- Email: [email protected]
Determination and pharmacokinetics of gastrodin and p-hydroxybenzylalcohol after oral administration of Gastrodia elata Bl. extract in rats by high-performance liquid chromatography–electrospray ionization mass spectrometric method
ARTICLE IN PRESS
Phytomedicine 15 (2008) 844–850 www.elsevier.de/phymed
Determination and pharmacokinetics of gastrodin and p-hydroxybenzylalcohol after oral administration of Gastrodia elata Bl. extract in rats by high-performance liquid chromatography–electrospray ionization mass spectrometric method W. Zhang, Y.X. Sheng, J.L. Zhang Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China
Abstract In this report, a high-performance liquid chromatography–electrospray ionization mass spectrometric (HPLC–MS) method was used to determine gastrodin (GAS) and p-hydroxybenzylalcohol (HBA) in rat plasma after oral administration of Gastrodia elata Bl. (Chinese name: Tianma) extract. Up to 200 ml of plasma containing GAS, HBA and pyromucic acid (as internal standard, IS) were deproteinized with six volumes of methanol. Calibration curves showed linearity within the concentration range tested 2.00–200.00 mg/ml for GAS and 0.832–104.00 mg/ml for HBA in plasma with a correlation coefficient (r) greater than 0.9997. The limit of quantification of 2.00 and 0.83 mg/ml for GAS and HBA had been achieved, respectively. The intra-day and inter-day precisions of the method were determined to be less than 17.82% for GAS and 10.21% for HBA. The recoveries were in the range of 91.12–108.64% with RSD less than 7.80% for GAS and 92.91–106.14% with RSD less than 4.30% for HBA. Evidence showed that a rapid, simple and reproducible LC–MS assay was established to determine GAS and HBA in rat plasma. r 2008 Elsevier GmbH. All rights reserved. Keywords: Gastrodia elata Bl; Gastrodin; p-Hydroxybenzylalcohol; HPLC–MS; Pharmacokinetics
Introduction Tianma, the dried rhizome of Gastrodia elata Bl. (Fam. Orchidaceae), is an important and notable Chinese herbal medicine which has been used for treatments of headaches, migraine, dizziness, epilepsy, rheumatism, neuralgia, paralysis and other neuralgic and nervous disorders for over 1000 years in China (Tang and Eisenbrand, 1992). Over the past several decades, various chemical and pharmacological studies Corresponding author. Tel.: +86 10 83154880; fax: +86 10 63017757. E-mail address: [email protected] (J.L. Zhang).
0944-7113/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2008.02.012
on Tianma have been carried out to demonstrate its bioactive compounds and pharmacological effects. It was reported that Tianma could improve D-galactoseinduced memory impairment, meliorate the deficit in senescent mice on the step-down passive avoidance task, prevent rat pheochromocytoma and have an anticonvulsive effect (Kao et al., 1994, 1995; Yun and Huang, 2004; Hsieh, 2007). Tianma also has been used as a health supplement and an ingredient in cosmetics. In addition, gastrodin (GAS) (Fig. 1A) and p-hydroxybenzylalcohol (HBA) (Fig. 1B) have been shown to exhibit a scavenging effect on free radicals and consolidate and retrieve memory (Liu and Mori, 1992, 1993; Hsieh, 1997). GAS and HBA are the main active constituents
ARTICLE IN PRESS W. Zhang et al. / Phytomedicine 15 (2008) 844–850
GluO
CH2OH HO
CH2OH
Fig. 1. The chemical structures of (A) GAS and (B) HBA.
of Tianma as reported (Feng, 1979). HBA is the aglycone of GAS. However, to our knowledge, there has been no report on the pharmacokinetic studies of Tianma extract in biological samples. Only three reports related to the pharmacokinetic studies we found were the study of single compound (gastrodin, vanillin, 4-hydroxybenzaldehyde or ligustrazine) in animal models (You et al., 1994; Yong et al., 1999; Li et al., 2006). In the present study, a reproducible and rapid LC–MS method was developed and applied to pharmacokinetic studies of GAS and HBA in rat plasma after oral administration of Tianma extract. The study of the pharmacokinetic parameters of the two active components might provide useful information for clinical treatment.
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with an ESI/APCI source (Thermo Electron Corporation, USA). The RP-C18 column (Zorbax SB-C18 2.1 100 mm, 3.5 mm) was equipped with a guard column (SB-C18 2.1 10 mm, 3.5 mm). The mobile phase consisted of acetonitrile and 0.1% acetic acid (4.5:95.5) and the flow rate was 0.2 ml/min. The injection volume was 1 ml. For the MS part, the mass spectra were recorded using ESI source in the negative mode with ion spray voltage at 3.0 KV, capillary temperature at 350 1C and cone voltage at 75 V. The scan mode was select ion monitoring (SIM). The deprotonated molecular ions for GAS, HBA and IS were set at m/z 285, 123 and 111, respectively (Fig. 4).
Preparation of Tianma extract Two hundred grams of powder was immersed in deionized water and extracted twice: once with 2000 ml deionized water and a second time with 1600 ml deionized water under sonication for 30 min. Then the supernatants were pooled together and concentrated to 100 ml under reduced pressure at 30 1C. The Tianma extract was obtained and the concentration was 2.0 g/ml.
Materials and methods
Stock and working solutions
Chemicals and reagents
Accurately weighed 4.0 mg of GAS, 5.2 mg of HBA and 3.2 mg of IS were dissolved with 2 ml of methanol (in a volumetric flask) and used as stock solution. The concentrations were approximately 2.0, 2.6 and 1.6 mg/ml, respectively. All stock solutions were stored at 4 1C before analysis. Different volumes of GAS and HBA stock solutions were dissolved with 1 ml of methanol as working solutions.
The dried rhizome of Tianma purchased from TongRenTang (Beijing, China) was ground to powder, and then passed through a 60-mesh sieve for preparation. The powder was stored at about 4 1C before use. Standards of GAS and furoic acid (as internal standard, IS) were obtained from the National Institute for the Control of Biological and Pharmaceutical Drugs (Beijing, China). HBA of HPLC grade was purchased from Fluka (Switzerland). Methanol and acetonitrile were of HPLC grade from Burdick & Jackson Company (Muskegon, MI, USA). Acetic acid and ethyl ether were of analytical grade from Peking Chemical Factory, and deionized water from a Milli-Q system (Millipore, Bedford, MA, USA) was used for all preparations. Physiological saline was purchased from Shijiazhuang Pharmaceutical Cooperation Factory (Hebei Province, China). Heparin sodium was from Changzhou Qianhong Biochemical Pharmaceutical Co. Ltd. Heparin sodium was dissolved in physiological saline at the ratio of 1–25. Heparin solution was used to rinse the test tubes prior to blood collection for plasma.
Instruments and conditions The experiments were performed on a FinniganTM Surveyor LC-MSQTM Plus mass spectrometer equipped
Preparation of standards and quality control (QC) samples To 200 ml blank plasma samples, 10 ml of working solution and 10 ml of IS solution were added to prepare biological calibration curves and quality control samples. The final concentrations of biological calibration standards were within the range of 2.00–200.00 mg/ml for GAS and 0.83–104.0 mg/ml for HBA. Quality control samples were prepared at concentrations of 2.00, 10.00 and 200.00 mg/ml for GAS and 0.83, 2.04 and 104.00 mg/ml for HBA.
Sample preparation To 200 ml plasma samples, 10 ml of IS solution was added. After being vortexed for 15 s, 1.2 ml of methanol was added and vortexed for 30 s to precipitate protein. The mixture was centrifuged at 5000 rpm for 5 min.
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W. Zhang et al. / Phytomedicine 15 (2008) 844–850
The supernatant was evaporated to dryness under a reduced pressure at 30 1C. The residue was reconstituted in 200 ml of methanol, and then was filtered through 0.45 mm filter for LC/MS analysis.
Validation of the method Linearity The calibration curve of GAS was constructed by calculating the peak area ratios (y) of GAS to IS against GAS concentrations (x). The calibration curve of HBA was processed the same as that of GAS. The limit of quantification (LOQ) was defined as the lowest limitation of quantification concentrations of GAS and HBA measured with an acceptable precision (less than 20%) and accuracy (within the range of 80–120%). Precision and accuracy The precision and accuracy of the method were performed by analyzing QC samples with low, medium and high concentrations. Intra-day precision and accuracy (each, n ¼ 6) were evaluated by analysis of QC samples at different times during the same day. Inter-day precision and accuracy were determined by repeated analysis of QC samples over 5 consecutive days (n ¼ 1 series per day). The measured concentrations of the QC samples were calculated from the biological calibration curves. The precision was evaluated by the intra-day and inter-day variability with relative standard deviation (RSD %). The accuracy was obtained by measured concentration of QC samples divided by spiked concentration. Recovery The recoveries of GAS and HBA were investigated by comparing the GAS/IS and HBA /IS peak area ratio of blank plasma spiked with standard solution with that of the standards spiked in biological samples at the concentrations of 2.00, 10.00 and 200.00 mg/ml for GAS and 0.83, 2.04 and 104.00 mg/ml for HBA. Stability The stability was investigated by determining the QC samples at 0, 2, 4, 8, 12 and 24 h at room temperature. The effects of three freeze–thaw cycles were evaluated by repeated analysis (n ¼ 3) of QC samples.
Pharmacokinetic study Forty male Wistar rats weighing 200710 g were provided by Institute of Laboratory Animal Science. Rats were fasted in metabolic cages for 24 h and maintained on physiological saline prior to oral administration of Tianma extract at the dose of 10 ml/kg body
weight. This approximately corresponded to 40 mg/kg of GAS and 4 mg/kg of HBA. After 5 min oral administration of pharmacological saline at the dose of 10 ml/kg body weight, four rats were anesthetized with ethyl ether and blank blood was collected from abdominal artery in clean heparinized glass tubes and used as blanks. Blank plasma was separated by centrifugation at 3500 rpm for 5 min. The remaining 36 rats were divided into 6 groups with 6 rats per group. Blood was collected at different time points (5, 15, 30, 45, 70, 100, 120, 150, 165, 180, 210 and 240) after oral administration of Tianma extract. Blood samples collected from rats given Tianma at different time points mentioned above were prepared for analysis by the methods cited above. The concentrations of GAS and HBA in plasma were determined at each time point. By using the software of WinNonlin, the pharmacokinetic parameters, such as the maximum plasma concentration (Cmax) and the corresponding time (Tmax), the absorption phase (k01), elimination phase (k10), plasma clearance (CL) as well as apparent volume of distribution (Vd), were calculated. The area under the plasma concentration–time curve (AUC0–t) was calculated using trapezoidal rule to the last point.
Results and discussion Extraction of GAS and HBA in rat plasma To reduce the endogenous-related substances in plasma the method of precipitation of protein was performed. Methanol was used as the precipitant and we tried various ratios of methanol and plasma. When the ratio was 6 to 1 (equal to 1.2 ml of methanol to 0.2 ml of plasma) the effect of protein precipitation was the best.
Optimization of HPLC–MS conditions The selection of the LC–MS conditions for assay was guided by requirement for obtaining chromatograms with resolution of tested constituents and IS in rat plasma. To obtain the LC portion with good resolution, column and mobile phase were investigated separately. A Zorbax SB-C18 (2.1 100 mm, 3.5 mm) was used. The mobile phase consisting of acetonitrile (4.5%) and water (95.5%) with acetic acid at 0.1% yielded the best separation and allowed for subsequent MS analysis. During the chromatographic run, the mass spectrometer was programmed for three SIM events, which allowed for optimal detection of each constituent. Finally the elution flow rate at 0.2 ml/min to ensure the LC–MS assay had good resolution within 10 min.
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Intensity
W. Zhang et al. / Phytomedicine 15 (2008) 844–850
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700 600 500 400 300 200 100 0 700 600 500 400 300 200 100 0 1400 1200 1000 800 600 400 200 0 0
1
2
3
4
5
6
7
8
9
Intensity
Time (min)
1400 1200 1000 800 600 400 200 0 3000 2500 2000 1500 1000 500 0 1400 1200 1000 800 600 400 200 0
IS
HBA
GAS
0
1
2
3
4
5
6
7
8
9
7
8
9
Time (min)
Intensity
1400 1200 1000 800 600 400 200 0 5000
IS
4000 3000
HBA
2000 1000 0 1400 1200 1000 800 600 400 200 0
GAS
0
1
2
3
4
5
6
Time (min)
Fig. 2. Representative chromatograms of LC–MS. (A) Blank plasma, (B) blank plasma spiked with GAS, HBA and IS, (C) plasma at 45 min after oral administration of Tianma extract; ‘‘a’’ represents the m/z of 111, ‘‘b’’ represents the m/z of 123 and ‘‘c’’ represents the m/z of 285.
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were linear within the concentration range of 2.00–200.00 mg/ml for GAS and 0.83–104.0 mg/ml for HBA, with the correlation coefficient more than 0.9997. The LOQ was defined as the lowest limitation of quantification concentrations of GAS and HBA measured with an acceptable precision (less than 20%) and accuracy (within the range of 80–120%). And the LOQs were 2.00 mg/ml for GAS and 0.83 mg/ml for HBA. The calculated results are given in Table 1.
Specificity The typical SIM chromatograms of reference standards of tested compounds and IS are shown in Fig. 2 together with the chromatogram of blank plasma. The identification of the GAS and HBA peaks was achieved by comparing retention time and mass spectra with commercial standards. The retention times were 4.6, 5.4 and 7.6 min for GAS, IS and HBA, respectively. All the major constituents were well separated and no interfering peak was found in the chromatogram of blank plasma.
Precision and accuracy The RSD of the overall intra- and inter-day precisions for GAS and HBA were less than 17.82%. The accuracy for GAS and HBA at low, middle and high concentrations was within the range of 82.0–105.8%. The results are shown in Table 2.
Calibration curves and limit of quantification (LOQ) Plasma spiked with six different concentrations of GAS and HBA were processed. The calibration curves
Table 1.
Calibration curves for GAS and HBA in rat plasma
Sample
Standard curve
Range (mg/ml)
Correlation coefficient (r)
GAS HBA
Y ¼ 0.073X0.07 Y ¼ 0.23X+0.20
2.00–200.00 0.83–104.00
0.9997 0.9998
The calibration curve in plasma. Y, peak area ratio (GAS or HBA/IS); X, concentration of GAS or HBA in blood samples (mg/ml).
Table 2. Sample
Intra-day and inter-day precision and accuracy of GAS and HBA in rat plasma Spiked (mg/ml)
Intra-day (n ¼ 6)
Inter-day (n ¼ 6)
Measured (mg/ml)a
RSD (%)
Accuracy (%)b
Measured (mg/ml)
RSD (%)
Accuracy (%)
GAS
2.00 10.00 200.00
1.6470.14 9.8870.54 187.0676.52
8.5 5.5 3.3
82.0 98.8 93.5
1.7270.31 9.8070.54 201.81711.68
17.8 5.5 5.8
86.0 98.0 100.9
HBA
0.83 2.08 104.00
0.8870.024 2.0370.16 103.3873.89
2.7 7.9 3.8
105.8 97.6 99.4
0.8770.09 2.0270.14 100.8076.98
10.2 6.9 6.9
104.6 97.1 96.9
a
Mean7SD. Accuracy ¼ (mean of measured concentration/spiked concentration) 100.
b
Table 3.
Recovery of GAS and HBA in rat plasma
Sample
Spiked (mg/ml)
Measured (mg/ml)
Recovery (%)a
RSD (%)
GAS
2.00 10.00 200.00
2.1770.14 9.1170.71 199.9179.54
108.677.2 91.177.1 99.974.8
6.7 7.8 4.8
HBA
0.83 2.08 104.00
106.172.9 92.974.0 94.271.9
2.7 4.3 1.9
a
0.8870.024 1.9370.083 99.9871.95
Recovery ¼ (mean of measured concentration/spiked concentration) 100.
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Recovery The overall recovery of GAS and HBA ranged from 91.1% to 108.6% with RSD less than 7.8%. The results are shown in Table 3.
Stability The results of the stability of GAS and HBA in plasma kept for 24 h at room temperature and after three freeze (10 1C)/thaw cycles are shown in Table 4. GAS and HBA were stable in plasma at room temperature or after three freeze–thaw cycles at 10 1C.
Application – pharmacokinetic study The described method was applied to analysis of plasma samples after oral administration of Tianma extract. Fig. 3 shows the mean plasma concentration– time profile of GAS (n ¼ 6). The mean pharmacokinetic parameters of GAS after oral administration of Tianma extract are shown in Table 5 and by fitting plasma data a two-compartment model was calculated. From the above data, we found that GAS in rat plasma was detected at 15 min after oral administration, and reached
Table 4. Stability of GAS and HBA in rat plasma kept for 24 h at room temperature and during three freeze–thaw cycles Sample
Spiked (mg/ml)
Mean of percentage remaining (%) 24 h at room temperature
Three freeze–thaw cycles
2.00 10.00 200.00
87.0 92.7 105.4
86.5 95.9 102.1
HBA
0.83 2.08 104.00
116.9 89.9 73.2
109.6 97.1 92.1
Concentration (µg/mL)
GAS
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the Cmax at 70 min. GAS was rapidly eliminated from the plasma and could be detected up to 240 min after oral administration by the established method. It suggested that GAS could not be easily accumulated in rat plasma. The plasma concentration of GAS started to increase again at 165 min and had a second peak at 180 min. It may indicate the enterohepatic circulation of GAS. But it still needs further investigation to prove whether the enterohepatic circulation exists. Another active constituent, HBA, could not be detected in rat plasma at the beginning time points by the established methods and detected only at 45, 70, 100 and 120 min after oral administration of Tianma extract. The results might be due to the very low content of HBA in Tianma extract. On the other hand, from the structures of GAS and HBA, HBA is the aglycone of GAS. HBA detected in rat plasma at later time points might be the metabolite of GAS (Fig. 4).
Conclusion A simple, specific, rapid and reproducible LC–MS method has been developed to determine the active constituents GAS and HBA in rat plasma after oral administration of Tianma extract. Sample preparation Table 5. Mean pharmacokinetic parameters of GAS in rat plasma (n ¼ 6) after oral administration Parameter
Unit
Value
Cmax Tmax AUC0t k01 k10 Vd CL
mg/ml min minmg/ml min min ml ml/min
13.48 70 1869.91 36.47 43.42 267.99 4.28
All pharmacokinetic parameters are calculated from the mean plasma concentrations.
20 18 16 14 12 10 8 6 4 2 0 0
50
100
150 Time (min)
200
250
300
Fig. 3. Mean plasma concentration–time profile of GAS in rat plasma after oral administration. Each point represents the mean7SD.
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Intensity
850 50000 48000 46000 44000 42000 40000 38000 36000 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0
GAS
HBA
0
1
2
3
4
5
6
7
8
9
Time (min)
Fig. 4. HPLC chromatogram of Tianma extract.
was simple and reliable. The pharmacokinetic parameters obtained here could provide more information for absorption studies of GAS. The results provide scientific data for the further study and scientific clinical use of Tianma extract.
Acknowledgement We thank the Ministry of Science and Technology of the People’s Republic of China (ke(2004-05)) and Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine (Peking Union Medical College) of Ministry of Education of the People’s Republic of China for financial support of this work.
References Feng, H.C., 1979. Studies on the constituents of Gastrodia elata. Acta Chim. Sin. 37, 175. Kao, N.N., Yu, S.Z., Liu, R.H., Hsu, C.T., 1994. Improving effects of Gastrodia elata BI on D-galactose-induced aging in mice. Chin. Trad. Herbal Drugs 25, 521–523. Kao, N.N., Yu, S.Z., H’su, C.T., 1995. Improving effects of Gastrodia elata B1 on learning and memory in senescent mice. China J. Chin. Mater. Med. 20 (9), 562–564. Liu, J., Mori, A., 1992. Antioxidant and free radical scavenging activities of Gastrodia elata B1 and Uncaria rhynchophylla (MIQ.). Neuropharmacology 31, 1287–1298. Liu, J., Mori, A., 1993. Antioxidant and pre-oxidant activities of p-hydroxybenzylalcohol and vanillin: effects of free
radicals, brain peroxidation and degradation of benzoate, deoxyribose, amino acid and DNA. Neuropharmacology 32, 659–669. Hsieh, M.T., 1997. Gastrodin and p-hydroxybenzylalcohol facilitated memory consolidation and retrieval, but not acquisition, on the passive avoidance task in rats. J. Ethnopharmacol. 56, 45–54. Hsieh, Ch.L., Lin, J.J., Chiang, S.Y., Su, Sh.Y., Tang, N.Y., Lin, G.G., Lin, I.H., Liu, Ch.H., Hsiang, Ch.Y., Chen, J.Ch., Ho, T.Y., 2007. Gastrodia elata modulated activator protein 1 via c-Jun N-terminal kinase signaling pathway in kainic acid-induced epilepsy in rats. J. Ethnopharmacol. 109 (2), 241–247. Tang, W., Eisenbrand, G., 1992. Chinese Drugs of Plant Origin, 71.Gastrodia elata Bl. Springer, Berlin, Heidelberg, pp. 545–548. Yong, C.S., Quan, Q.Z., Kim, J.A., Ha, J.H., Lee, D.U., Keun, H., 1999. Pharmacokinetics of 4-hydroxy-3-methoxybenzaldehyde and phydroxybenzaldehyde, constituents of Gastrodia elata, in rats. Yakche Hakhoechi (College of Pharmacy, Yeungnam University, Gyongsan) 29 (1), 47–53. You, J.H., Tan, T.Z., Kuang, A.R., Zhong, Y.G., He, S., 1994. The distribution and metabolism of 3H-gastrodin and 3Hgastrodigenin in mice. J. West China Univ. Med. Sci. 25, 325–328. Yun, L.L., Huang, N.K., 2004. Gastrodia elata prevents rat pheochromocytoma cells from serum-deprived apoptosis: the role of the MAPK family. Life Sci. 75, 1649–1657. Li, L.L., Zhang, Z.R., Gong, T., He, L.L., Deng, L., 2006. Simultaneous determination of gastrodin and ligustrazine hydrochloride in dog plasma by gradient high-performance liquid chromatography. J. Pharmaceut. Biomed. Anal. 41, 1083–1087.
Phytomedicine 15 (2008) 844–850 www.elsevier.de/phymed
Determination and pharmacokinetics of gastrodin and p-hydroxybenzylalcohol after oral administration of Gastrodia elata Bl. extract in rats by high-performance liquid chromatography–electrospray ionization mass spectrometric method W. Zhang, Y.X. Sheng, J.L. Zhang Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China
Abstract In this report, a high-performance liquid chromatography–electrospray ionization mass spectrometric (HPLC–MS) method was used to determine gastrodin (GAS) and p-hydroxybenzylalcohol (HBA) in rat plasma after oral administration of Gastrodia elata Bl. (Chinese name: Tianma) extract. Up to 200 ml of plasma containing GAS, HBA and pyromucic acid (as internal standard, IS) were deproteinized with six volumes of methanol. Calibration curves showed linearity within the concentration range tested 2.00–200.00 mg/ml for GAS and 0.832–104.00 mg/ml for HBA in plasma with a correlation coefficient (r) greater than 0.9997. The limit of quantification of 2.00 and 0.83 mg/ml for GAS and HBA had been achieved, respectively. The intra-day and inter-day precisions of the method were determined to be less than 17.82% for GAS and 10.21% for HBA. The recoveries were in the range of 91.12–108.64% with RSD less than 7.80% for GAS and 92.91–106.14% with RSD less than 4.30% for HBA. Evidence showed that a rapid, simple and reproducible LC–MS assay was established to determine GAS and HBA in rat plasma. r 2008 Elsevier GmbH. All rights reserved. Keywords: Gastrodia elata Bl; Gastrodin; p-Hydroxybenzylalcohol; HPLC–MS; Pharmacokinetics
Introduction Tianma, the dried rhizome of Gastrodia elata Bl. (Fam. Orchidaceae), is an important and notable Chinese herbal medicine which has been used for treatments of headaches, migraine, dizziness, epilepsy, rheumatism, neuralgia, paralysis and other neuralgic and nervous disorders for over 1000 years in China (Tang and Eisenbrand, 1992). Over the past several decades, various chemical and pharmacological studies Corresponding author. Tel.: +86 10 83154880; fax: +86 10 63017757. E-mail address: [email protected] (J.L. Zhang).
0944-7113/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2008.02.012
on Tianma have been carried out to demonstrate its bioactive compounds and pharmacological effects. It was reported that Tianma could improve D-galactoseinduced memory impairment, meliorate the deficit in senescent mice on the step-down passive avoidance task, prevent rat pheochromocytoma and have an anticonvulsive effect (Kao et al., 1994, 1995; Yun and Huang, 2004; Hsieh, 2007). Tianma also has been used as a health supplement and an ingredient in cosmetics. In addition, gastrodin (GAS) (Fig. 1A) and p-hydroxybenzylalcohol (HBA) (Fig. 1B) have been shown to exhibit a scavenging effect on free radicals and consolidate and retrieve memory (Liu and Mori, 1992, 1993; Hsieh, 1997). GAS and HBA are the main active constituents
ARTICLE IN PRESS W. Zhang et al. / Phytomedicine 15 (2008) 844–850
GluO
CH2OH HO
CH2OH
Fig. 1. The chemical structures of (A) GAS and (B) HBA.
of Tianma as reported (Feng, 1979). HBA is the aglycone of GAS. However, to our knowledge, there has been no report on the pharmacokinetic studies of Tianma extract in biological samples. Only three reports related to the pharmacokinetic studies we found were the study of single compound (gastrodin, vanillin, 4-hydroxybenzaldehyde or ligustrazine) in animal models (You et al., 1994; Yong et al., 1999; Li et al., 2006). In the present study, a reproducible and rapid LC–MS method was developed and applied to pharmacokinetic studies of GAS and HBA in rat plasma after oral administration of Tianma extract. The study of the pharmacokinetic parameters of the two active components might provide useful information for clinical treatment.
845
with an ESI/APCI source (Thermo Electron Corporation, USA). The RP-C18 column (Zorbax SB-C18 2.1 100 mm, 3.5 mm) was equipped with a guard column (SB-C18 2.1 10 mm, 3.5 mm). The mobile phase consisted of acetonitrile and 0.1% acetic acid (4.5:95.5) and the flow rate was 0.2 ml/min. The injection volume was 1 ml. For the MS part, the mass spectra were recorded using ESI source in the negative mode with ion spray voltage at 3.0 KV, capillary temperature at 350 1C and cone voltage at 75 V. The scan mode was select ion monitoring (SIM). The deprotonated molecular ions for GAS, HBA and IS were set at m/z 285, 123 and 111, respectively (Fig. 4).
Preparation of Tianma extract Two hundred grams of powder was immersed in deionized water and extracted twice: once with 2000 ml deionized water and a second time with 1600 ml deionized water under sonication for 30 min. Then the supernatants were pooled together and concentrated to 100 ml under reduced pressure at 30 1C. The Tianma extract was obtained and the concentration was 2.0 g/ml.
Materials and methods
Stock and working solutions
Chemicals and reagents
Accurately weighed 4.0 mg of GAS, 5.2 mg of HBA and 3.2 mg of IS were dissolved with 2 ml of methanol (in a volumetric flask) and used as stock solution. The concentrations were approximately 2.0, 2.6 and 1.6 mg/ml, respectively. All stock solutions were stored at 4 1C before analysis. Different volumes of GAS and HBA stock solutions were dissolved with 1 ml of methanol as working solutions.
The dried rhizome of Tianma purchased from TongRenTang (Beijing, China) was ground to powder, and then passed through a 60-mesh sieve for preparation. The powder was stored at about 4 1C before use. Standards of GAS and furoic acid (as internal standard, IS) were obtained from the National Institute for the Control of Biological and Pharmaceutical Drugs (Beijing, China). HBA of HPLC grade was purchased from Fluka (Switzerland). Methanol and acetonitrile were of HPLC grade from Burdick & Jackson Company (Muskegon, MI, USA). Acetic acid and ethyl ether were of analytical grade from Peking Chemical Factory, and deionized water from a Milli-Q system (Millipore, Bedford, MA, USA) was used for all preparations. Physiological saline was purchased from Shijiazhuang Pharmaceutical Cooperation Factory (Hebei Province, China). Heparin sodium was from Changzhou Qianhong Biochemical Pharmaceutical Co. Ltd. Heparin sodium was dissolved in physiological saline at the ratio of 1–25. Heparin solution was used to rinse the test tubes prior to blood collection for plasma.
Instruments and conditions The experiments were performed on a FinniganTM Surveyor LC-MSQTM Plus mass spectrometer equipped
Preparation of standards and quality control (QC) samples To 200 ml blank plasma samples, 10 ml of working solution and 10 ml of IS solution were added to prepare biological calibration curves and quality control samples. The final concentrations of biological calibration standards were within the range of 2.00–200.00 mg/ml for GAS and 0.83–104.0 mg/ml for HBA. Quality control samples were prepared at concentrations of 2.00, 10.00 and 200.00 mg/ml for GAS and 0.83, 2.04 and 104.00 mg/ml for HBA.
Sample preparation To 200 ml plasma samples, 10 ml of IS solution was added. After being vortexed for 15 s, 1.2 ml of methanol was added and vortexed for 30 s to precipitate protein. The mixture was centrifuged at 5000 rpm for 5 min.
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W. Zhang et al. / Phytomedicine 15 (2008) 844–850
The supernatant was evaporated to dryness under a reduced pressure at 30 1C. The residue was reconstituted in 200 ml of methanol, and then was filtered through 0.45 mm filter for LC/MS analysis.
Validation of the method Linearity The calibration curve of GAS was constructed by calculating the peak area ratios (y) of GAS to IS against GAS concentrations (x). The calibration curve of HBA was processed the same as that of GAS. The limit of quantification (LOQ) was defined as the lowest limitation of quantification concentrations of GAS and HBA measured with an acceptable precision (less than 20%) and accuracy (within the range of 80–120%). Precision and accuracy The precision and accuracy of the method were performed by analyzing QC samples with low, medium and high concentrations. Intra-day precision and accuracy (each, n ¼ 6) were evaluated by analysis of QC samples at different times during the same day. Inter-day precision and accuracy were determined by repeated analysis of QC samples over 5 consecutive days (n ¼ 1 series per day). The measured concentrations of the QC samples were calculated from the biological calibration curves. The precision was evaluated by the intra-day and inter-day variability with relative standard deviation (RSD %). The accuracy was obtained by measured concentration of QC samples divided by spiked concentration. Recovery The recoveries of GAS and HBA were investigated by comparing the GAS/IS and HBA /IS peak area ratio of blank plasma spiked with standard solution with that of the standards spiked in biological samples at the concentrations of 2.00, 10.00 and 200.00 mg/ml for GAS and 0.83, 2.04 and 104.00 mg/ml for HBA. Stability The stability was investigated by determining the QC samples at 0, 2, 4, 8, 12 and 24 h at room temperature. The effects of three freeze–thaw cycles were evaluated by repeated analysis (n ¼ 3) of QC samples.
Pharmacokinetic study Forty male Wistar rats weighing 200710 g were provided by Institute of Laboratory Animal Science. Rats were fasted in metabolic cages for 24 h and maintained on physiological saline prior to oral administration of Tianma extract at the dose of 10 ml/kg body
weight. This approximately corresponded to 40 mg/kg of GAS and 4 mg/kg of HBA. After 5 min oral administration of pharmacological saline at the dose of 10 ml/kg body weight, four rats were anesthetized with ethyl ether and blank blood was collected from abdominal artery in clean heparinized glass tubes and used as blanks. Blank plasma was separated by centrifugation at 3500 rpm for 5 min. The remaining 36 rats were divided into 6 groups with 6 rats per group. Blood was collected at different time points (5, 15, 30, 45, 70, 100, 120, 150, 165, 180, 210 and 240) after oral administration of Tianma extract. Blood samples collected from rats given Tianma at different time points mentioned above were prepared for analysis by the methods cited above. The concentrations of GAS and HBA in plasma were determined at each time point. By using the software of WinNonlin, the pharmacokinetic parameters, such as the maximum plasma concentration (Cmax) and the corresponding time (Tmax), the absorption phase (k01), elimination phase (k10), plasma clearance (CL) as well as apparent volume of distribution (Vd), were calculated. The area under the plasma concentration–time curve (AUC0–t) was calculated using trapezoidal rule to the last point.
Results and discussion Extraction of GAS and HBA in rat plasma To reduce the endogenous-related substances in plasma the method of precipitation of protein was performed. Methanol was used as the precipitant and we tried various ratios of methanol and plasma. When the ratio was 6 to 1 (equal to 1.2 ml of methanol to 0.2 ml of plasma) the effect of protein precipitation was the best.
Optimization of HPLC–MS conditions The selection of the LC–MS conditions for assay was guided by requirement for obtaining chromatograms with resolution of tested constituents and IS in rat plasma. To obtain the LC portion with good resolution, column and mobile phase were investigated separately. A Zorbax SB-C18 (2.1 100 mm, 3.5 mm) was used. The mobile phase consisting of acetonitrile (4.5%) and water (95.5%) with acetic acid at 0.1% yielded the best separation and allowed for subsequent MS analysis. During the chromatographic run, the mass spectrometer was programmed for three SIM events, which allowed for optimal detection of each constituent. Finally the elution flow rate at 0.2 ml/min to ensure the LC–MS assay had good resolution within 10 min.
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Intensity
W. Zhang et al. / Phytomedicine 15 (2008) 844–850
847
700 600 500 400 300 200 100 0 700 600 500 400 300 200 100 0 1400 1200 1000 800 600 400 200 0 0
1
2
3
4
5
6
7
8
9
Intensity
Time (min)
1400 1200 1000 800 600 400 200 0 3000 2500 2000 1500 1000 500 0 1400 1200 1000 800 600 400 200 0
IS
HBA
GAS
0
1
2
3
4
5
6
7
8
9
7
8
9
Time (min)
Intensity
1400 1200 1000 800 600 400 200 0 5000
IS
4000 3000
HBA
2000 1000 0 1400 1200 1000 800 600 400 200 0
GAS
0
1
2
3
4
5
6
Time (min)
Fig. 2. Representative chromatograms of LC–MS. (A) Blank plasma, (B) blank plasma spiked with GAS, HBA and IS, (C) plasma at 45 min after oral administration of Tianma extract; ‘‘a’’ represents the m/z of 111, ‘‘b’’ represents the m/z of 123 and ‘‘c’’ represents the m/z of 285.
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were linear within the concentration range of 2.00–200.00 mg/ml for GAS and 0.83–104.0 mg/ml for HBA, with the correlation coefficient more than 0.9997. The LOQ was defined as the lowest limitation of quantification concentrations of GAS and HBA measured with an acceptable precision (less than 20%) and accuracy (within the range of 80–120%). And the LOQs were 2.00 mg/ml for GAS and 0.83 mg/ml for HBA. The calculated results are given in Table 1.
Specificity The typical SIM chromatograms of reference standards of tested compounds and IS are shown in Fig. 2 together with the chromatogram of blank plasma. The identification of the GAS and HBA peaks was achieved by comparing retention time and mass spectra with commercial standards. The retention times were 4.6, 5.4 and 7.6 min for GAS, IS and HBA, respectively. All the major constituents were well separated and no interfering peak was found in the chromatogram of blank plasma.
Precision and accuracy The RSD of the overall intra- and inter-day precisions for GAS and HBA were less than 17.82%. The accuracy for GAS and HBA at low, middle and high concentrations was within the range of 82.0–105.8%. The results are shown in Table 2.
Calibration curves and limit of quantification (LOQ) Plasma spiked with six different concentrations of GAS and HBA were processed. The calibration curves
Table 1.
Calibration curves for GAS and HBA in rat plasma
Sample
Standard curve
Range (mg/ml)
Correlation coefficient (r)
GAS HBA
Y ¼ 0.073X0.07 Y ¼ 0.23X+0.20
2.00–200.00 0.83–104.00
0.9997 0.9998
The calibration curve in plasma. Y, peak area ratio (GAS or HBA/IS); X, concentration of GAS or HBA in blood samples (mg/ml).
Table 2. Sample
Intra-day and inter-day precision and accuracy of GAS and HBA in rat plasma Spiked (mg/ml)
Intra-day (n ¼ 6)
Inter-day (n ¼ 6)
Measured (mg/ml)a
RSD (%)
Accuracy (%)b
Measured (mg/ml)
RSD (%)
Accuracy (%)
GAS
2.00 10.00 200.00
1.6470.14 9.8870.54 187.0676.52
8.5 5.5 3.3
82.0 98.8 93.5
1.7270.31 9.8070.54 201.81711.68
17.8 5.5 5.8
86.0 98.0 100.9
HBA
0.83 2.08 104.00
0.8870.024 2.0370.16 103.3873.89
2.7 7.9 3.8
105.8 97.6 99.4
0.8770.09 2.0270.14 100.8076.98
10.2 6.9 6.9
104.6 97.1 96.9
a
Mean7SD. Accuracy ¼ (mean of measured concentration/spiked concentration) 100.
b
Table 3.
Recovery of GAS and HBA in rat plasma
Sample
Spiked (mg/ml)
Measured (mg/ml)
Recovery (%)a
RSD (%)
GAS
2.00 10.00 200.00
2.1770.14 9.1170.71 199.9179.54
108.677.2 91.177.1 99.974.8
6.7 7.8 4.8
HBA
0.83 2.08 104.00
106.172.9 92.974.0 94.271.9
2.7 4.3 1.9
a
0.8870.024 1.9370.083 99.9871.95
Recovery ¼ (mean of measured concentration/spiked concentration) 100.
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Recovery The overall recovery of GAS and HBA ranged from 91.1% to 108.6% with RSD less than 7.8%. The results are shown in Table 3.
Stability The results of the stability of GAS and HBA in plasma kept for 24 h at room temperature and after three freeze (10 1C)/thaw cycles are shown in Table 4. GAS and HBA were stable in plasma at room temperature or after three freeze–thaw cycles at 10 1C.
Application – pharmacokinetic study The described method was applied to analysis of plasma samples after oral administration of Tianma extract. Fig. 3 shows the mean plasma concentration– time profile of GAS (n ¼ 6). The mean pharmacokinetic parameters of GAS after oral administration of Tianma extract are shown in Table 5 and by fitting plasma data a two-compartment model was calculated. From the above data, we found that GAS in rat plasma was detected at 15 min after oral administration, and reached
Table 4. Stability of GAS and HBA in rat plasma kept for 24 h at room temperature and during three freeze–thaw cycles Sample
Spiked (mg/ml)
Mean of percentage remaining (%) 24 h at room temperature
Three freeze–thaw cycles
2.00 10.00 200.00
87.0 92.7 105.4
86.5 95.9 102.1
HBA
0.83 2.08 104.00
116.9 89.9 73.2
109.6 97.1 92.1
Concentration (µg/mL)
GAS
849
the Cmax at 70 min. GAS was rapidly eliminated from the plasma and could be detected up to 240 min after oral administration by the established method. It suggested that GAS could not be easily accumulated in rat plasma. The plasma concentration of GAS started to increase again at 165 min and had a second peak at 180 min. It may indicate the enterohepatic circulation of GAS. But it still needs further investigation to prove whether the enterohepatic circulation exists. Another active constituent, HBA, could not be detected in rat plasma at the beginning time points by the established methods and detected only at 45, 70, 100 and 120 min after oral administration of Tianma extract. The results might be due to the very low content of HBA in Tianma extract. On the other hand, from the structures of GAS and HBA, HBA is the aglycone of GAS. HBA detected in rat plasma at later time points might be the metabolite of GAS (Fig. 4).
Conclusion A simple, specific, rapid and reproducible LC–MS method has been developed to determine the active constituents GAS and HBA in rat plasma after oral administration of Tianma extract. Sample preparation Table 5. Mean pharmacokinetic parameters of GAS in rat plasma (n ¼ 6) after oral administration Parameter
Unit
Value
Cmax Tmax AUC0t k01 k10 Vd CL
mg/ml min minmg/ml min min ml ml/min
13.48 70 1869.91 36.47 43.42 267.99 4.28
All pharmacokinetic parameters are calculated from the mean plasma concentrations.
20 18 16 14 12 10 8 6 4 2 0 0
50
100
150 Time (min)
200
250
300
Fig. 3. Mean plasma concentration–time profile of GAS in rat plasma after oral administration. Each point represents the mean7SD.
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Intensity
850 50000 48000 46000 44000 42000 40000 38000 36000 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0
GAS
HBA
0
1
2
3
4
5
6
7
8
9
Time (min)
Fig. 4. HPLC chromatogram of Tianma extract.
was simple and reliable. The pharmacokinetic parameters obtained here could provide more information for absorption studies of GAS. The results provide scientific data for the further study and scientific clinical use of Tianma extract.
Acknowledgement We thank the Ministry of Science and Technology of the People’s Republic of China (ke(2004-05)) and Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine (Peking Union Medical College) of Ministry of Education of the People’s Republic of China for financial support of this work.
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