmacokinetic studies have examined its oral administration. The goal of this study was to investigate the pharmacokinetic behavior of patchouli alcohol following single-dose oral administration in rats; the influence of other patchouli oil components on the pharmacokinetic profile of patchouli alcohol was also examined. In this study, a simple and selective GC/MS method was developed and validated to measure the level of patchouli alcohol in rat plasma. The study revealed that the pharmacokinetics profile was linear in both the patchouli alcohol and patchouli oil groups. The Cmax and AUC0–t of patchouli alcohol were greater in all three doses of patchouli alcohol compared to corresponding patchouli oil doses. Additionally, the Tmax values were significantly greater in the patchouli oil group. These results suggest that the other ingredients in patchouli oil influence the pharmacokinetic behavior of patchouli alcohol during its absorption. The results provide a meaningful basis for evaluating
& Cheng Peng [email protected] Ruoqi Zhang [email protected] 1
Pharmacy College, Chengdu University of Traditional Chinese Medicine, No. 1166, Liutai Avenue, Chengdu 611137, People’s Republic of China
the clinical application of patchouli oil and patchouli alcohol. Keywords Patchouli alcohol Pogostemon cablin (Blanco) Benth GC/MS Oral administration Pharmacokinetics
1 Introduction Pogostemonis herba is the aerial part of dried Pogostemon cablin (Blanco) Benth (Pogostemon, Lamiaceae). It is used in traditional Chinese medicine to remove dampness, relieve sunstroke, stop vomiting and increase appetite [The Pharmacopoeia Commission of People’s Republic of China (PRC) 2010]. Patchouli oil is the essential oil of pogostemonis herba, and it has been widely used to relieve clinical sunstroke (The Pharmacopoeia Commission of PRC 2010). Recent studies have demonstrated that patchouli oil has various pharmacological activities, including antiemetic properties and trypanocidal, anti-bacterial, anti-fungal, and Ca2? antagonist activities; it also repels mosquitoes (Pohlit et al. 2011; Yang et al. 1999, 2000; Kiuchi et al. 2004; Ichikawa et al. 1989). In addition, our previous work has demonstrated that patchouli oil can inhibit the proliferation of and induce apoptosis in PC3 human prostate cancer cells (Cai et al. 2013). Patchouli alcohol (PA, Fig. 1a) comprises 20–26 % of patchouli oil by weight and is the primary active ingredient. PA is the phytochemical marker used to determine the quality of pogostemonis herba and patchouli oil in traditional Chinese medicine (The Pharmacopoeia Commission of PRC 2010). PA has various pharmacological activities, acting as an inhibitor of asexual fungal propagation (Weide et al. 2006), inflammation (Li et al. 2011), the influenza
Eur J Drug Metab Pharmacokinet Fig. 1 Chemical structures and ion spectrum of PA (a) and IS (b)
virus (Lai et al. 2009) and tumorigenesis (Jeong et al. 2013). Thus far, studies have focused on its pharmacology, synthesis (Na¨f et al. 1981), pharmaceutics (You et al. 2010), and clinical use (Wang et al. 2013; Wan et al. 2013; Cui 2013; Fan et al. 2012; Yang et al. 2012). Because patents already cover new PA applications (Lai et al. 2012; Peng et al. a, b) and pogostemonis herbal drugs are generally well accepted, preclinical research has become necessary to promote the development of new PA therapeutic strategies. The low number of pharmacokinetic studies on PA is detrimental to potential new applications. Only one pharmacokinetic study (Yang et al. 2004) on PA, which examined an intravenous injection in rats, has been reported, but all pogostemonis herbal drugs are administered orally in clinical settings. It is therefore essential to study the compound’s pharmacokinetic behavior after oral administration to illustrate its absorption mechanisms and to provide information for clinical use. In addition, patchouli oil is commonly used clinically (Wang et al. 2013; Wan et al. 2013; Cui 2013) as the main form of pogostemonis herba, and thus, its other components (approximately 74–80 %), such as b-patchoulene,
caryophyllene, a-guaiene, seychellene, b-guaiene, and pogostone (Hu et al. 2006), may influence PA’s action. This paper examines the pharmacokinetics of increasing doses of PA and patchouli oil by single-dose oral administration in rats.
2 Experimental 2.1 Chemicals and reagents Pogostemon cablin (Blanco) Benth was collected from the pogostemonis herba Good Agriculture Practice for Chinese Crude Drugs Planting Base in the Guangzhou province of China in August 2012. According to the Pharmacopoeia of P. R. China, pogostemonis herba was identified by Dr. Xiong, a member of our research group (The Pharmacopoeia Commission of PRC 2010). Patchouli oil from pogostemonis herba was obtained by steam distillation (The Pharmacopoeia Commission of PRC 2010). PA was identified by gas chromatography–mass spectrometry (GC/ MS) (Hu et al. 2006) and accounted for 20 % of the oil
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composition based on the area normalization method. PA [98 %purity by GC/MS analysis (Hu et al. 2006)] was isolated from the patchouli oil as previously reported (Zhu et al. 2013). Isoalantolactone, used as an internal standard (IS, Fig. 1b), was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China, purity [98 %). Methanol was purchased from the TEDIA Company (Fairfield, IA, USA), and ethyl acetate was obtained from ARK Chemical (Chengdu, China). The water used was distilled in glass and passed through a Milli-Q water purification system (Millipore, Bedford, MA, USA). Methanol and water were HPLC grade. Ethyl acetate was analytically pure and redistilled. Other reagents were also analytically pure. 2.2 Equipment and GC/MS conditions GC/MS was performed with an Agilent 7890A GC instrument, an Agilent 5975C MS and Agilent ChemStation software (Agilent Technologies, Palo Alto, CA, USA). The injector was operated in the splitless mode at 250 °C (1 lL injection volume). Compounds were separated on an Agilent HP-5 ms capillary column (30 m 9 0.25 mm i.d. 9 0.25 lm coated with 5 % phenyl methyl siloxane film). The column temperature was set to 100 °C for injection and held for 1 min followed by increasing the temperature by 14 °C min-1 to 240 °C, a 1-min hold, and then a 35 °C min-1 increase to 280 °C and another 1-min hold. The spectrometer was operated in electron-impact (EI) mode with an ionization energy of 70 eV and a scan rate of 0.30 s per scan. The inlet ionization source temperature was 280 °C. The parent ion and product ion mass spectra of the base ion peaks at 222.2 and 190.1 m/z for PA and IS, respectively, were acquired with selected ion monitoring. 2.3 Preparation of standards and quality control samples Stock solutions of PA and IS were prepared in methanol at concentrations of approximately 20 and 100 lg/mL, respectively. A 5-, 10- and 20-fold dilution of stock solution of PA were prepared as working solution þ (4000 ng/mL), Þ (2000 ng/mL) and ˜ (1000 ng/mL). A 5- and 10-fold dilution of working solution ˜ were prepared as ˆ (200 ng/mL) and ´ (100 ng/mL). A 5- and 10-fold dilution of working solution ´ were prepared as ` (20 ng/mL) and (10 ng/mL). All the working standard solutions were prepared on the day of analysis by diluting the stock solution with methanol. The working concentration of IS was approximately 10 lg/mL. Each day, before extraction, the plasma calibration curve was prepared by spiking blank plasma (100 lL) with
known amounts of PA. The standard curve in plasma was created with 5, 10, 50, 100, 500, 1000, and 2000 ng/mL. The concentration of IS in plasma was 2 lg/mL. The quality control (QC) samples were prepared fresh in bulk on the day of analysis at four concentrations: 5 (LLQC), 15 (LQC), 500 (MQC), and 1500 (HQC) ng/mL. 2.4 Sample treatment Control (blank) plasma was obtained from untreated rats for calibration, and bulk spiked QC samples were removed from the freezer and allowed to thaw to room temperature before extraction. Control plasma with a volume of 170 lL (100 lL of blank plasma spiked with 50 lL of a working standard PA solution and 20 lL of IS solution) and sample plasma with a volume of 170 lL (100 lL of sample plasma added to 70 lL of methanol) were prepared for extraction. Then, a sample was extracted using 2 mL of ethyl acetate in a vortex shaker (Vortex Genius 3, IKAWerke GmbH & Co., Staufen, Germany). After centrifugation, the upper organic phase was transferred and evaporated to dryness under a stream of nitrogen at 30 °C (N-EVAP 11155, Organomation Associates, Inc., Berlin, Massachusetts, USA). The residue was reconstituted in 100 lL of methanol. The sample was transferred to a glass autosampler vial insert, and 1 lL was injected into the chromatographic system. 2.5 Method validation The assay method was validated for its selectivity, linearity, sensitivity, accuracy, precision, extraction recovery and stability. The selectivity was determined by analyzing six different blank plasma samples. No interference from endogenous or exogenous materials was present at the retention times of either PA or IS. The calibration curves were constructed over a linear range of 5–2000 ng/mL using the analyte/IS peak area ratio (y-axis) vs. the nominal concentration of the analyte (xaxis), and the curves were fitted by linear least-squares regression analysis with a weighting factor of 1/x2. A correlation coefficient (r) of 0.99 was considered to be acceptable for all calibration curves. The sensitivity of the method was evaluated in terms of the lower limit of quantification (LLOQ), at which point the analyte response must be at least five times the blank response. The intra-batch accuracy and precision of the method were evaluated by analyzing five replicates at four different QC levels, and the inter-batch accuracy and precision were evaluated by analyzing the QC samples in three different batches. The accuracy was expressed as a percentage of the nominal concentration (%). The mean accuracy should not
Eur J Drug Metab Pharmacokinet Fig. 2 Chromatograms of the base ion peak at m/z 222.2 of PA and the base ion peak at m/z 190.1 of IS. a Blank plasma; b blank plasma spiked with standard and IS; c plasma sample
Table 1 Precision, accuracy and extraction recovery for the analysis of PA in rat plasma Nominal concentration (ng/mL)
Intra-batch (n = 5)
Inter-batch (n = 3)
Extraction recovery (%)
RSD (%)
Accuracy (%)
RSD (%)
Accuracy (%)
PA
IS
5 15
6.49 5.95
98.23 ± 6.42 97.58 ± 5.79
6.09 6.27
100.58 ± 6.11 100.53 ± 6.31
77.63 ± 6.60
74.26 ± 7.01
500
5.86
99.48 ± 6.03
6.43
98.29 ± 6.47
72.43 ± 7.35
1500
4.72
99.07 ± 4.66
5.26
100.04 ± 5.25
79.25 ± 5.49
deviate by more than 15 %, except for the LLQC (±20 %). The precision was expressed as a relative standard deviation (RSD) of the replicate samples and should not exceed 15 %, except for the LLQC (20 %). The extraction recoveries for PA and IS were evaluated by assaying two groups of samples: (1) neat standard solutions of PA and IS in a solution of methanol, and (2) extracts from plasma spiked with PA and IS. Samples of each group were prepared at three QC levels: LQC, MQC and HQC. By comparing the absolute peak areas of the
analytes obtained in Groups 1 and 2, the extraction recoveries of the analytes were calculated as follows: extraction recovery (%) = 100 9 Group 2/Group 1. The stability of PA was assessed during sample collection and handling and after short-term (at room temperature for 14 h) and long-term (at -20 °C for 21 days) storage, after three freeze and thaw cycles from -20 °C to 25 °C, and after being kept in the autosampler for 12 h at 25 °C. The samples were processed and analyzed with the freshly prepared samples.
Eur J Drug Metab Pharmacokinet Table 2 Stability of PA in rat plasma (n = 3) Analyte concentration (ng/mL)
Bias (%) Short-term stability
Long-term stability
Three freeze–thaw cycle stability
Autosampler stability
15
4.99
10.81
8.23
3.56
500
3.88
4.32
4.23
3.6
1500
4.09
4.82
3.43
2.06
Bias (%) = (the absolute value of mean measured concentration of stability sample minus freshly prepared sample/measured concentration of freshly prepared sample) 9 100
(International Business Machines Corporation, USA). The PA group was administered orally at three doses (10, 30 and 100 mg/kg, dissolved in 0.5 % sodium carboxymethyl cellulose). The oil group was gavaged with equivalent amounts of PA as those used in the PA group (50, 150 and 500 mg/kg, dissolved in 0.5 % sodium carboxymethyl cellulose). Six rats were used for each dose. All rats were fasted for 12 h before dosing and for 4 h after dosing. Water was provided ad libitum. Blood samples (approximately 0.3 mL) were withdrawn from the orbital veins of each rat before dosing and 5, 10, 20 and 40 min and 1, 2, 4, 6, 10, 16, 24, 36 and 48 h after dosing. The heparinized blood was centrifuged at 4500 rpm for 10 min, and the plasma in the upper layer was transferred and stored at -20 °C until analysis. 2.7 Data analysis
Fig. 3 Mean plasma concentration–time profiles in rats administered PA (a) and patchouli oil (b) (n = 6)
All stability experiments were performed at the three QC levels of LQC, MQC and HQC in triplicate. The samples were considered stable if the deviations from the calculated concentrations of freshly prepared QC samples were within 15 %. 2.6 Pharmacokinetic study The animal protocol was approved by the Committee of Scientific Research and the Committee of Animal Care of the Chengdu University of Traditional Chinese Medicine (Chengdu, China). Thirty-six Sprague Dawley rats (SPF, females) weighing 200–220 g were purchased from the Animal Experiment Center of Chengdu University of Traditional Chinese Medicine (Chengdu, China). All rats were randomly allocated to either the PA group (n = 18) or the oil group (n = 18) using PASW Statistics 18.0
All data were processed using Phoenix WinNonlin 6.3 (Pharsight Corporation, USA) to construct pharmacokinetic profiles. The Newman–Keuls test was used to evaluate the differences in AUC0–t/dose, Cmax/dose, and t1/2z among the 3 doses administered. The statistical significance of t1/2z, Cmax, and AUC0–t between the PA and oil groups was evaluated using the t test. The Wilcoxon t rank-sum test was used to analyze the differences in Tmax. A value of P \ 0.05 was considered statistically significant. 2.8 Results and discussion The GC/MS analyses of PA and IS are shown in Fig. 1a, b, respectively. Using proper procedure, an accurate identification of PA and an optimal chromatographic peak were satisfactorily acquired from the rat plasma samples. The selectivity of this method toward the endogenous plasma matrix was assessed using the plasma of all rats. The typical chromatograms are shown in Fig. 2. The retention times of PA and IS were approximately 8.6 and 10.8 min, respectively (Fig. 2a, b). The matrix effect of the analytes was considered, and no signal interfered under these conditions. The peak area ratios of PA to IS vs. the nominal concentrations exhibited acceptable linearity in plasma over the concentration range of 5–2000 ng/mL. The typical
Eur J Drug Metab Pharmacokinet Table 3 Pharmacokinetic parameters [mean (SD)] of PA and oil groups in rat plasma (n = 6) Parameter
PA group (mg/kg) 10
Oil group (mg/kg) 30
100
50
150
500
AUC0–t, ng/mLh
1110.40 (476.74)
4434.36 (1700.03)
15,203.61 (4615.59)
810.38 (257.68)
2576.08 (654.33)
9352.50 (2601.90)
AUC0–?, ng/mLh
1563.84 (529.08)
5005.28 (1441.81)
17,120.06 (5566.02)
960.05 (281.71)
3015.50 (770.86)
12,845.02 (1588.04)
MRT0–t, h
14.02 (3.89)
14.74 (1.72)
13.04 (1.24)
12.78 (2.61)
14.10 (1.82)
14.23 (3.98)
MRT0–?, h
29.58 (11.42)
23.44 (8.39)
19.97 (7.08)
20.29 (4.20)
23.03 (8.21)
39.49 (20.96)
t1/2z, h
21.51 (8.46)
17.81 (7.52)
17.82 (9.29)
15.28 (3.68)
17.83 (7.10)
26.72 (12.26)
Tmax, h
0.83 (0.39)
0.72 (0.25)
1.56 (0.68)
3.33 (1.03)
2.67 (1.03)
3.44 (1.36)
Vz/F, L CLz/F, L/h
41.85 (25.60) 1.49 (0.53)
36.30 (24.13) 1.29 (0.38)
30.77 (13.03) 1.31 (0.53)
47.42 (9.64) 2.25 (0.70)
52.40 (22.38) 2.08 (0.45)
81.00 (34.82) 1.58 (0.19)
Cmax, ng/mL
99.83 (28.33)
367.46 (155.38)
1167.93 (221.34)
48.00 (10.96)
161.91 (35.02)
674.82 (258.40)
Fig. 4 Statistical results of AUC0–t/dose, Cmax/dose, t1/2z and Tmax among the 3 doses administered in PA (10, 30 and 100 mg/kg) and patchouli oil groups (50, 150 and 500 mg/kg)
regression equation was y = 0.2775x - 0.0033 with r = 0.9979. The LLOQ (signal-to-noise ratio, S/N, C5) of PA was 5 ng/mL. Table 1 shows the intra-batch and inter-batch precision and accuracy of PA. The intra-batch and inter-batch precision RSD were 4.72–6.49 % and 5.26–6.43 %, respectively. The intra- and inter-batch accuracy ranged from 97.58 to 99.48 % and from 98.29 to 100.58 %, respectively. The deviations of mean accuracy were within 15 %. The extraction recoveries for PA and IS are shown in Table 1. The mean recovery of PA was 76.44 % (n = 15). The mean recovery of IS was 74.26 % (n = 15). The RSDs of these values were less than 15 %. The stability tests found that the plasma samples at the three QC concentrations were stable (Table 2). The PA in the reconstitution solution was also stable under the autosampler conditions (25 °C) for 12 h.
The results showed that the established method was sensitive enough to detect PA following oral administration to rats at a dose of 10–100 mg/kg. Figure 3 depicts the mean plasma PA concentration vs. time profiles in both groups at each of three doses. The pharmacokinetic parameters of PA are shown in Table 3. The statistical results for the pharmacokinetic parameters among the 3 PA doses administered alone and in patchouli oil are shown in Fig. 4. The mean Cmax and AUC0–t values were both linear in the range of the doses administered. The pharmacokinetic parameters of AUC0–t/dose, Cmax/dose, t1/2z and Tmax were similar between three doses in the PA and patchouli oil groups. The Cmax, Tmax, t1/2z, and AUC0–t values were compared between the PA and oil groups, and the statistical results are shown in Fig. 5. The Cmax values were significantly higher in the PA group than in the oil group (mean [SD] 99.83
Eur J Drug Metab Pharmacokinet
Fig. 5 Statistical results of AUC0–t, t1/2z, Cmax and Tmax between the PA and patchouli oil groups (the contents of PA were equal in PA group, both recorded with doses of 10, 30 and 100 mg/kg). **P \ 0.01 and *P \ 0.05 vs. the oil group
Table 4 A comparative pharmacokinetic results (mean ± SD) of PA and oil groups in rat plasma (n = 6) between oral and intravenous administration Dose(mg/ kg) PA group (ig) Oil group (ig) PA group (iv) Oil group (iv)
AUC0–t, ng/mLh
t1/2, h
10
1110.40 ± 476.74
21.51 ± 8.46
30
4434.36 ± 1700.03
17.81 ± 7.52
100
15,203.61 ± 4615.59
50
810.38 ± 257.68
17.82 ± 9.29 15.28 ± 3.68
150
2576.08 ± 654.33
17.83 ± 7.10
500
9352.50 ± 2601.90
26.72 ± 12.26
283.97 ± 19.55
0.67 ± 0.05
2 4
528.37 ± 15.77
0.60 ± 0.05
8
1014.38 ± 123.88
0.59 ± 0.01
5
435.38 ± 45.35
0.83 ± 0.08
10 20
950.67 ± 96.73 1845.68 ± 185.05
0.83 ± 0.10 0.83 ± 0.10
[28.33] vs. 48.00 [10.96] ng/mL, P = 0.005, for a 10 mg/ kg dose; 367.46 [155.38] vs. 161.91 [35.02] ng/mL, P = 0.022, for a 30 mg/kg dose; and 1167.93 [221.34] vs. 674.82 [258.40] ng/mL, P = 0.005, for a 100 mg/kg dose). The values of the apparent t1/2 were significantly larger after oral administration than those after iv administration (Lai et al. 2009; Yang et al. 2004). The Tmax values were significantly greater after oil administration at all doses compared with PA alone. The AUC0–t values at the 30 and 100 mg/kg doses were significantly greater in the PA group
than in the oil group (mean [SD] 4434.36 [1700.03] vs. 2576.08 [654.33] ng/mLh, P = 0.044; and 15,203.61 [4615.59] vs. 9352.50 [2601.90] ng/mLh, P = 0.027, respectively), which disagrees with the previous pharmacokinetic study (Yang et al. 2004). The comparative pharmacokinetics results between this study and the previous pharmacokinetic study (Yang et al. 2004) are summarized in Table 4. The differences in the rate and degree of absorption may have attributed to the saturation of some enzymes and to transporter competition between PA and some other ingredient in the volatile oil. In this rat model, a simple and selective GC/MS method for the quantification of PA in rat plasma was developed and validated. The desired sensitivity was achieved with an LLOQ of 5 ng/mL. This method was successfully applied to a pharmacokinetic study of single-dose oral PA administration (10–100 mg/kg) in rats. The system’s response to PA appeared to be dose proportional over the range of 10–100 mg/kg. The results indicated that there was a significant difference in the resulting plasma concentration between the oral administration of PA and that of patchouli oil. The ingredients in patchouli oil decreased the rate and amount of PA. These findings will aid the development of new therapeutic strategies using PA. This study highlighted the potential effects that the other components in patchouli oil can have on PA bioactivity. Acknowledgments The authors gratefully acknowledge the assistance of Feng Wan and Doctor Liang Xiong in the extraction of PA
Eur J Drug Metab Pharmacokinet and patchouli oil. We thank Associate Professor of Pharmacy Yun-tao Jia and Dr. Lin Song (Department of Pharmacy, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China) for reviewing this article and advices. This study was supported by science and technology support program (No. 2014SZ0071) by Sichuan Province. Conflict of interest interest.
All the authors have declared no conflict of
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