Accepted Manuscript Title: Simultaneous determination and pharmacokinetic study of eight components in rat plasma by UHPLC–MS/MS after oral administration of Hypericum japonicum Thunb extract Author: Qian Pang Yuanyuan Tian Jianping Mi Jin Wang Yuanjin Xu PII: DOI: Reference:
S0731-7085(15)30200-4 http://dx.doi.org/doi:10.1016/j.jpba.2015.10.027 PBA 10303
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
18-6-2015 10-10-2015 19-10-2015
Please cite this article as: Qian Pang, Yuanyuan Tian, Jianping Mi, Jin Wang, Yuanjin Xu, Simultaneous determination and pharmacokinetic study of eight components in rat plasma by UHPLCndashMS/MS after oral administration of Hypericum japonicum Thunb extract, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.10.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous determination and pharmacokinetic study of eight components in rat plasma by UHPLC–MS/MS after oral administration of Hypericum japonicum Thunb extract a,b
a,b
a,c
a,b
Qian Pang , Yuanyuan Tian , Jianping Mi , Jin Wang ， Yuanjin Xu
a,b*
[email protected]
a
State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004,
China b
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China, Beihai Center for Disease Control
and Prevention, Beihai 536000,China ∗Corresponding author at: State Key Laboratory of Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi
University, Nanning 530004, China. Tel.: +860771-3237743.
1
Graphical Abstract
2
Highlights Simultaneous determination of eight active constituents in rat plasma Pharmacokinetic study of eight active constituents by UHPLC–MS/MS The first report on pharmacokinetic study of Hypericum japonicum Thunb by UHPLC–MS/MS
3
Abstract
A rapid and sensitive assay based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) was established and validated for the simultaneous determination of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin in rat plasma using catechin and daidzein as the internal standards (IS). Plasma samples added internal standards were acidified with formic acid then pretreated by direct protein precipitation with acetonitrile. The separation of eight constituents was achieved on a C18 column with gradient elution using methanol and 0.2% acetic acid aqueous solution as the mobile phase and detected by multiple reaction monitoring using electrospray ionization source in the positive-negative ionization mode. The method was validated for sufficient specificity, precision, accuracy, and sensitivity over the concentration range of 10–6000 ng·mL-1 for gallic acid, 1.5–3000 ng·mL-1 for protocatechuic acid, 10–15000 ng·mL-1 for vanillic acid, 2–3600 ng·mL-1 for caffeic acid, 1.5–3600 ng·mL-1 for epicatechin, 4–6000 ng·mL-1 for isoquercitrin, 2–9000 ng·mL-1 for vincetoxicoside B, and 20–18000 ng·mL-1 for quercetin. The overall intra-run precision and the inter-run precision were showed in the range of 1.0%–14.2% and 2.8%–12.9%, respectively, and the accuracy was no more than 12.8%. This analytical method was successfully applied to investigate the pharmacokinetics of eight ingredients in rats after oral administration of Hypericum japonicum Thunb extract. Keywords: Hypericum japonicum Thunb; Pharmacokinetics; UHPLC–MS/MS; Rat plasma
4
1. Introduction Hypericum japonicum Thunb, a member of genus Hypericum L. (Clusiaceae/Hypericaceae), is an annual or perennial herb widely distributed in Asia, North America and Oceania [1]. It has been widely used for the therapy of the infectious hepatitis, hemostasis, detumescence, dysentery and relieving internal heat or fever
[1-2]
.Many pharmacological studies have demonstrated that its
dried entire plant possesses antibacterial, antioxidant, antihypoxic and hepatoprotective activities [3-5]
. Phytochemical investigations of Hypericum japonicum Thunb have indicated many
ingredients such as flavonoids, phenolic acids, xanthones, phloroglucinols and chromones
[6]
.
Among them, phenolic acids and flavonoids are the main bioactive constituents which contribute to the pharmacological efficacy in Hypericum japonicum Thunb. Based on a tremendous amount of previous pharmacological researches, phenolic acids (gallic acid, protocatechuic acid, vanillic acid and caffeic acid) and flavonoids (epicatechin, isoquercitrin, vincetoxicoside B and quercetin) strikingly manifest a variety of pharmacological properties, including antioxidant, antimicrobial, anti-inflammatory and antitumour functions
[7-11]
. In addition, it has displayed good effect in
clinical application of hepatitis [12]. Most of previous studies have been concentrated on the phytochemical and other pharmacological investigation of Hypericum japonicum Thunb. However, apart from quercitrin and isoquercitrin [13], no research reports on pharmacokinetics of other components of Hypericum japonicum Thunb was available. It is well known that Traditional Chinese Medicine (TCM) has thousands of years of history in China that play a crucial role in the disease protection, control, and treatment. But there is a considerable lack in therapeutic mechanism understanding due to its manifold and complicated constituents. Pharmacokinetic data may help to illustrate and foresee
5
efficacy and toxicity of the herb medicine and to optimize dose regiment and reduce adverse effects [14]. In this study, a rapid, sensitive and robust ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) was established for the simultaneous determination of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin of Hypericum japonicum Thunb in rat plasma using catechin and daidzein as the internal standards. The method was fully validated and successfully applied to the pharmacokinetics after oral administration of Hypericum japonicum Thunb extract to rats. The results may supply some valuable references for the apprehension of pharmacological action mechanism of Hypericum japonicum Thunb. 2. Experimental 2.1. Chemicals, reagents and animals The standards of gallic acid, protocatechuic acid, caffeic acid and epicatechin were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Standards substances of isoquercitrin, vincetoxicoside B, quercetin and catechin (IS) were obtained from Chengdu Pufeide Biological Technology Co., Ltd. (Sichuan, China). The standards of vanillic acid and daidzein (IS) were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Sichuan, China).The purity of each standard substance was more than 98%. Methanol and acetonitrile of HPLC grade were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Ultra-pure water used in the experiment was generated by a Milli-Q water purification system (Millipore Corporation, Billerica, USA). Other chemicals were all analytical reagents.
6
Ultra-pure water was used throughout and all solutions were passed through 0.22 μm pore size filters. Hypericum japonicum Thunb (Batch No.: 140701) was obtained from Nanning Shengyuan Chinese Traditional Medicine Co., Ltd. (Guangxi, China) and was authenticated by professor Xuejian Li in Guangxi University of Chinese Medicine. We kept the voucher specimens in reserve in State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University. Sprague-Dawley (SD) rats (280 ± 20 g) were obtained from the Animal Center of Guangxi Medical University (Guangxi, China). The rats were maintained in an air-conditioned room under the constant room temperature (23 ± 2 °C), relative humidity level (50 ± 15%), and lighting (12 h light/dark cycle) with free access to food and water for 7 days before the experiment. Before the herb extract administration, the rats were deprived of food for 12 h but had access to water spontaneously. The experimental protocols were reviewed and approved by Laboratory Animal Care Committee of Guangxi Department of Science and Technology. 2.2. UHPLC–MS/MS apparatus and operation conditions
The UHPLC–MS/MS is composed of an Agilent 1290 ultra high performance liquid chromatography system (Agilent Co., USA), equipped with a binary pump solvent delivery system, an autosampler, and an online degasser. The chromatographic separation was achieved on a ZORBAX RRHD Eclipse Plus C18 column (2.1 x 50 mm, 1.8 μm) [Guangzhou Dexiang Science and Technology Co., Ltd. (Guangdong, China)] at the temperature of 25 °C. The mobile phase was consisted of 0.2% acetic acid aqueous solution (A) and methanol (B) using a gradient elution as follow: 0–6 min, linear 7
changed from 10% to 18% B, 6–6.5 min, linear changed from 18% to 52% B, 6.5–9 min, linear changed from 52% to 56% B, 9–9.5 min, linear changed from 56% to 68% B, 9.5–10.5 min, linear changed from 68% to 80% B, 10.5–12 min, linear changed from 80% to 100% B, 12–17 min, isocratic elution with 100% B, and then quickly returned to the initial condition. The flow rate was set at 0.3 mL·min-1 and the injection volume was 1 μL for analysis. Mass spectrometry detection of all the analytes, including the IS, was performed using triple quadrupole 6460 mass spectrometer (Agilent Co., USA) with an electrospray ionization source (ESI) source interface set in positive and negative ionization mode. Multiple reaction monitoring (MRM) using the precursor–product ion transitions of m/z 169.1–125.1, 153.1–109.1, 169.0–92.9, 179.1–135.1,
291.1–139.1,
463.1–300.1,
447.0–301.0,
303.0–152.9,
291.1–138.9
and
255.1–137.1 were employed for the quantification of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B, quercetin, catechin (IS) and daidzein (IS), respectively. The optimal MS parameters were as follow: capillary voltage 4000 V (ESI +), 3000V (ESI−); nozzle voltage 0 V; spray voltage 4000 V and nebulizer gas (nitrogen) pressure 2.8 x 105 Pa. Nitrogen was used as the desolvation and sheath gas with a flow rate of 10 L min-1 and 12 L min-1, respectively. The desolvation gas was heated to 300 °C and the temperature of the sheath gas was set at 360 °C. High purity nitrogen was served as the collision gas. 2.3. Preparation of the Hypericum japonicum Thunb extract The dried herb of Hypericum japonicum Thunb were powdered to a homogeneous size in a mill, passed through a 50-mesh sieve. 90 g of accurately weighed powder was soaked in 60% ethanol (1:50, w/v ) for 12 h, and then extracted in an ultrasonic bath (200 W, 40 kHz) for 45 min. The mixed extract was subjected to centrifugate at 5000 x g for 40 min. The supernatant was
8
evaporated to dryness using the evaporator at a constant temperature of 36 °C. To calculate the administered dose, the contents of eight components in Hypericum japonicum Thunb were measured quantitatively by an external standard assay using the same chromatography conditions as described above. The contents of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin were 0.69, 4.33, 1.22, 0.59, 4.60, 46.52, 82.12 and 9.48 mg·g-1 in extract respectively. 2.4. Preparation of calibration standard and quality control (QC) samples Stock solutions of eight analytes and IS at the concentration of 1000 μg·mL-1 for each were prepared by dissolving the accurately weighed reference standards in methanol, respectively. A serious of standard working solutions was obtained by future diluting the stock solutions. Mixed calibration standard samples containing gallic acid 10, 30, 150, 750, 1500, 3000, 4500, and 6000 ng·mL-1, protocatechuic acid 1.5, 5, 25, 120, 360, 720, 1500, and 3000 ng·mL-1, vanillic acid 10, 90, 450, 1800, 3600, 5400, 8100, and 15000 ng·mL-1, caffeic acid 2, 6, 30, 150, 450, 900, 1800, and 3600 ng·mL-1, epicatechin 1.5, 15, 45, 150, 450, 900, 1800, and 3600 ng·mL-1, isoquercitrin 4, 10, 50, 250, 750, 1500, 3000, and 6000 ng·mL-1, vincetoxicoside B 2, 15, 50, 250, 750, 1500, 3000, and 9000 ng·mL-1, quercetin 20, 150, 600, 1800, 5400, 8100, 12000, and 18000 ng·mL-1 were obtained by spiking 100 μL aliquots of blank plasma with 10μL working solutions of corresponding concentrations. For the validation and pharmacokinetic study of the assay, four concentrations of the standard solution, including gallic acid (30, 300, 800, and 3000 ng·mL-1), protocatechuic acid (4, 80, 400, and 2000 ng·mL-1), vanillic acid (30, 600, 2400, and 12000 ng·mL-1), caffeic acid (6, 100, 400, and 2000 ng·mL-1), epicatechin (3.5, 90, 500, and 2700 ng·mL-1), isoquercitrin (10, 120, 600, and 3000 ng·mL-1), vincetoxicoside B (5, 300, 1500, and
9
7500 ng·mL-1), and quercetin (60, 600, 3000, and 12000 ng·mL-1) were applied to prepared the quality control (QC) samples. The standards and quality control (QC) samples were extracted on each analysis day with the same processes for plasma samples preparation as described below. 2.5. Preparation of plasma samples All of the plasma samples were executed by direct protein precipitation with acetonitrile. Concretely, a 100 μL aliquot of blank plasma were spiked with 10 μL of the IS solution, 10 μL of the methanol (volume of the corresponding working solution for the calibration curve and the quality control samples) in a 1.5 mL Eppendorf tube. The sample was extracted with 250 μL of acetonitrile containing 0.1% formic acid by vortex mixing for 1 min and centrifugated at 12000 x g for 20 min. The supernatant was transferred into another Eppendorf tube and evaporated to dryness by thickener at room temperature. The obtained residue was reconstituted with 100μL methanol and centrifugated at 12000 x g for another 20 min. Finally, the supernatant was transferred into an autosample vial and an aliquot of 1μL of the sample solution was injected into the UHPLC–MS/MS system for analysis. 2.6. Method validation The assay was validated for specificity, selectivity, calibration curve, sensitivity, accuracy, precision, stability, matrix effect and recovery, in accordance with the USA Food and Drug Administration (FDA) bioanalytical method validation guidance [15]. 2.6.1. Specificity and selectivity Selectivity is the ability of an analytical method to differentiate and quantify the analyte in the presence of other components in the sample
[15]
. In our study, the selectivity and specificity
toward the endogenous plasma matrix constituents was ascertained by comparing the
10
chromatograms of blank rat plasma from six sources, blank plasma spiked with IS and the analytes at the concentrations of the lower limit of quantification (LLOQ) and the plasma samples from the rats after oral administration of Hypericum japonicum Thunb extract. 2.6.2. Linearity and sensitivity The linearity of the assay was generated by analysis of eight plasma calibration curves consisted of eight concentration levels. The plasma calibration curves were determined by plotting the peak-area ratios (y) of eight analytes to IS versus the corresponding nominal concentration (x) of analytes using the weighted (1/x2) least least-squares linear regression model. The linear regression analysis with 1/x2 weighting factor was performed to determine the intercept, slope and correlation coefficient (r). The sensitivity of the method was evaluated by determining the lower limit of quantification (LLOQ) and limit of detection (LOD). LLOQ and LOD were determined via the signal-to-noise ratio (S/N) of 10:1 and 3:1, respectively. The LLOQ was defined as the lowest concentration on the calibration curve that could be ascertained with an acceptable accuracy within ±20% and a precision less than 20%, which was evaluated based on six replicated samples. 2.6.3. Precision and accuracy For the sake of evaluating the intra- and inter-day precision and accuracy, QC samples at four concentration levels of the eight analytes were analyzed in six replicates on the same day and on four separate days, respectively. The precision in determination was expressed as the relative standard deviation (RSD) and the accuracy was estimated by means of calculating the percent deviation of the measured mean value that observed in the analysis of QC samples deviated from the nominal value and defined as relative error (RE).
11
2.6.4. Extraction recovery and matrix effect The extraction recoveries of the analytes in this assay were estimated by comparing peak area ratios from QC samples regularly pretreated with those obtained from post-extracted supernatants spiked with the pure reference standards at the same concentrations. The matrix effects for the constituents were assessed and calculated by the ratio of the detector response for acetonitrile precipitated-blank boi-samples spiked with standard solutions to the response for the corresponding standard solutions prepared by dilution in methanol. The extraction recoveries and matrix effects of the IS were determined in the similar procedures. 2.6.5. Stability The QC plasma samples at four concentrations were subjected to analyze under the various circumstances containing short-term stability (storage for 12 h at ambient temperature), long-term stability (storage for 30 days at −20 °C), post-treatment stability (storage for 24 h in an auto-sampler) and freeze–thaw stability (three freezes at −20 °C and thaw cycles). The results were utilized to assess the stability. 2.7. Pharmacokinetic study in rat plasma The proposed method was applied to determine the plasma concentration of eight active components for a pharmacokinetic study conducted in six healthy Sprague–Dawley rats weighing 280 ± 20 g. The rats were fasted in the last 12 hours before the experiment with the exception of free access to water. The dosing solution was prepared by dissolving appropriate of Hypericum japonicum Thunb extract in normal saline solution. Hypericum japonicum Thunb extract were given orally to the rats at a dose of 2.5 g·kg-1 body weight (equivalent to 1.73 mg·kg-1 of gallic acid, 10.83 mg·kg-1 of protocatechuic acid, 3.05 mg·kg-1 of vanillic acid, 1.48 mg·kg-1 of caffeic
12
acid, 11.50 mg·kg-1 of epicatechin, 116.30 mg·kg-1 of isoquercitrin, 205.30 mg·kg-1 of vincetoxicoside B and 23.70 mg·kg-1 of quercetin). Retro-orbital blood samples (250 μL) were collected into the sodium heparinized tubes before drug administration and at different time intervals post-dosing (0.083, 0.25, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h). The heparinized blood samples were centrifuged at 1800 x g at a constant temperature of 4 °C for 30 min to obtain plasma, which was stored at −20 °C until analysis. All the pharmacokinetic parameters were calculated by compartmental analysis using 3P97 pharmacokinetic program (Mathematical Pharmacology Professional Committee of China, Shanghai, China). 3. Results and discussion 3.1. Assay validation 3.1.1. Selectivity and specificity The selectivity and specificity were assessed using independent plasma samples derived from six different rats. The typical chromatograms for the drug-free plasma, drug-free with analytes at LLOQ and IS and an in vivo rat plasma sample after oral administration of Hypericum japonicum Thunb are shown in Figure 1. As shown in Figure 1, all the peaks can be confirmed and there is no significant interference from the endogenous substances observed at the retention time of each component. 3.1.2. Linearity and sensitivity As for plasma calibration curve, the ratios of the peak areas (analytes/IS) as ordinate variables were plotted versus the corresponding nominal concentration of analytes as abscissa. After comparing three weighting models (x, 1/x and 1/x2), the 1/x2 weighing factor was chosen to achieve the best linear fit and least-square residual for the calibration curve. The ratios of the peak
13
areas (analytes/IS) as ordinate variables were plotted versus the corresponding nominal concentration of analytes as abscissa. All the eight ingredients exhibited good linearity with correlation coefficients (r) within the range of 0.9989–0.9995. 3.1.3. Precision and accuracy The results of the intra- and inter-day precision and accuracy of the eight analytes are summarized in Table 1. In the present assay, the overall intra- precision and the inter-day precision were showed in the range of 1.0%–14.2% and 2.8%–12.9%, respectively, and the accuracy was no more than 12.8%. The results indicated that the method was reliable for the determination of Sprague-Dawley rat plasma samples. 3.1.4. Extraction recovery and matrix effect Table 2 summarized the extraction recovery and matrix effects of eight analytes. Based on the data displayed in Table 2, the extraction recovery of the investigated components in plasma at four concentration levels ranged from 76.6% to 113.7%. The assessment of matrix effect of the assay was performed systematically and all the ratios of the analytes were found to be within the acceptable range (84.9%–116.2%). The results indicated that there was no significant ion suppression or enhancement from plasma for this assay. 3.1.5. Stability The stability of the assay was conducted strictly under different circumstances. The results indicated that eight analytes were stable in plasma samples after three freeze–thaw cycles (RE: −11.8% to 11.5%, RSD < 9.6%) and at room temperature for 12 h (RE: −14.6% to 14.0%, RSD < 10.9%). Post-preparative stability of the analytes also declared that no obvious loss occurred when the extracted samples were stored in an auto-sampler for 24 h (RE: −14.2% to 7.9%, RSD
vanillic acid > caffeic acid > gallic acid. Following a single oral administration of total flavonoids from Hypericum japonicum Thunb, double peaks were emerged in curves of mean plasma concentration for epicatechin and quercetin. This phenomenon had been confirmed in the previous literature
[16-17]
. It may be explained by distribution, re-absorption and enterohepatic
15
circulation. Meanwhile, it should be pointed out that the area under concentration–time curve (AUC) of quercetin was much higher than other components. The factor that may contribute to this phenomenon can be explained by flavonoid glycosides (isoquercitrin and vincetoxicoside) were released as the aglycone in the intestinal tract by hydrolytic action of the bacteria
[18]
. In
general, the information described in present study would be informative for future application of herbal medicine in clinical therapy. 4. Conclusions In the present study, a sensitive and reliable UHPLC–MS/MS method was established for simultaneous determination of eight components following oral administration of Hypericum japonicum Thunb extract in rat plasma. The analysis assay is simple and fast because of the straightforward sample pre-treatment procedure and its relative short analysis running time. This is the first report of pharmacokinetic studies of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin in vivo after oral administration of Hypericum japonicum Thunb extract. This assay could offer a scientific basis for the further research of Hypericum japonicum Thunb. Acknowledgements This research was supported by the National Natural Science foundation of China (No. 20865001), the Guangxi Province Natural Science foundation (0832034).
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References [1] Editorial board of Flora of China, Flora of China, Vol. 50, Science Press, Beijing, China, 1990, pp. 47–48. [2] Jiangsu New Medical College, Dictionary of Chinese Traditional Medicine (Zhong Yao Da Ci Dian), ShangHai People’s Publishing House, ShangHai, China, 1977, pp. 813–814. [3] P.V. Samaga, V.R. Rai, Evaluation of pharmacological properties and phenolic profile of Hypericum japonicum Thunb from Western Ghats of India, J. Pharm. Res. 7 (2013) 626–632. [4] X.W. Wang, Y. Mao, N.L. Wang, X.Sh. Yao, A New Phloroglucinol Diglycoside Derivative from Hypericum japonicum Thunb., Molecules 13 (2008) 2796–2803. [5] N. Wang, P.B. Li, Y.G. Wang, W. Peng, Zh. Wu, S.Y. Tan, Sh.L. Liang, X. Shen, W.W. Su, Hepatoprotective effect of Hypericum japonicum extract and its fractions, J. Ethnopharmacol. 116 (2008) 1–6. [6] W.N. Gao, J.G. Luo, L.Y. Kong, Quality evaluation of Hypericum japonicum by using high-performance liquid chromatography coupled with photodiode array detector and electrospray ionization tandem mass spectrometry, Biomed. Chromatogr. 23 (2009) 1022–1030. [7] M. Cotoras, H. Vivanco, R. Melo, M. Aguirre, E. Silva, L. Mendoza, In Vitro and in Vivo Evaluation of the Antioxidant and Prooxidant Activity of Phenolic Compounds Obtained from Grape (Vitis vinifera) Pomace, Molecules 19 (2014) 21154–21167. [8] F. Fratianni, R. Pepe, F. Nazzaro, Polyphenol Composition, Antioxidant, Antimicrobial and Quorum Quenching Activity of the “Carciofo di Montoro” (Cynara cardunculus var.
17
scolymus) Global Artichoke of the Campania Region, Southern Italy, Food Nutr. Sci. 5 (2014) 2053–2062. [9] E.O. Sousa, C.M.B.A. Miranda, C.B. Nobre, A.A. Boligon, M.L. Athayde, J.G.M. Costa, Phytochemical analysis and antioxidant activities of Lantana camara and Lantana motevidensis extracts, Ind. Crops Prod. 70 (2015) 7–15. [10] C.H. Lu, Y.Y. Li, L.J. Li, L.Y. Liang, Y.M. Shen, Anti-inflammatory activities of fractions from Geranium nepalense and related polyphenols, Drug Discoveries Ther. 6 (2012) 194–197. [11] Q. Chen, P. Li, P. Li, Y. Xu, Y. Li, B. Tang, Isoquercitrin inhibits the progression of pancreatic cancer in vivo and in vitro by regulating opioid receptors and the mitogen-activated protein kinase signalling pathway, Oncol. Rep. 33 (2015) 840–848. [12] H. Lin, Q.X. Mei, X.L. Kong, Y.T. Wang, Z.L. Chen, The clinical application of Hypericum japonicum Thunb in hepatopathy and its research survey on pharmacological action, Pharmacy Today. 21 (2011) 550–552. [13] J. Li, Zh.W. Wang, L. Zhang, X. Liu, X.H. Chen, K.Sh. Bi, HPLC analysis and pharmacokinetic study of quercitrin and isoquercitrin in rat plasma after administration of Hypericum japonicum thunb· extract, Biomed. Chromatogr. 22 (2008) 374–378. [14] Y. Wang, Ch.H. Xu, P. Wang, X.Y. Lin, Y. Yang, D.H. Li, H.F. Li, X.Zh. Wu, H.B. Liu, Pharmacokinetic comparisons of different combinations of Shaoyao-Gancao-Decoction in Rats: Simultaneous determination of ten active constituents by HPLC–MS/MS, J. Chromatogr. B 932 (2013) 76–87. [15] FDA, Guidance for Industry: Bioanalytical Method Validation, Center for Drug
18
Evaluation and Research, US Department of Health and Human Services, US, FDA, Rockville, MD, 2001. [16] K.F. Duan, Zh.F. Yuan, W. Guo, Y. Meng, Y. Cui, D.Zh. Kong, L.T. Zhang, N. Wang, LC–MS/MS determination and pharmacokinetic study of five flavone components after solvent extraction/acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract, J. Ethnopharmacol. 135 (2011) 201–208. [17] Q.H. Zhang, W.B. Wang, J. Li, Y.X. Chang, Y.F. Wang, J.Sh. Zhang, B.L. Zhang, X.M. Gao, Simultaneous determination of catechin, epicatechin and epicatechin gallate in rat plasma by LC–ESI-MS/MS for pharmacokinetic studies after oral administration of Cynomotium songaricum extract, J. Chromatogr. B 880 (2012) 168–171. [18] L.L. Lu, D.W. Qian, J. Yang, Sh. Jiang, J.M. Guo, E.X. Shang, J.A. Duan, Identification of isoquercitrin metabolites produced by human intestinal bacteria using UPLC–Q-TOF/MS, Biomed. Chromatogr. 27 (2013) 509–514.
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Figure Captions Fig.1. Representative MRM chromatograms of gallic acid (1), protocatechuic acid (2), vanillic acid (3), caffeic acid (4), epicatechin (5), isoquercitrin (6), vincetoxicoside B (7), quercetin (8), catechin (9,IS) and daidzein (10,IS) in rat plasma: (A) blank plasma; (B) blank plasma with eight analytes in LLOQ and IS; (C) plasma sample at 45 min following oral administration of Hypericum japonicum Thunb extract in rats.
20
21
Fig.2. The mean plasma concentration–time curve profile of eight analytes following single administration of Hypericum japonicum Thunb extract to rats (n = 6, mean ± SD).
22
Tables Table 1. The intra-day and inter-day precisions and accuracies of the eight analytes in rat plasma at four concentration levels (n = 4 days, 6 replicates per day). Intra-day
Inter-day
Cnom Analytes
Gallic acid
Protocatechuic acid
Vanillic acid
Caffeic acid
Epicatechin
Isoquercitrin
Vincetoxicoside B
Quercetin
(ng·mL-1)
Cdet (ng·mL-1)
Precision
Accuracy
(RSD %)
(RE %)
Cdet (ng·mL-1)
Precision
Accuracy
(RSD %)
(RE %)
30.0
30.1 ± 1.5
4.9
0.4
28.8 ± 1.7
5.8
−4.1
300.0
303.7 ± 8.6
2.8
1.2
264.8 ± 30.9
11.7
−11.7
800.0
774.3 ± 13.1
1.7
−3.2
840.5 ± 56.6
6.7
5.1
3000.0
2580.7 ± 43.1
1.7
−14.0
3129.8 ± 402.9
12.9
4.3
4.0
3.9 ± 0.4
10.4
−1.6
3.7 ± 0.3
8.1
−6.5
80.0
74.5 ± 3.6
4.8
−6.9
78.3 ± 5.1
6.6
−2.2
400.0
372.2 ± 24.3
6.5
−7.0
418.1 ± 33.6
8.0
4.5
2000.0
2249.3 ± 116.9
5.2
12.5
2023.4 ± 153.2
7.6
11.7
30.0
27.8 ± 2.4
8.4
−7.3
27.9 ± 2.3
8.4
−6.9
600.0
549.0 ± 10.3
1.9
−8.5
597.3 ± 75.4
12.6
−0.5
2400.0
2425.1 ± 127.0
5.2
1.0
2436.1 ± 113.8
4.7
1.5
12000.0
13286.5 ± 224.9
1.7
10.7
13329.2 ± 380.3
2.8
11.1
6.0 ± 0.4
7.5
0.5
6.7
−7.8
100.0
89.34± 6.6
7.4
−10.7
96.8 ± 6.3
6.5
−3.2
400.0
343.5 ± 3.4
1.0
−14.1
383.6 ± 41.8
10.9
−4.1
2000.0
1978.0 ± 110.4
5.6
−1.0
2033.8 ± 126.2
6.2
1.7
3.5
3.5 ± 0.2
6.8
−0.1
3.4 ± 0.2
6.6
−3.8
90.0
88.7 ± 9.5
10.7
−1.4
88.3 ± 9.5
10.8
−1.9
500.0
563.9 ± 9.6
1.7
12.8
531.2 ± 31.5
5.9
6.2
2700.0
2417.2 ± 166.0
6.9
−10.5
2612.8 ± 222.7
8.5
−3.2
10.0
8.8 ± 0.3
3.1
−11.8
9.8 ± 0.9
9.2
−1.6
120.0
108.1 ± 6.7
6.2
−9.9
110.3 ± 8.8
8.0
−8.1
600.0
640.3 ± 33.9
5.3
6.7
626.7 ± 49.4
7.9
4.4
3000.0
2827.8 ± 340.7
12.0
−5.7
3030.8 ± 257.8
8.5
1.0
5.4 ± 0.1
1.4
8.2
4.8 ± 0.4
8.9
−0.7
300.0
314.0 ± 44.5
14.2
4.7
297.9 ± 35.7
12.0
−0.7
1500.0
1551.7 ± 138.5
8.9
3.4
1491.4 ± 98.0
6.6
−0.6
7500.0
7891.3 ± 561.9
7.1
5.2
8105.5 ± 538.8
6.6
8.1
60.0
58.1 ± 6.4
10.9
−3.2
58.6 ± 5.1
8.6
−2.4
600.0
557.5 ± 36.4
6.5
−7.1
577.6 ± 44.9
7.8
−3.7
3000.0
2885.5 ± 125.7
4.4
−3.8
3103.2 ± 227.3
7.3
3.4
12000.0
12251.5 ± 1142.4
9.3
2.1
11953.6 ± 1121.4
9.4
−0.4
6.0
5.0
Cnom: nominal concentration; Cdet: mean value of the detected concentration
23
5.5 ± 0.4
Table 2. The extraction recoveries and matrix effects of the eight analytes in rat plasma at four concentration levels (n = 6) Analytes
Extraction recovery
C nom (ng·mL-1)
Mean (%) Gallic acid
Protocatechuic acid
Vanillic acid
Caffeic acid
Epicatechin
Isoquercitrin
Vincetoxicoside B
Quercetin
RSD (%)
Matrix effect Mean (%)
RSD (%)
30.0
93.4
3.0
89.3
1.7
300.0
113.7
4.6
88.8
2.6
800.0
92.2
7.4
111.6
1.4
3000.0
90.7
3.7
116.2
3.5
4.0
86.5
4.7
88.2
1.8
80.0
82.5
2.2
104.9
1.8
400.0
102.8
3.4
104.0
3.2
2000.0
81.7
1.8
98.3
1.2
30.0
88.4
0.6
87.8
5.4
600.0
84.5
3.0
110.5
7.2
2400.0
101.7
1.7
98.6
1.8
12000.0
113.0
2.8
113.1
1.9
6.0
82.5
3.2
89.1
1.8
100.0
76.6
6.8
98.0
3.5
400.0
88.8
3.8
93.4
6.6
2000.0
78.6
3.9
97.6
3.3
3.5
88.6
1.2
89.2
2.4
90.0
100.7
2.2
84.9
2.6
500.0
108.6
1.4
107.9
0.6
2700.0
98.1
5.5
103.7
2.4
10.0
86.1
2.4
102.1
7.8
120.0
84.0
2.3
89.5
4.9
600.0
79.2
3.1
94.4
6.4
3000.0
89.9
6.8
107.7
5.7
5.0
82.5
3.1
96.0
6.9
300.0
92.7
6.1
105.7
7.7
1500.0
85.7
5.3
96.6
5.8
7500.0
94.7
7.9
107.9
4.1
60.0
84.0
1.8
103.9
3.5
600.0
103.3
5.6
93.8
6.2
3000.0
82.5
7.0
103.3
9.0
12000.0
85.6
1.7
105.7
6.0
Cnom: nominal concentration
24
Table 3. Pharmacokinetic parameters of eight analytes after oral administration of Hypericum japonicum Thumb extract (n = 6, mean ± SD). Parameters
Gallic acid
t1/2α (h)
0.53 ± 0.029
t1/2β (h)
Vanillic acid
Caffeic acid
0.52 ± 0.07
0.36 ± 0.08
1.78 ± 0.23
3.23 ± 0.45
7.24 ± 0.68
5.99 ± 0.52
4.55 ± 0.59
K21 (1/h)
0.84 ± 0.072
0.21 ± 0.04
0.44 ± 0.008
0.19 ± 0.003
K10 (1/h)
0.54 ± 0.014
0.56 ± 0.06
0.59 ± 0.009
0.26 ± 0.08
0.56 ± 0.046
0.50 ± 0.02
1.29 ± 0.007
0.42 ± 0.071
0.0028 ± 0.0007
0.004 ± 0.0005
0.002 ± 0.0001
0.0018 ± 0.0003
K12 (1/h) -1
-1
CL (L h g ) Tpeak (h)
0.39 ± 0.024
Cmax (ng·mL-1)
228.31 ± 18.74
V/F (L g-1)
0.005 ± 0.001
Protocatechuic acid
0.54 ± 0.03
0.46 ± 0.05
930.76 ± 59.39
607.57 ± 85.81
0.44 ± 0.032 164.37 ± 11.38
0.007 ± 0.0008
0.003 ± 0.0004
0.0074 ± 0.0007
671.37 ± 207.22
3003.49 ± 358.63
2074.88 ± 225.52
871.13 ± 167.69
AUC0–t (ng·h mL )
555.68 ± 121.54
2921.58 ± 401.50
1667.08 ± 346.30
843.36 ± 177.91
MRT0–∞ (h)
3.53 ± 1.73
6.48 ± 0.95
5.95 ± 0.70
5.80 ± 0.63
MRT0–t (h)
2.13 ± 0.40
4.72 ± 0.18
3.15 ± 0.85
5.07 ± 0.81
-1
-1
AUC0–∞ (ng·h mL )
25
Table 4. Pharmacokinetic parameters of eight analytes after oral administration of Hypericum japonicum Thumb extract (n = 6, mean ± SD). Parameters
Epicatechin
Isoquercitrin
Vincetoxicoside B
Quercetin
t1/2α (h)
0.40 ± 0.07
0.92 ± 0.042
0.54 ± 0.10
0.38 ± 0.005
t1/2β (h)
7.93 ± 0.23
3.39 ± 0.53
3.01 ± 0.35
29.21 ± 3.86
K21 (1/h)
0.98 ± 0.14
0.36 ± 0.05
0.50 ± 0.016
0.14 ± 0.01
K10 (1/h)
0.16 ± 0.04
0.52 ± 0.02
0.64 ± 0.005
0.32 ± 0.04
0.91 ± 0.13
0.23 ± 0.01
0.41 ± 0.002
1.38 ± 0.04
0.001 ± 0.00
0.02 ± 0.001
0.02 ± 0.003
0.002 ± 0.0001
K12 (1/h) -1
-1
CL (L h g ) Tpeak (h)
0.75 ± 0.07
Cmax (ng·mL-1)
1151.71 ± 72.56
V/F (L g-1)
0.01 ± 0.002
0.69 ± 0.03
0.34 ± 0.02
0.52 ± 0.01
2087.70 ± 101.93
5372.34 ± 927.96
1684.46 ± 187.38
0.04 ± 0.005
0.03 ± 0.004
0.006 ± 0.0005
-1
11984.34 ± 257.81
7152.76 ± 1844.41
12329.48 ± 2442.48
16108.89 ± 314.23
-1
AUC0–t (ng·h mL )
10700.63 ± 158.91
7126.23 ± 1848.15
12298.17 ± 2431.17
15286.88 ± 290.92
MRT0–∞ (h)
10.39 ± 0.29
3.65 ± 0.18
3.42 ± 0.40
9.77 ± 0.11
MRT0–t (h)
7.48 ± 0.08
3.59 ± 0.20
3.36 ± 0.38
8.63 ± 0.064
AUC0–∞ (ng·h mL )
26
S0731-7085(15)30200-4 http://dx.doi.org/doi:10.1016/j.jpba.2015.10.027 PBA 10303
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
18-6-2015 10-10-2015 19-10-2015
Please cite this article as: Qian Pang, Yuanyuan Tian, Jianping Mi, Jin Wang, Yuanjin Xu, Simultaneous determination and pharmacokinetic study of eight components in rat plasma by UHPLCndashMS/MS after oral administration of Hypericum japonicum Thunb extract, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.10.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous determination and pharmacokinetic study of eight components in rat plasma by UHPLC–MS/MS after oral administration of Hypericum japonicum Thunb extract a,b
a,b
a,c
a,b
Qian Pang , Yuanyuan Tian , Jianping Mi , Jin Wang ， Yuanjin Xu
a,b*
[email protected]
a
State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004,
China b
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China, Beihai Center for Disease Control
and Prevention, Beihai 536000,China ∗Corresponding author at: State Key Laboratory of Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi
University, Nanning 530004, China. Tel.: +860771-3237743.
1
Graphical Abstract
2
Highlights Simultaneous determination of eight active constituents in rat plasma Pharmacokinetic study of eight active constituents by UHPLC–MS/MS The first report on pharmacokinetic study of Hypericum japonicum Thunb by UHPLC–MS/MS
3
Abstract
A rapid and sensitive assay based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) was established and validated for the simultaneous determination of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin in rat plasma using catechin and daidzein as the internal standards (IS). Plasma samples added internal standards were acidified with formic acid then pretreated by direct protein precipitation with acetonitrile. The separation of eight constituents was achieved on a C18 column with gradient elution using methanol and 0.2% acetic acid aqueous solution as the mobile phase and detected by multiple reaction monitoring using electrospray ionization source in the positive-negative ionization mode. The method was validated for sufficient specificity, precision, accuracy, and sensitivity over the concentration range of 10–6000 ng·mL-1 for gallic acid, 1.5–3000 ng·mL-1 for protocatechuic acid, 10–15000 ng·mL-1 for vanillic acid, 2–3600 ng·mL-1 for caffeic acid, 1.5–3600 ng·mL-1 for epicatechin, 4–6000 ng·mL-1 for isoquercitrin, 2–9000 ng·mL-1 for vincetoxicoside B, and 20–18000 ng·mL-1 for quercetin. The overall intra-run precision and the inter-run precision were showed in the range of 1.0%–14.2% and 2.8%–12.9%, respectively, and the accuracy was no more than 12.8%. This analytical method was successfully applied to investigate the pharmacokinetics of eight ingredients in rats after oral administration of Hypericum japonicum Thunb extract. Keywords: Hypericum japonicum Thunb; Pharmacokinetics; UHPLC–MS/MS; Rat plasma
4
1. Introduction Hypericum japonicum Thunb, a member of genus Hypericum L. (Clusiaceae/Hypericaceae), is an annual or perennial herb widely distributed in Asia, North America and Oceania [1]. It has been widely used for the therapy of the infectious hepatitis, hemostasis, detumescence, dysentery and relieving internal heat or fever
[1-2]
.Many pharmacological studies have demonstrated that its
dried entire plant possesses antibacterial, antioxidant, antihypoxic and hepatoprotective activities [3-5]
. Phytochemical investigations of Hypericum japonicum Thunb have indicated many
ingredients such as flavonoids, phenolic acids, xanthones, phloroglucinols and chromones
[6]
.
Among them, phenolic acids and flavonoids are the main bioactive constituents which contribute to the pharmacological efficacy in Hypericum japonicum Thunb. Based on a tremendous amount of previous pharmacological researches, phenolic acids (gallic acid, protocatechuic acid, vanillic acid and caffeic acid) and flavonoids (epicatechin, isoquercitrin, vincetoxicoside B and quercetin) strikingly manifest a variety of pharmacological properties, including antioxidant, antimicrobial, anti-inflammatory and antitumour functions
[7-11]
. In addition, it has displayed good effect in
clinical application of hepatitis [12]. Most of previous studies have been concentrated on the phytochemical and other pharmacological investigation of Hypericum japonicum Thunb. However, apart from quercitrin and isoquercitrin [13], no research reports on pharmacokinetics of other components of Hypericum japonicum Thunb was available. It is well known that Traditional Chinese Medicine (TCM) has thousands of years of history in China that play a crucial role in the disease protection, control, and treatment. But there is a considerable lack in therapeutic mechanism understanding due to its manifold and complicated constituents. Pharmacokinetic data may help to illustrate and foresee
5
efficacy and toxicity of the herb medicine and to optimize dose regiment and reduce adverse effects [14]. In this study, a rapid, sensitive and robust ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) was established for the simultaneous determination of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin of Hypericum japonicum Thunb in rat plasma using catechin and daidzein as the internal standards. The method was fully validated and successfully applied to the pharmacokinetics after oral administration of Hypericum japonicum Thunb extract to rats. The results may supply some valuable references for the apprehension of pharmacological action mechanism of Hypericum japonicum Thunb. 2. Experimental 2.1. Chemicals, reagents and animals The standards of gallic acid, protocatechuic acid, caffeic acid and epicatechin were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Standards substances of isoquercitrin, vincetoxicoside B, quercetin and catechin (IS) were obtained from Chengdu Pufeide Biological Technology Co., Ltd. (Sichuan, China). The standards of vanillic acid and daidzein (IS) were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Sichuan, China).The purity of each standard substance was more than 98%. Methanol and acetonitrile of HPLC grade were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Ultra-pure water used in the experiment was generated by a Milli-Q water purification system (Millipore Corporation, Billerica, USA). Other chemicals were all analytical reagents.
6
Ultra-pure water was used throughout and all solutions were passed through 0.22 μm pore size filters. Hypericum japonicum Thunb (Batch No.: 140701) was obtained from Nanning Shengyuan Chinese Traditional Medicine Co., Ltd. (Guangxi, China) and was authenticated by professor Xuejian Li in Guangxi University of Chinese Medicine. We kept the voucher specimens in reserve in State Key Laboratory of Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University. Sprague-Dawley (SD) rats (280 ± 20 g) were obtained from the Animal Center of Guangxi Medical University (Guangxi, China). The rats were maintained in an air-conditioned room under the constant room temperature (23 ± 2 °C), relative humidity level (50 ± 15%), and lighting (12 h light/dark cycle) with free access to food and water for 7 days before the experiment. Before the herb extract administration, the rats were deprived of food for 12 h but had access to water spontaneously. The experimental protocols were reviewed and approved by Laboratory Animal Care Committee of Guangxi Department of Science and Technology. 2.2. UHPLC–MS/MS apparatus and operation conditions
The UHPLC–MS/MS is composed of an Agilent 1290 ultra high performance liquid chromatography system (Agilent Co., USA), equipped with a binary pump solvent delivery system, an autosampler, and an online degasser. The chromatographic separation was achieved on a ZORBAX RRHD Eclipse Plus C18 column (2.1 x 50 mm, 1.8 μm) [Guangzhou Dexiang Science and Technology Co., Ltd. (Guangdong, China)] at the temperature of 25 °C. The mobile phase was consisted of 0.2% acetic acid aqueous solution (A) and methanol (B) using a gradient elution as follow: 0–6 min, linear 7
changed from 10% to 18% B, 6–6.5 min, linear changed from 18% to 52% B, 6.5–9 min, linear changed from 52% to 56% B, 9–9.5 min, linear changed from 56% to 68% B, 9.5–10.5 min, linear changed from 68% to 80% B, 10.5–12 min, linear changed from 80% to 100% B, 12–17 min, isocratic elution with 100% B, and then quickly returned to the initial condition. The flow rate was set at 0.3 mL·min-1 and the injection volume was 1 μL for analysis. Mass spectrometry detection of all the analytes, including the IS, was performed using triple quadrupole 6460 mass spectrometer (Agilent Co., USA) with an electrospray ionization source (ESI) source interface set in positive and negative ionization mode. Multiple reaction monitoring (MRM) using the precursor–product ion transitions of m/z 169.1–125.1, 153.1–109.1, 169.0–92.9, 179.1–135.1,
291.1–139.1,
463.1–300.1,
447.0–301.0,
303.0–152.9,
291.1–138.9
and
255.1–137.1 were employed for the quantification of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B, quercetin, catechin (IS) and daidzein (IS), respectively. The optimal MS parameters were as follow: capillary voltage 4000 V (ESI +), 3000V (ESI−); nozzle voltage 0 V; spray voltage 4000 V and nebulizer gas (nitrogen) pressure 2.8 x 105 Pa. Nitrogen was used as the desolvation and sheath gas with a flow rate of 10 L min-1 and 12 L min-1, respectively. The desolvation gas was heated to 300 °C and the temperature of the sheath gas was set at 360 °C. High purity nitrogen was served as the collision gas. 2.3. Preparation of the Hypericum japonicum Thunb extract The dried herb of Hypericum japonicum Thunb were powdered to a homogeneous size in a mill, passed through a 50-mesh sieve. 90 g of accurately weighed powder was soaked in 60% ethanol (1:50, w/v ) for 12 h, and then extracted in an ultrasonic bath (200 W, 40 kHz) for 45 min. The mixed extract was subjected to centrifugate at 5000 x g for 40 min. The supernatant was
8
evaporated to dryness using the evaporator at a constant temperature of 36 °C. To calculate the administered dose, the contents of eight components in Hypericum japonicum Thunb were measured quantitatively by an external standard assay using the same chromatography conditions as described above. The contents of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin were 0.69, 4.33, 1.22, 0.59, 4.60, 46.52, 82.12 and 9.48 mg·g-1 in extract respectively. 2.4. Preparation of calibration standard and quality control (QC) samples Stock solutions of eight analytes and IS at the concentration of 1000 μg·mL-1 for each were prepared by dissolving the accurately weighed reference standards in methanol, respectively. A serious of standard working solutions was obtained by future diluting the stock solutions. Mixed calibration standard samples containing gallic acid 10, 30, 150, 750, 1500, 3000, 4500, and 6000 ng·mL-1, protocatechuic acid 1.5, 5, 25, 120, 360, 720, 1500, and 3000 ng·mL-1, vanillic acid 10, 90, 450, 1800, 3600, 5400, 8100, and 15000 ng·mL-1, caffeic acid 2, 6, 30, 150, 450, 900, 1800, and 3600 ng·mL-1, epicatechin 1.5, 15, 45, 150, 450, 900, 1800, and 3600 ng·mL-1, isoquercitrin 4, 10, 50, 250, 750, 1500, 3000, and 6000 ng·mL-1, vincetoxicoside B 2, 15, 50, 250, 750, 1500, 3000, and 9000 ng·mL-1, quercetin 20, 150, 600, 1800, 5400, 8100, 12000, and 18000 ng·mL-1 were obtained by spiking 100 μL aliquots of blank plasma with 10μL working solutions of corresponding concentrations. For the validation and pharmacokinetic study of the assay, four concentrations of the standard solution, including gallic acid (30, 300, 800, and 3000 ng·mL-1), protocatechuic acid (4, 80, 400, and 2000 ng·mL-1), vanillic acid (30, 600, 2400, and 12000 ng·mL-1), caffeic acid (6, 100, 400, and 2000 ng·mL-1), epicatechin (3.5, 90, 500, and 2700 ng·mL-1), isoquercitrin (10, 120, 600, and 3000 ng·mL-1), vincetoxicoside B (5, 300, 1500, and
9
7500 ng·mL-1), and quercetin (60, 600, 3000, and 12000 ng·mL-1) were applied to prepared the quality control (QC) samples. The standards and quality control (QC) samples were extracted on each analysis day with the same processes for plasma samples preparation as described below. 2.5. Preparation of plasma samples All of the plasma samples were executed by direct protein precipitation with acetonitrile. Concretely, a 100 μL aliquot of blank plasma were spiked with 10 μL of the IS solution, 10 μL of the methanol (volume of the corresponding working solution for the calibration curve and the quality control samples) in a 1.5 mL Eppendorf tube. The sample was extracted with 250 μL of acetonitrile containing 0.1% formic acid by vortex mixing for 1 min and centrifugated at 12000 x g for 20 min. The supernatant was transferred into another Eppendorf tube and evaporated to dryness by thickener at room temperature. The obtained residue was reconstituted with 100μL methanol and centrifugated at 12000 x g for another 20 min. Finally, the supernatant was transferred into an autosample vial and an aliquot of 1μL of the sample solution was injected into the UHPLC–MS/MS system for analysis. 2.6. Method validation The assay was validated for specificity, selectivity, calibration curve, sensitivity, accuracy, precision, stability, matrix effect and recovery, in accordance with the USA Food and Drug Administration (FDA) bioanalytical method validation guidance [15]. 2.6.1. Specificity and selectivity Selectivity is the ability of an analytical method to differentiate and quantify the analyte in the presence of other components in the sample
[15]
. In our study, the selectivity and specificity
toward the endogenous plasma matrix constituents was ascertained by comparing the
10
chromatograms of blank rat plasma from six sources, blank plasma spiked with IS and the analytes at the concentrations of the lower limit of quantification (LLOQ) and the plasma samples from the rats after oral administration of Hypericum japonicum Thunb extract. 2.6.2. Linearity and sensitivity The linearity of the assay was generated by analysis of eight plasma calibration curves consisted of eight concentration levels. The plasma calibration curves were determined by plotting the peak-area ratios (y) of eight analytes to IS versus the corresponding nominal concentration (x) of analytes using the weighted (1/x2) least least-squares linear regression model. The linear regression analysis with 1/x2 weighting factor was performed to determine the intercept, slope and correlation coefficient (r). The sensitivity of the method was evaluated by determining the lower limit of quantification (LLOQ) and limit of detection (LOD). LLOQ and LOD were determined via the signal-to-noise ratio (S/N) of 10:1 and 3:1, respectively. The LLOQ was defined as the lowest concentration on the calibration curve that could be ascertained with an acceptable accuracy within ±20% and a precision less than 20%, which was evaluated based on six replicated samples. 2.6.3. Precision and accuracy For the sake of evaluating the intra- and inter-day precision and accuracy, QC samples at four concentration levels of the eight analytes were analyzed in six replicates on the same day and on four separate days, respectively. The precision in determination was expressed as the relative standard deviation (RSD) and the accuracy was estimated by means of calculating the percent deviation of the measured mean value that observed in the analysis of QC samples deviated from the nominal value and defined as relative error (RE).
11
2.6.4. Extraction recovery and matrix effect The extraction recoveries of the analytes in this assay were estimated by comparing peak area ratios from QC samples regularly pretreated with those obtained from post-extracted supernatants spiked with the pure reference standards at the same concentrations. The matrix effects for the constituents were assessed and calculated by the ratio of the detector response for acetonitrile precipitated-blank boi-samples spiked with standard solutions to the response for the corresponding standard solutions prepared by dilution in methanol. The extraction recoveries and matrix effects of the IS were determined in the similar procedures. 2.6.5. Stability The QC plasma samples at four concentrations were subjected to analyze under the various circumstances containing short-term stability (storage for 12 h at ambient temperature), long-term stability (storage for 30 days at −20 °C), post-treatment stability (storage for 24 h in an auto-sampler) and freeze–thaw stability (three freezes at −20 °C and thaw cycles). The results were utilized to assess the stability. 2.7. Pharmacokinetic study in rat plasma The proposed method was applied to determine the plasma concentration of eight active components for a pharmacokinetic study conducted in six healthy Sprague–Dawley rats weighing 280 ± 20 g. The rats were fasted in the last 12 hours before the experiment with the exception of free access to water. The dosing solution was prepared by dissolving appropriate of Hypericum japonicum Thunb extract in normal saline solution. Hypericum japonicum Thunb extract were given orally to the rats at a dose of 2.5 g·kg-1 body weight (equivalent to 1.73 mg·kg-1 of gallic acid, 10.83 mg·kg-1 of protocatechuic acid, 3.05 mg·kg-1 of vanillic acid, 1.48 mg·kg-1 of caffeic
12
acid, 11.50 mg·kg-1 of epicatechin, 116.30 mg·kg-1 of isoquercitrin, 205.30 mg·kg-1 of vincetoxicoside B and 23.70 mg·kg-1 of quercetin). Retro-orbital blood samples (250 μL) were collected into the sodium heparinized tubes before drug administration and at different time intervals post-dosing (0.083, 0.25, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h). The heparinized blood samples were centrifuged at 1800 x g at a constant temperature of 4 °C for 30 min to obtain plasma, which was stored at −20 °C until analysis. All the pharmacokinetic parameters were calculated by compartmental analysis using 3P97 pharmacokinetic program (Mathematical Pharmacology Professional Committee of China, Shanghai, China). 3. Results and discussion 3.1. Assay validation 3.1.1. Selectivity and specificity The selectivity and specificity were assessed using independent plasma samples derived from six different rats. The typical chromatograms for the drug-free plasma, drug-free with analytes at LLOQ and IS and an in vivo rat plasma sample after oral administration of Hypericum japonicum Thunb are shown in Figure 1. As shown in Figure 1, all the peaks can be confirmed and there is no significant interference from the endogenous substances observed at the retention time of each component. 3.1.2. Linearity and sensitivity As for plasma calibration curve, the ratios of the peak areas (analytes/IS) as ordinate variables were plotted versus the corresponding nominal concentration of analytes as abscissa. After comparing three weighting models (x, 1/x and 1/x2), the 1/x2 weighing factor was chosen to achieve the best linear fit and least-square residual for the calibration curve. The ratios of the peak
13
areas (analytes/IS) as ordinate variables were plotted versus the corresponding nominal concentration of analytes as abscissa. All the eight ingredients exhibited good linearity with correlation coefficients (r) within the range of 0.9989–0.9995. 3.1.3. Precision and accuracy The results of the intra- and inter-day precision and accuracy of the eight analytes are summarized in Table 1. In the present assay, the overall intra- precision and the inter-day precision were showed in the range of 1.0%–14.2% and 2.8%–12.9%, respectively, and the accuracy was no more than 12.8%. The results indicated that the method was reliable for the determination of Sprague-Dawley rat plasma samples. 3.1.4. Extraction recovery and matrix effect Table 2 summarized the extraction recovery and matrix effects of eight analytes. Based on the data displayed in Table 2, the extraction recovery of the investigated components in plasma at four concentration levels ranged from 76.6% to 113.7%. The assessment of matrix effect of the assay was performed systematically and all the ratios of the analytes were found to be within the acceptable range (84.9%–116.2%). The results indicated that there was no significant ion suppression or enhancement from plasma for this assay. 3.1.5. Stability The stability of the assay was conducted strictly under different circumstances. The results indicated that eight analytes were stable in plasma samples after three freeze–thaw cycles (RE: −11.8% to 11.5%, RSD < 9.6%) and at room temperature for 12 h (RE: −14.6% to 14.0%, RSD < 10.9%). Post-preparative stability of the analytes also declared that no obvious loss occurred when the extracted samples were stored in an auto-sampler for 24 h (RE: −14.2% to 7.9%, RSD
vanillic acid > caffeic acid > gallic acid. Following a single oral administration of total flavonoids from Hypericum japonicum Thunb, double peaks were emerged in curves of mean plasma concentration for epicatechin and quercetin. This phenomenon had been confirmed in the previous literature
[16-17]
. It may be explained by distribution, re-absorption and enterohepatic
15
circulation. Meanwhile, it should be pointed out that the area under concentration–time curve (AUC) of quercetin was much higher than other components. The factor that may contribute to this phenomenon can be explained by flavonoid glycosides (isoquercitrin and vincetoxicoside) were released as the aglycone in the intestinal tract by hydrolytic action of the bacteria
[18]
. In
general, the information described in present study would be informative for future application of herbal medicine in clinical therapy. 4. Conclusions In the present study, a sensitive and reliable UHPLC–MS/MS method was established for simultaneous determination of eight components following oral administration of Hypericum japonicum Thunb extract in rat plasma. The analysis assay is simple and fast because of the straightforward sample pre-treatment procedure and its relative short analysis running time. This is the first report of pharmacokinetic studies of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin in vivo after oral administration of Hypericum japonicum Thunb extract. This assay could offer a scientific basis for the further research of Hypericum japonicum Thunb. Acknowledgements This research was supported by the National Natural Science foundation of China (No. 20865001), the Guangxi Province Natural Science foundation (0832034).
16
References [1] Editorial board of Flora of China, Flora of China, Vol. 50, Science Press, Beijing, China, 1990, pp. 47–48. [2] Jiangsu New Medical College, Dictionary of Chinese Traditional Medicine (Zhong Yao Da Ci Dian), ShangHai People’s Publishing House, ShangHai, China, 1977, pp. 813–814. [3] P.V. Samaga, V.R. Rai, Evaluation of pharmacological properties and phenolic profile of Hypericum japonicum Thunb from Western Ghats of India, J. Pharm. Res. 7 (2013) 626–632. [4] X.W. Wang, Y. Mao, N.L. Wang, X.Sh. Yao, A New Phloroglucinol Diglycoside Derivative from Hypericum japonicum Thunb., Molecules 13 (2008) 2796–2803. [5] N. Wang, P.B. Li, Y.G. Wang, W. Peng, Zh. Wu, S.Y. Tan, Sh.L. Liang, X. Shen, W.W. Su, Hepatoprotective effect of Hypericum japonicum extract and its fractions, J. Ethnopharmacol. 116 (2008) 1–6. [6] W.N. Gao, J.G. Luo, L.Y. Kong, Quality evaluation of Hypericum japonicum by using high-performance liquid chromatography coupled with photodiode array detector and electrospray ionization tandem mass spectrometry, Biomed. Chromatogr. 23 (2009) 1022–1030. [7] M. Cotoras, H. Vivanco, R. Melo, M. Aguirre, E. Silva, L. Mendoza, In Vitro and in Vivo Evaluation of the Antioxidant and Prooxidant Activity of Phenolic Compounds Obtained from Grape (Vitis vinifera) Pomace, Molecules 19 (2014) 21154–21167. [8] F. Fratianni, R. Pepe, F. Nazzaro, Polyphenol Composition, Antioxidant, Antimicrobial and Quorum Quenching Activity of the “Carciofo di Montoro” (Cynara cardunculus var.
17
scolymus) Global Artichoke of the Campania Region, Southern Italy, Food Nutr. Sci. 5 (2014) 2053–2062. [9] E.O. Sousa, C.M.B.A. Miranda, C.B. Nobre, A.A. Boligon, M.L. Athayde, J.G.M. Costa, Phytochemical analysis and antioxidant activities of Lantana camara and Lantana motevidensis extracts, Ind. Crops Prod. 70 (2015) 7–15. [10] C.H. Lu, Y.Y. Li, L.J. Li, L.Y. Liang, Y.M. Shen, Anti-inflammatory activities of fractions from Geranium nepalense and related polyphenols, Drug Discoveries Ther. 6 (2012) 194–197. [11] Q. Chen, P. Li, P. Li, Y. Xu, Y. Li, B. Tang, Isoquercitrin inhibits the progression of pancreatic cancer in vivo and in vitro by regulating opioid receptors and the mitogen-activated protein kinase signalling pathway, Oncol. Rep. 33 (2015) 840–848. [12] H. Lin, Q.X. Mei, X.L. Kong, Y.T. Wang, Z.L. Chen, The clinical application of Hypericum japonicum Thunb in hepatopathy and its research survey on pharmacological action, Pharmacy Today. 21 (2011) 550–552. [13] J. Li, Zh.W. Wang, L. Zhang, X. Liu, X.H. Chen, K.Sh. Bi, HPLC analysis and pharmacokinetic study of quercitrin and isoquercitrin in rat plasma after administration of Hypericum japonicum thunb· extract, Biomed. Chromatogr. 22 (2008) 374–378. [14] Y. Wang, Ch.H. Xu, P. Wang, X.Y. Lin, Y. Yang, D.H. Li, H.F. Li, X.Zh. Wu, H.B. Liu, Pharmacokinetic comparisons of different combinations of Shaoyao-Gancao-Decoction in Rats: Simultaneous determination of ten active constituents by HPLC–MS/MS, J. Chromatogr. B 932 (2013) 76–87. [15] FDA, Guidance for Industry: Bioanalytical Method Validation, Center for Drug
18
Evaluation and Research, US Department of Health and Human Services, US, FDA, Rockville, MD, 2001. [16] K.F. Duan, Zh.F. Yuan, W. Guo, Y. Meng, Y. Cui, D.Zh. Kong, L.T. Zhang, N. Wang, LC–MS/MS determination and pharmacokinetic study of five flavone components after solvent extraction/acid hydrolysis in rat plasma after oral administration of Verbena officinalis L. extract, J. Ethnopharmacol. 135 (2011) 201–208. [17] Q.H. Zhang, W.B. Wang, J. Li, Y.X. Chang, Y.F. Wang, J.Sh. Zhang, B.L. Zhang, X.M. Gao, Simultaneous determination of catechin, epicatechin and epicatechin gallate in rat plasma by LC–ESI-MS/MS for pharmacokinetic studies after oral administration of Cynomotium songaricum extract, J. Chromatogr. B 880 (2012) 168–171. [18] L.L. Lu, D.W. Qian, J. Yang, Sh. Jiang, J.M. Guo, E.X. Shang, J.A. Duan, Identification of isoquercitrin metabolites produced by human intestinal bacteria using UPLC–Q-TOF/MS, Biomed. Chromatogr. 27 (2013) 509–514.
19
Figure Captions Fig.1. Representative MRM chromatograms of gallic acid (1), protocatechuic acid (2), vanillic acid (3), caffeic acid (4), epicatechin (5), isoquercitrin (6), vincetoxicoside B (7), quercetin (8), catechin (9,IS) and daidzein (10,IS) in rat plasma: (A) blank plasma; (B) blank plasma with eight analytes in LLOQ and IS; (C) plasma sample at 45 min following oral administration of Hypericum japonicum Thunb extract in rats.
20
21
Fig.2. The mean plasma concentration–time curve profile of eight analytes following single administration of Hypericum japonicum Thunb extract to rats (n = 6, mean ± SD).
22
Tables Table 1. The intra-day and inter-day precisions and accuracies of the eight analytes in rat plasma at four concentration levels (n = 4 days, 6 replicates per day). Intra-day
Inter-day
Cnom Analytes
Gallic acid
Protocatechuic acid
Vanillic acid
Caffeic acid
Epicatechin
Isoquercitrin
Vincetoxicoside B
Quercetin
(ng·mL-1)
Cdet (ng·mL-1)
Precision
Accuracy
(RSD %)
(RE %)
Cdet (ng·mL-1)
Precision
Accuracy
(RSD %)
(RE %)
30.0
30.1 ± 1.5
4.9
0.4
28.8 ± 1.7
5.8
−4.1
300.0
303.7 ± 8.6
2.8
1.2
264.8 ± 30.9
11.7
−11.7
800.0
774.3 ± 13.1
1.7
−3.2
840.5 ± 56.6
6.7
5.1
3000.0
2580.7 ± 43.1
1.7
−14.0
3129.8 ± 402.9
12.9
4.3
4.0
3.9 ± 0.4
10.4
−1.6
3.7 ± 0.3
8.1
−6.5
80.0
74.5 ± 3.6
4.8
−6.9
78.3 ± 5.1
6.6
−2.2
400.0
372.2 ± 24.3
6.5
−7.0
418.1 ± 33.6
8.0
4.5
2000.0
2249.3 ± 116.9
5.2
12.5
2023.4 ± 153.2
7.6
11.7
30.0
27.8 ± 2.4
8.4
−7.3
27.9 ± 2.3
8.4
−6.9
600.0
549.0 ± 10.3
1.9
−8.5
597.3 ± 75.4
12.6
−0.5
2400.0
2425.1 ± 127.0
5.2
1.0
2436.1 ± 113.8
4.7
1.5
12000.0
13286.5 ± 224.9
1.7
10.7
13329.2 ± 380.3
2.8
11.1
6.0 ± 0.4
7.5
0.5
6.7
−7.8
100.0
89.34± 6.6
7.4
−10.7
96.8 ± 6.3
6.5
−3.2
400.0
343.5 ± 3.4
1.0
−14.1
383.6 ± 41.8
10.9
−4.1
2000.0
1978.0 ± 110.4
5.6
−1.0
2033.8 ± 126.2
6.2
1.7
3.5
3.5 ± 0.2
6.8
−0.1
3.4 ± 0.2
6.6
−3.8
90.0
88.7 ± 9.5
10.7
−1.4
88.3 ± 9.5
10.8
−1.9
500.0
563.9 ± 9.6
1.7
12.8
531.2 ± 31.5
5.9
6.2
2700.0
2417.2 ± 166.0
6.9
−10.5
2612.8 ± 222.7
8.5
−3.2
10.0
8.8 ± 0.3
3.1
−11.8
9.8 ± 0.9
9.2
−1.6
120.0
108.1 ± 6.7
6.2
−9.9
110.3 ± 8.8
8.0
−8.1
600.0
640.3 ± 33.9
5.3
6.7
626.7 ± 49.4
7.9
4.4
3000.0
2827.8 ± 340.7
12.0
−5.7
3030.8 ± 257.8
8.5
1.0
5.4 ± 0.1
1.4
8.2
4.8 ± 0.4
8.9
−0.7
300.0
314.0 ± 44.5
14.2
4.7
297.9 ± 35.7
12.0
−0.7
1500.0
1551.7 ± 138.5
8.9
3.4
1491.4 ± 98.0
6.6
−0.6
7500.0
7891.3 ± 561.9
7.1
5.2
8105.5 ± 538.8
6.6
8.1
60.0
58.1 ± 6.4
10.9
−3.2
58.6 ± 5.1
8.6
−2.4
600.0
557.5 ± 36.4
6.5
−7.1
577.6 ± 44.9
7.8
−3.7
3000.0
2885.5 ± 125.7
4.4
−3.8
3103.2 ± 227.3
7.3
3.4
12000.0
12251.5 ± 1142.4
9.3
2.1
11953.6 ± 1121.4
9.4
−0.4
6.0
5.0
Cnom: nominal concentration; Cdet: mean value of the detected concentration
23
5.5 ± 0.4
Table 2. The extraction recoveries and matrix effects of the eight analytes in rat plasma at four concentration levels (n = 6) Analytes
Extraction recovery
C nom (ng·mL-1)
Mean (%) Gallic acid
Protocatechuic acid
Vanillic acid
Caffeic acid
Epicatechin
Isoquercitrin
Vincetoxicoside B
Quercetin
RSD (%)
Matrix effect Mean (%)
RSD (%)
30.0
93.4
3.0
89.3
1.7
300.0
113.7
4.6
88.8
2.6
800.0
92.2
7.4
111.6
1.4
3000.0
90.7
3.7
116.2
3.5
4.0
86.5
4.7
88.2
1.8
80.0
82.5
2.2
104.9
1.8
400.0
102.8
3.4
104.0
3.2
2000.0
81.7
1.8
98.3
1.2
30.0
88.4
0.6
87.8
5.4
600.0
84.5
3.0
110.5
7.2
2400.0
101.7
1.7
98.6
1.8
12000.0
113.0
2.8
113.1
1.9
6.0
82.5
3.2
89.1
1.8
100.0
76.6
6.8
98.0
3.5
400.0
88.8
3.8
93.4
6.6
2000.0
78.6
3.9
97.6
3.3
3.5
88.6
1.2
89.2
2.4
90.0
100.7
2.2
84.9
2.6
500.0
108.6
1.4
107.9
0.6
2700.0
98.1
5.5
103.7
2.4
10.0
86.1
2.4
102.1
7.8
120.0
84.0
2.3
89.5
4.9
600.0
79.2
3.1
94.4
6.4
3000.0
89.9
6.8
107.7
5.7
5.0
82.5
3.1
96.0
6.9
300.0
92.7
6.1
105.7
7.7
1500.0
85.7
5.3
96.6
5.8
7500.0
94.7
7.9
107.9
4.1
60.0
84.0
1.8
103.9
3.5
600.0
103.3
5.6
93.8
6.2
3000.0
82.5
7.0
103.3
9.0
12000.0
85.6
1.7
105.7
6.0
Cnom: nominal concentration
24
Table 3. Pharmacokinetic parameters of eight analytes after oral administration of Hypericum japonicum Thumb extract (n = 6, mean ± SD). Parameters
Gallic acid
t1/2α (h)
0.53 ± 0.029
t1/2β (h)
Vanillic acid
Caffeic acid
0.52 ± 0.07
0.36 ± 0.08
1.78 ± 0.23
3.23 ± 0.45
7.24 ± 0.68
5.99 ± 0.52
4.55 ± 0.59
K21 (1/h)
0.84 ± 0.072
0.21 ± 0.04
0.44 ± 0.008
0.19 ± 0.003
K10 (1/h)
0.54 ± 0.014
0.56 ± 0.06
0.59 ± 0.009
0.26 ± 0.08
0.56 ± 0.046
0.50 ± 0.02
1.29 ± 0.007
0.42 ± 0.071
0.0028 ± 0.0007
0.004 ± 0.0005
0.002 ± 0.0001
0.0018 ± 0.0003
K12 (1/h) -1
-1
CL (L h g ) Tpeak (h)
0.39 ± 0.024
Cmax (ng·mL-1)
228.31 ± 18.74
V/F (L g-1)
0.005 ± 0.001
Protocatechuic acid
0.54 ± 0.03
0.46 ± 0.05
930.76 ± 59.39
607.57 ± 85.81
0.44 ± 0.032 164.37 ± 11.38
0.007 ± 0.0008
0.003 ± 0.0004
0.0074 ± 0.0007
671.37 ± 207.22
3003.49 ± 358.63
2074.88 ± 225.52
871.13 ± 167.69
AUC0–t (ng·h mL )
555.68 ± 121.54
2921.58 ± 401.50
1667.08 ± 346.30
843.36 ± 177.91
MRT0–∞ (h)
3.53 ± 1.73
6.48 ± 0.95
5.95 ± 0.70
5.80 ± 0.63
MRT0–t (h)
2.13 ± 0.40
4.72 ± 0.18
3.15 ± 0.85
5.07 ± 0.81
-1
-1
AUC0–∞ (ng·h mL )
25
Table 4. Pharmacokinetic parameters of eight analytes after oral administration of Hypericum japonicum Thumb extract (n = 6, mean ± SD). Parameters
Epicatechin
Isoquercitrin
Vincetoxicoside B
Quercetin
t1/2α (h)
0.40 ± 0.07
0.92 ± 0.042
0.54 ± 0.10
0.38 ± 0.005
t1/2β (h)
7.93 ± 0.23
3.39 ± 0.53
3.01 ± 0.35
29.21 ± 3.86
K21 (1/h)
0.98 ± 0.14
0.36 ± 0.05
0.50 ± 0.016
0.14 ± 0.01
K10 (1/h)
0.16 ± 0.04
0.52 ± 0.02
0.64 ± 0.005
0.32 ± 0.04
0.91 ± 0.13
0.23 ± 0.01
0.41 ± 0.002
1.38 ± 0.04
0.001 ± 0.00
0.02 ± 0.001
0.02 ± 0.003
0.002 ± 0.0001
K12 (1/h) -1
-1
CL (L h g ) Tpeak (h)
0.75 ± 0.07
Cmax (ng·mL-1)
1151.71 ± 72.56
V/F (L g-1)
0.01 ± 0.002
0.69 ± 0.03
0.34 ± 0.02
0.52 ± 0.01
2087.70 ± 101.93
5372.34 ± 927.96
1684.46 ± 187.38
0.04 ± 0.005
0.03 ± 0.004
0.006 ± 0.0005
-1
11984.34 ± 257.81
7152.76 ± 1844.41
12329.48 ± 2442.48
16108.89 ± 314.23
-1
AUC0–t (ng·h mL )
10700.63 ± 158.91
7126.23 ± 1848.15
12298.17 ± 2431.17
15286.88 ± 290.92
MRT0–∞ (h)
10.39 ± 0.29
3.65 ± 0.18
3.42 ± 0.40
9.77 ± 0.11
MRT0–t (h)
7.48 ± 0.08
3.59 ± 0.20
3.36 ± 0.38
8.63 ± 0.064
AUC0–∞ (ng·h mL )
26
