Special issue paper Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3206
Determination of protein-unbound rhynchiphylline brain distribution by microdialysis and ultra-performance liquid chromatography with tandem mass spectrometry Chia-Jung Leea,b†, Thomas Y. Hsueha,b†, Lie-Chwen Lina,c and Tung-Hu Tsaia,b,d* ABSTRACT: The stem with hook of Uncaria rhynchophylla (Chinese herbal name Gou-Teng) is a traditional Chinese medicine that has been ethnopharmacologically used to extinguish wind and clean interior heat. Rhynchophylline (RHY), a tetracyclic oxindole alkaloid isolated from U. rhynchophylla, displays significant antineuroinflammatory effects. However, there is no evidence to indicate that rhynchophylline can cross the blood–brain barrier and be detected in the brain. In this study, an in vivo microdialysis sampling method coupled with UPLC/MS/MS was employed for the continuous simultaneous monitoring of unbound RHY in rat blood and brain. The precursor ion → product ion transition at m/z 385.2 → 160.0 for rhynchophylline was monitored. A calibration curve gave good linearity (r > 0.996) over the concentration range from 0.5 to 1000 ng/mL. The results demonstrated that rhynchophylline could be detected in the brain and plasma from 15 min to 6 h after its administration (1 or 10 mg/kg, i.v.). All the pharmacokinetic parameters of rhynchophylline in the brain and plasma were obtained. These results show that rhynchophylline can cross the blood–brain barrier and they provide useful clinical information. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: rhynchiphylline; microdialysis; UPLC/MS/MS; blood–brain barrier; pharmacokinetic parameters
Introduction
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* Correspondence to: T.-H. Tsai, Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. Email: [email protected] †
The first two authors contributed equally.
a
Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
b
Department of Education and Research, Taipei City Hospital, Taipei, Taiwan
c
National Research Institute of Chinese Medicine, Taipei, Taiwan
d
Graduate Institute of Acupuncture Science, China Medical University, Taichung, Taiwan Abbreviations used: ACD, anticoagulant citrate dextrose; ESI, electrospray ionization; RHY, rhynchophylline
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The plant species, stem with hook of Uncaria rhynchophylla (Gou-Teng), is a traditional Chinese medicine and it is ethnopharmacologically used to extinguish ‘wind’ and clean ‘interior heat’. Previous investigations of U. rhynchophylla have focused on its sedative, anticonvulsive and analgesic effects (Hsieh et al., 1999; Lee et al., 2003; Jung et al., 2010), although U. rhynchophylla has also been used to treat cerebrovascular diseases such as hypotension and stroke (Ndagijimana et al., 2013). According to a study by Lin et al., the oral administration of U. rhynchophylla extract successfully reduces kainic acid-induced neuronal death and discharges in hippocampal CA1 pyramidal neurons (Lin and Hsieh, 2011). Similar results were presented by Shim et al. (2009), indicating that the U. rhynchophylla extract has neuroprotective activity against 6-OHDA-induced toxicity through its antioxidative and antiapoptotic activities in Parkinson’s disease models. Furthermore, it has been suggested that U. rhynchophylla extract ameliorates cognitive deficits in a D-galactose-induced mouse model of Alzheimer’s disease. In addition, rhynchophylline and isorhynchophylline, the active components in U. rhynchophylla, significantly reduce Aβ-induced cell death, intracellular calcium overloading and tau protein hyperphosphorylation in PC12 cells (Xian et al., 2012). The chemical constituents of U. rhynchophylla, which include alkaloids, sterols, flavonoids and tannins, have been extensively studied. Among these constituents, alkaloids, such as rhynchophylline, isorhynchophylline, hirsutine, corynoxeine and isocorynoxeine,
play important bioactive roles. Rhynchophylline, a tetracyclic oxindole alkaloid isolated from U. rhynchophylla, markedly reduces the production of nitric oxide, prostaglandins E2, monocyte chemoattractant protein, tumor necrosis factor-α and interleukin1β to protect against neuroinflammation in an lipopolysaccharide (LPS)-activated microglia model (Song et al., 2012). Rhynchophylline also suppresses GluA2/3 expression in ketamineinduced PC12 cells and displays anti-addiction effects through down-regulating GluN1 expression (Zhou et al., 2013). The active ingredients of U. rhynchophylla responsible for its proposed activity in controlling Alzheimer’s disease have been identified as
C.-J. Lee et al. rhynchophylline and isorhynchophylline (Xian et al., 2012). Therefore, rhynchophylline is a potential neuroprotective compound and it is necessary to understand its pharmacokinetics in the brain. Pharmacokinetic studies can help evaluate the absorption, distribution, metabolism and elimination of drugs, which is very important in designing clinically rational treatment regimes. However, there have been few reports on the pharmacokinetics of rhynchophylline. A study by Cai et al. established a liquid chromatography mass spectrometry method for the determination of the concentration of rhynchophylline in rat plasma, finding that, after oral administration of a single dosage of 15 mg/kg rhynchophylline, the half-life of rhynchophylline is 1.72 h (Wang et al., 2010; Cai et al., 2013). However, there has been no evidence that rhynchophylline can cross the blood– brain barrier and be identified in the brain by in vivo examination (Imamura et al., 2011). For this task, microdialysis sampling is a powerful technique for monitoring molecules in the extracellular environment. As a continuous sampling method, it can simultaneously monitor multiple sites in biological fluids and most tissues, such as blood or brain. This method is useful for the dynamic sampling and monitoring of the unbound drug in a single animal. Our hypothesis is that rhynchophylline may penetrate the blood–brain barrier to produce a pharmacological effect in the central nervous system. To investigate this hypothesis, microdialysis probes were inserted into the jugular vein and brain of Sprague–Dawley rats to analyze the pharmacokinetcis of rhynchophylline in the peripheral and central nervous system. An in vivo microdialysis sampling method coupled with ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) was employed for the continuous simultaneous monitoring of protein-unbound rhynchophylline in rat blood and brain.
Experimental Chemicals and reagents A reference standard of rhynchophylline (purity >98%) was obtained from Wako (Osaka, Japan). HPLC-grade methanol was obtained from the J.T. Baker Company (Phillipsburg, NJ, USA). Water was prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). Formic acid and other reagents were of analytical grade and purchased from Merck (Darmstadt, Germany).
Animals All experimental protocols involving animals were reviewed and approved by the Institutional Animal Experimental Committee of National Yang-Ming University, Taipei, Taiwan (approval number 981272). Six-week-old male Sprague–Dawley rats (220 ± 20 g body weight) were purchased from the laboratory animal center of National Yang-Ming University. All experimental animals were housed in standard rat cages on a 12 h light/dark cycle under institutional guidelines and had free access to food and water.
Preparation of U. rhynchophylla extract
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A crude sample of U. rhynchophylla (4 kg) obtained from our previous study (Wu et al., 2013) was immersed in 80 L ethanol and refluxed twice for 4 h. The extraction solution was filtered through filter paper (Whatman no. 5, Maidstone, UK) and concentrated to give about 0.5 kg of the U. rhynchophylla extract. The U. rhynchophylla extract (1 g) was dissolved in 100 mL ethanol and then centrifuged at 13,200 rpm
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(Eppendorf Centrifuge 5415R, Kent City, MI, USA) for 10 min to remove the dissolved part. An external standard method was used to construct the calibration curve (1–100 ng/mL) to determine the content of rhynchophylline in the U. rhynchophylla extract. The amount of rhynchophylline was determined to be 1.21 ± 0.01 mg/g (n = 3). The cali2 bration curve of this analysis was: y = 5595.4x + 905.72; R 0.9998.
Instrumentation and analytical conditions Chromatographic analysis was carried out on an Acquity UPLC system (Waters Corp., Milford, MA, USA), which consisted of a chromatographic pump and an Acquity UPLC BEH C18 column (50 × 2.1 mm i.d., 1.7 μm; Waters Corp.), combined with an in-line pre-column (KrudKatcher Ultra, Phenomenex, Torrance, CA, USA). The analysis temperature was maintained at 40°C. The mobile phase consisted of (A) 0.1% (v/v) formic acid and (B) methanol containing 0.1% formic acid and delivered at 0.3 mL/min. Isocratic elution was A:B = 65:35. A Valco valve (Valco Intruments, Houston, TX, USA) was used to divert the first minute of the eluent to waste in order to elute salt in the dialysate. The injection volume was 5 μL. Mass spectrometric detection was performed with a Xevo-TQ triple quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) with an electrospray ionization (ESI) interface. Pure argon was used as the collision gas and pure nitrogen was used as the nebulizing gas. The ESI source was operated in positive ion mode. Multiple-reaction monitoring conditions were optimized by the infusion analysis of the compound (10 ng/mL) dissolved in methanol at 20 μL/min. The precursor ion–product ion transitions of the analyte are given in Table 1. Masslynx V4.1 software (Waters Corp., Milford, MA, USA) was used for data acquisition and treatment.
Microdialysis experiment The blood and brain microdialysis systems consisted of a CMA/100 microinjection pump, CMA/140 microfraction collector (CMA, Stockholm, Sweden) and in-house prepared microdialysis probes. The silica capillary had a concentric design and active lengths of 10 and 3 mm for blood and brain, respectively. The tips of the capillaries were covered by a dialysis membrane (150 μm outer diameter with a nominal molecular weight cut-off of 13,000 Da; Spectrum Co., Laguna Hills, CA, USA; Shaw and Tsai, 2012). The rats were anesthetized with ethyl carbamate (1 g/mL) and α-chloralose (0.1 g/mL) at a dose of 1 mL/kg throughout the experiment. The right femoral vein cannula (PE-50 tubing) was tunneled beneath the skin in the anesthetized experimental rat for drug administration. The blood microdialysis probe was inserted into the jugular vein toward the right atrium and then perfused with anticoagulant citrate dextrose (ACD) solution (citric acid 3.5 mM, sodium citrate 7.5 mM and dextrose 13.6 mM). The brain microdialysis probe was implanted in the striatum zone and perfused with Ringer’s solution (consisting of NaCl
Table 1. Mass spectrometric conditions of rhynchophylline Parameters
Rhynchophylline
Ionization mode Capillary voltage (kV) Cone voltage (V) Collision energy (eV) Source temperature (°C) Desolvation temperature (°C) Desolvation gas flow rate (L/h) Precursor-product ion reaction monitoring
Copyright © 2014 John Wiley & Sons, Ltd.
ESI (+) 2.7 46 28 150 550 1000 m/z 385.2 → 160.0
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Determination of protein-unbound rhynchiphylline brain distribution 8.6 g; KCl 0.3 g; CaCl2 0.33 g in 10 00 mL H2O; pH 7.0). The flow rates of the ACD and Ringer’s solution were set at 2.0 μL/min by a microinjection. The dialysates were collected every 15 min for 6 h and preserved at 20 °C in a refrigerator.
Method validation The proposed method was validated according to the US Food and Drug Administration guidelines. A stock solution of rhynchophylline was prepared in methanol at a concentration of 1 mg/mL. The working standard solution was serially diluted by the mobile phase (35% MeOH). The calibration standards were prepared by adding different concentrations of working standards (5 μL) and blank blood or brain dialysates (45 μL) to give 5, 10, 25, 50, 100, 250 and 500 ng/mL solutions of rhynchophylline. The calibration curves were constructed by least-squares linear regression of the peak-area ratios vs the concentrations of rhynchophylline. The lower limit of quantification (LLOQ) was defined as the lowest concentration of the calibration range having an accuracy within ±20% and a precision within 20%. The intra-day precision and accuracy of the analytical method were assessed by three concentrations (1, 10 and 100 ng/mL) of quality control samples in six replicates. The inter-day precision was determined across three concentrations on six different days, and the mean concentrations and the coefficient of variation were calculated. A study of the matrix effect was conducted to evaluate selectivity, by comparing the peak areas of the analytes in those samples spiked with the analyte. Three concentrations of the analytes, each in six replicates, were studied. When the peak area ratio (A/B × 100)% of the analytes and the internal standard solution was between 80 and 120%, the matrix effect could be considered as negligible. The freeze–thaw stability was assessed over three freeze–thaw cycles.
Recovery of microdialysates The recovery of the microdialysates was estimated by in vivo assay. Three different concentrations (0.1, 1 and 10 μg/mL) of rhynchophylline were dissolved in ACD and Ringer’s solution for blood and brain probes, respectively. After balancing for 2 h, the ACD and Ringer’s solution containing rhynchophylline was perfused through the microdialysis probes. The perfusion flow rate was constant at 2 μL/min. The in vivo recovery (R) of rhynchophylline was calculated as R = (Cperf – Cdial)/Cdial, where Cperf is the concentration of the perfusate and Cdial the concentration of the collected dialysate.
Figure 1. Chemical structures of rhynchophylline. Molecular weight = 384.
Figure 2. Product ion spectrum of protonated rhynchophylline.
[C9H8N2O]+. Electron ionization mass spectrometry revealed that rhynchophylline underwent loss of hydroxyl, methyl or methoxyl groups from the molecular ion, and cleavage of the spiran ring C, in agreement with a previous report (Saxton, 1965). The quantification of rhynchophylline was performed by montoring the m/z 385.2 → 160.0 transition. The mobile phases were water (A) and methanol (B), both of which contained 0.1% formic acid. An isocratic elution, A:B = 65:35 for 4 min, was chosen to achieve a short run time. The retention time of rhynchophylline with this system was 1.74 min.
Animal preparation The ethanolic extract of U. rhynchophylla (5 g) was dissolved in 100 mL of double-distilled water with PEG 400 (1:1), and sonicated (Branson 2510, Danbury, CT, USA) for 10 min. The final concentration was 50 mg/mL. The drug solution was given to rats via gastric gavage in the group for oral administration (dose 1000 mg/kg, equal to 1.21 mg/kg rhynchophylline, n = 5). For the other groups, rhynchophylline was dissolved in PEG 400 and intravenously administered at three different concentrations (1 or 10 mg/kg, i.v.). The microdialysis sample was stored at 20°C before analysis. WinNonlin Standard Edition version 1.1 software (Pharsight Corp., Mountain View, CA, USA) was used to calculate each individual set of data by a noncompartmental model.
Results and discussion Method development
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Selectivity. The specificity of this method was evaluated by analyzing individual blank microdialysis samples from six different rats. No significant interference from endogenous components was found at the retention times of rhynchophylline. Figure 3 shows UPLC/MS/MS chromatograms of blank microdialysate, blank microdialysate spiked with rhynchophylline, and a microdialysate sample collected 15 min after the i.v. administration of rhynchophylline. Linearity and LLOQ. The linearity was evaluated on six separate occasions with two sets of calibration curves per occasion. The calibration curves indicated that the linearity was good over the concentration range 0.5–1000 ng/mL for rhynchophylline. Typical linear regression equations for the calibration curves were y = 4266.7x + 2754.8 (r = 0.9991), with relative standard deviation (RSD) of the slope 5.6% and RSD of the intercept 1.5% for ACD, and y = 4759x + 5957.2 (r = 0.9957), with RSD of the slope 0.9% and RSD of the intercept 3.5% for Ringer’s solution. The LLOQ of the assay was 0.5 ng/mL for both ACD and Ringer’s solution, for which the signal-to-noise ratios were
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The chemical structure of rhynchophylline is shown in Fig. 1. The optimized ESI conditions for rhynchophylline are shown in Table 1, and the major peak detected for rhynchophylline in positive ion mode was the [M + H]+ ion at m/z 385.2. Fragmentation of m/z 385.2 produced a major product ion at m/z 160 (Fig. 2), which was assigned to the oxindole structure
Method validation
C.-J. Lee et al.
Figure 3. UPLC/MS/MS chromatograms of rhynchophylline: (A) a blank rat plasma sample; (B) blank plasma spiked with rhynchophylline standard solution (100 ng/mL); (C) rat blood sample (72.8 ng/mL) collected at 15 min after rhynchophylline (1 mg/kg i.v.) administration; and (D) rat sample brain (88.7 ng/mL) collected at 15 min after rhynchophylline (1 mg/kg i.v.) administration.
both >10. These data demonstrated that this method was sensitive enough for the pharmacokinetic study of rhynchophylline in vivo. Accuracy and precision. The intra-day and inter-day accuracy and precision for rhynchophylline in different perfusates are summarized in Table 2. All the results for the samples tested were within the acceptable criterion of ±20%, and the matrix effect ranged from 114 to 121% (Table 3), which indicates a stable matrix effect for rhynchophylline. Pharmacokinetic applications. The validated method was successfully applied to the pharmacokinetic study of rhynchophylline. The concentrations of rhynchophylline in the rat plasma and brain were monitored by the validated UPLC/MS/MS method after rhynchophylline adiministration. After oral administration of U.
rhynchophylla extract at 1 g/kg, rhynchophylline was undetectable in plasma or brain samples, which may be due to the low rhynchophylline content (1.21 ± 0.01 mg/g) in the herbal extract. In previous research, Cai et al. (2013) gave a single high dosage of rhynchophylline 15 mg/kg, and Wang et al. (2010) gave a single dosage of 37.5 mg/kg, so that the concentrations of rhynchophylline and its metabolites could be measured in plasma. The mean blood and brain concentration–time profiles after rhynchophylline administration (1 or 10 mg/kg, i.v.) dosedependently are shown in Fig. 4. The plasma concentrations appeared to decline after either a low dose or after a high dose with rapid distribution and elimination. The data demonstrated that rhynchonphylline easily penetrates the blood–brain barrier, and can be detected at the first microdialysis collection time point within 15 min of administration. Comparing the rhynchophylline
Table 2. Precision and accuracy for analysis of rhynchophylline in different dialysate Spiked concentration (ng/mL)
Intra-day (n = 6) Observed concentration (ng/mL)
ACD (blood)
Ringer’s solution (brain)
1 10 100 1 10 100
0.85 ± 0.03 10.03 ± 0.12 99.48 ± 0.96 0.89 ± 0.04 9.92 ± 0.14 99.28 ± 1.22
Precision (RSD, %) 2.7 1.24 0.96 4.3 1.37 1.22
Inter-day (n = 6) Accuracy (RE, %)
Observed concentration (ng/mL)
84.27 100.29 99.48 89.41 99.18 99.28
0.81 ± 0.08 10.38 ± 0.91 99.17 ± 1.02 0.92 ± 0.05 10.05 ± 0.23 99.18 ± 2.24
Precision (RSD, %) 7.74 9.07 1.02 4.82 2.34 2.24
Accuracy (RE, %) 80.75 103.79 99.17 92.17 100.52 99.18
ACD, Anticoagulant citrate dextrose.
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Mean extraction recovery (n = 6) Mean (%) RSD (%) 6.3 2.6 0.8 3.3 4.4 0.7
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114.13 ± 15.62 15.59 ± 7.87 1.64 ± 0.26 489.92 ± 70.80 92.08 ± 14.22 15.90 108.87 ± 44.85 732.90 ± 220.68 81.66 ± 9.27 160.66 ± 33.15 28.09 ± 1.14
10 mg/kg
37.13 ± 8.34 76.42 ± 21.38 10.31 ± 2.91 125.36 ± 19.04 16.88 ± 3.56
1 mg/kg
Blood Parameters
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levels in blood and in brain after rhynchophylline administration (10 mg/kg, i.v.), the AUC (area under the curve) of rhynchophylline in blood was 81.66 ± 9.27 min μg/mL, which was higher than the AUC of rhynchophylline in the brain (14.01 ± 3.13 min μg/mL) (Table 4). It is suggested that rhynchophylline may contribute to the permeability of the blood–brain barrier. The AUCbrain/blood ratio of 17.15% was calculated from the results of the rhynchophylline administration (10 mg/kg, i.v.) (Table 4).
Table 4. Pharmacokinetic parameters of blood and brain after rhynchophylline administration (1 and 10 mg/kg, i.v.)
Figure 4. Blood (A) and brain (B) drug concentration–time curves of rhynchophylline after i.v. administration of rhynchophylline in rats.
t1/2 (min) C0 (ng/mL) AUC (min μg/mL) Cl (mL/min/kg) Vss (L/kg) Brain–blood ratio (%)
121.07 121.30 114.06 113.53 126.78 115.04
1 mg/kg
Ringer’s solution (Brain)
1 10 100 1 10 100
Brain
ACD (blood)
Data are expressed as the mean ± standard deviation (n = 6). AUC, area under the plasma concentration versus the time curve; t1/2, half-life; Tmax, time to reach the drug concentration in plasma; C0, maximal drug concentration in plasma; Vss, volume of distribution at steady state.
Spiked concentration (ng/mL)
10 mg/kg
Table 3. Precision, accuracy, matrix effect, and recovery for analysis of rhynchophylline in rat plasma
146.01 ± 15.68 337.16 ± 183.69 14.01 ± 3.13 609.25 ± 125.30 82.64 ± 12.06 17.15
Determination of protein-unbound rhynchiphylline brain distribution
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Conclusion We have developed and validated a combined UPLC/MS/MS microdialysis sampling method to concurrently monitor the concentrations of rhynchophylline in rat plasma and brain. This is the first time that rhynchophylline has been monitored in the brain, and it provided the first evidence that rhynchophylline can cross the blood–brain barrier.
Acknowledgments This study was supported by National Science Council grants (NSC102-2113-M-010-001-MY3), post-doctoral training grant (NSC 100-2811-M-010-005 and NSC 101-2811-M-010-003) and TCH 10202; 10102-62-084 from Taipei City Hospital, Taipei, Taiwan.
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apoptosis in rat hippocampal slices. Neuroscience Letters 2003; 348: 51–55. Lin YW and Hsieh CL. Oral Uncaria rhynchophylla (UR) reduces kainic acid-induced epileptic seizures and neuronal death accompanied by attenuating glial cell proliferation and S100B proteins in rats. Journal of Ethnopharmacology 2011; 135: 313–320. Ndagijimana A, Wang X, Pan G, Zhang F, Feng H and Olaleye O. A review on indole alkaloids isolated from Uncaria rhynchophylla and their pharmacological studies. Fitoterapia 2013; 86: 35–47. Saxton JE. Alkaloids of Mitragyna and Ourouparia species. In The Alkaloids: Chemistry and Physiology, Vol. 8, Manske RHF (ed.). Academic Press: London, 1965; 59–91. Shaw LH and Tsai TH. Simultaneous determination and pharmacokinetics of protein unbound aspirin and salicylic acid in rat blood and brain by microdialysis: an application to herbal–drug interaction. Journal of Chromatography B 2012; 895–896: 31–38. Shim JS, Kim HG, Ju MS, Choi JG, Jeong SY and Oh MS. Effects of the hook of Uncaria rhynchophylla on neurotoxicity in the 6-hydroxydopamine model of Parkinson’s disease. Journal of Ethnopharmacology 2009; 126: 361–365. Song Y, Qu R, Zhu S, Zhang R and Ma S. Rhynchophylline attenuates LPSinduced pro-inflammatory responses through down-regulation of MAPK/NF-kappaB signaling pathways in primary microglia. Phytotherapy Research 2012; 26: 1528–1533. Wang W, Ma CM and Hattori M. Metabolism and pharmacokinetics of rhynchophylline in rats. Biological and Pharmaceutical Bulletin 2010; 33: 669–676. Wu YT, Lin LC and Tsai TH. Determination of rhynchophylline and hirsutine in rat plasma by UPLC-MS/MS after oral administration of Uncaria rhynchophylla extract. Biomedical Chromatography 2013; doi: 10.1002/bmc.3052. Xian YF, Lin ZX, Mao QQ, Hu Z, Zhao M, Che CT and Ip SP. Bioassayguided isolation of neuroprotective compounds from Uncaria rhynchophylla against beta-amyloid-induced neurotoxicity. Evidence-Based Complementary and Alternative Medicine 2012; 2012: 802625. Zhou JY, Chen J, Zhou SW and Mo ZX. Individual and combined effects of rhynchophylline and ketamine on proliferation, NMDAR1 and GluA2/3 protein expression in PC12 cells. Fitoterapia 2013; 85: 125–129.
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Biomed. Chromatogr. 2014; 28: 901–906
(wileyonlinelibrary.com) DOI 10.1002/bmc.3206
Determination of protein-unbound rhynchiphylline brain distribution by microdialysis and ultra-performance liquid chromatography with tandem mass spectrometry Chia-Jung Leea,b†, Thomas Y. Hsueha,b†, Lie-Chwen Lina,c and Tung-Hu Tsaia,b,d* ABSTRACT: The stem with hook of Uncaria rhynchophylla (Chinese herbal name Gou-Teng) is a traditional Chinese medicine that has been ethnopharmacologically used to extinguish wind and clean interior heat. Rhynchophylline (RHY), a tetracyclic oxindole alkaloid isolated from U. rhynchophylla, displays significant antineuroinflammatory effects. However, there is no evidence to indicate that rhynchophylline can cross the blood–brain barrier and be detected in the brain. In this study, an in vivo microdialysis sampling method coupled with UPLC/MS/MS was employed for the continuous simultaneous monitoring of unbound RHY in rat blood and brain. The precursor ion → product ion transition at m/z 385.2 → 160.0 for rhynchophylline was monitored. A calibration curve gave good linearity (r > 0.996) over the concentration range from 0.5 to 1000 ng/mL. The results demonstrated that rhynchophylline could be detected in the brain and plasma from 15 min to 6 h after its administration (1 or 10 mg/kg, i.v.). All the pharmacokinetic parameters of rhynchophylline in the brain and plasma were obtained. These results show that rhynchophylline can cross the blood–brain barrier and they provide useful clinical information. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: rhynchiphylline; microdialysis; UPLC/MS/MS; blood–brain barrier; pharmacokinetic parameters
Introduction
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* Correspondence to: T.-H. Tsai, Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. Email: [email protected] †
The first two authors contributed equally.
a
Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
b
Department of Education and Research, Taipei City Hospital, Taipei, Taiwan
c
National Research Institute of Chinese Medicine, Taipei, Taiwan
d
Graduate Institute of Acupuncture Science, China Medical University, Taichung, Taiwan Abbreviations used: ACD, anticoagulant citrate dextrose; ESI, electrospray ionization; RHY, rhynchophylline
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The plant species, stem with hook of Uncaria rhynchophylla (Gou-Teng), is a traditional Chinese medicine and it is ethnopharmacologically used to extinguish ‘wind’ and clean ‘interior heat’. Previous investigations of U. rhynchophylla have focused on its sedative, anticonvulsive and analgesic effects (Hsieh et al., 1999; Lee et al., 2003; Jung et al., 2010), although U. rhynchophylla has also been used to treat cerebrovascular diseases such as hypotension and stroke (Ndagijimana et al., 2013). According to a study by Lin et al., the oral administration of U. rhynchophylla extract successfully reduces kainic acid-induced neuronal death and discharges in hippocampal CA1 pyramidal neurons (Lin and Hsieh, 2011). Similar results were presented by Shim et al. (2009), indicating that the U. rhynchophylla extract has neuroprotective activity against 6-OHDA-induced toxicity through its antioxidative and antiapoptotic activities in Parkinson’s disease models. Furthermore, it has been suggested that U. rhynchophylla extract ameliorates cognitive deficits in a D-galactose-induced mouse model of Alzheimer’s disease. In addition, rhynchophylline and isorhynchophylline, the active components in U. rhynchophylla, significantly reduce Aβ-induced cell death, intracellular calcium overloading and tau protein hyperphosphorylation in PC12 cells (Xian et al., 2012). The chemical constituents of U. rhynchophylla, which include alkaloids, sterols, flavonoids and tannins, have been extensively studied. Among these constituents, alkaloids, such as rhynchophylline, isorhynchophylline, hirsutine, corynoxeine and isocorynoxeine,
play important bioactive roles. Rhynchophylline, a tetracyclic oxindole alkaloid isolated from U. rhynchophylla, markedly reduces the production of nitric oxide, prostaglandins E2, monocyte chemoattractant protein, tumor necrosis factor-α and interleukin1β to protect against neuroinflammation in an lipopolysaccharide (LPS)-activated microglia model (Song et al., 2012). Rhynchophylline also suppresses GluA2/3 expression in ketamineinduced PC12 cells and displays anti-addiction effects through down-regulating GluN1 expression (Zhou et al., 2013). The active ingredients of U. rhynchophylla responsible for its proposed activity in controlling Alzheimer’s disease have been identified as
C.-J. Lee et al. rhynchophylline and isorhynchophylline (Xian et al., 2012). Therefore, rhynchophylline is a potential neuroprotective compound and it is necessary to understand its pharmacokinetics in the brain. Pharmacokinetic studies can help evaluate the absorption, distribution, metabolism and elimination of drugs, which is very important in designing clinically rational treatment regimes. However, there have been few reports on the pharmacokinetics of rhynchophylline. A study by Cai et al. established a liquid chromatography mass spectrometry method for the determination of the concentration of rhynchophylline in rat plasma, finding that, after oral administration of a single dosage of 15 mg/kg rhynchophylline, the half-life of rhynchophylline is 1.72 h (Wang et al., 2010; Cai et al., 2013). However, there has been no evidence that rhynchophylline can cross the blood– brain barrier and be identified in the brain by in vivo examination (Imamura et al., 2011). For this task, microdialysis sampling is a powerful technique for monitoring molecules in the extracellular environment. As a continuous sampling method, it can simultaneously monitor multiple sites in biological fluids and most tissues, such as blood or brain. This method is useful for the dynamic sampling and monitoring of the unbound drug in a single animal. Our hypothesis is that rhynchophylline may penetrate the blood–brain barrier to produce a pharmacological effect in the central nervous system. To investigate this hypothesis, microdialysis probes were inserted into the jugular vein and brain of Sprague–Dawley rats to analyze the pharmacokinetcis of rhynchophylline in the peripheral and central nervous system. An in vivo microdialysis sampling method coupled with ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) was employed for the continuous simultaneous monitoring of protein-unbound rhynchophylline in rat blood and brain.
Experimental Chemicals and reagents A reference standard of rhynchophylline (purity >98%) was obtained from Wako (Osaka, Japan). HPLC-grade methanol was obtained from the J.T. Baker Company (Phillipsburg, NJ, USA). Water was prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). Formic acid and other reagents were of analytical grade and purchased from Merck (Darmstadt, Germany).
Animals All experimental protocols involving animals were reviewed and approved by the Institutional Animal Experimental Committee of National Yang-Ming University, Taipei, Taiwan (approval number 981272). Six-week-old male Sprague–Dawley rats (220 ± 20 g body weight) were purchased from the laboratory animal center of National Yang-Ming University. All experimental animals were housed in standard rat cages on a 12 h light/dark cycle under institutional guidelines and had free access to food and water.
Preparation of U. rhynchophylla extract
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A crude sample of U. rhynchophylla (4 kg) obtained from our previous study (Wu et al., 2013) was immersed in 80 L ethanol and refluxed twice for 4 h. The extraction solution was filtered through filter paper (Whatman no. 5, Maidstone, UK) and concentrated to give about 0.5 kg of the U. rhynchophylla extract. The U. rhynchophylla extract (1 g) was dissolved in 100 mL ethanol and then centrifuged at 13,200 rpm
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(Eppendorf Centrifuge 5415R, Kent City, MI, USA) for 10 min to remove the dissolved part. An external standard method was used to construct the calibration curve (1–100 ng/mL) to determine the content of rhynchophylline in the U. rhynchophylla extract. The amount of rhynchophylline was determined to be 1.21 ± 0.01 mg/g (n = 3). The cali2 bration curve of this analysis was: y = 5595.4x + 905.72; R 0.9998.
Instrumentation and analytical conditions Chromatographic analysis was carried out on an Acquity UPLC system (Waters Corp., Milford, MA, USA), which consisted of a chromatographic pump and an Acquity UPLC BEH C18 column (50 × 2.1 mm i.d., 1.7 μm; Waters Corp.), combined with an in-line pre-column (KrudKatcher Ultra, Phenomenex, Torrance, CA, USA). The analysis temperature was maintained at 40°C. The mobile phase consisted of (A) 0.1% (v/v) formic acid and (B) methanol containing 0.1% formic acid and delivered at 0.3 mL/min. Isocratic elution was A:B = 65:35. A Valco valve (Valco Intruments, Houston, TX, USA) was used to divert the first minute of the eluent to waste in order to elute salt in the dialysate. The injection volume was 5 μL. Mass spectrometric detection was performed with a Xevo-TQ triple quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) with an electrospray ionization (ESI) interface. Pure argon was used as the collision gas and pure nitrogen was used as the nebulizing gas. The ESI source was operated in positive ion mode. Multiple-reaction monitoring conditions were optimized by the infusion analysis of the compound (10 ng/mL) dissolved in methanol at 20 μL/min. The precursor ion–product ion transitions of the analyte are given in Table 1. Masslynx V4.1 software (Waters Corp., Milford, MA, USA) was used for data acquisition and treatment.
Microdialysis experiment The blood and brain microdialysis systems consisted of a CMA/100 microinjection pump, CMA/140 microfraction collector (CMA, Stockholm, Sweden) and in-house prepared microdialysis probes. The silica capillary had a concentric design and active lengths of 10 and 3 mm for blood and brain, respectively. The tips of the capillaries were covered by a dialysis membrane (150 μm outer diameter with a nominal molecular weight cut-off of 13,000 Da; Spectrum Co., Laguna Hills, CA, USA; Shaw and Tsai, 2012). The rats were anesthetized with ethyl carbamate (1 g/mL) and α-chloralose (0.1 g/mL) at a dose of 1 mL/kg throughout the experiment. The right femoral vein cannula (PE-50 tubing) was tunneled beneath the skin in the anesthetized experimental rat for drug administration. The blood microdialysis probe was inserted into the jugular vein toward the right atrium and then perfused with anticoagulant citrate dextrose (ACD) solution (citric acid 3.5 mM, sodium citrate 7.5 mM and dextrose 13.6 mM). The brain microdialysis probe was implanted in the striatum zone and perfused with Ringer’s solution (consisting of NaCl
Table 1. Mass spectrometric conditions of rhynchophylline Parameters
Rhynchophylline
Ionization mode Capillary voltage (kV) Cone voltage (V) Collision energy (eV) Source temperature (°C) Desolvation temperature (°C) Desolvation gas flow rate (L/h) Precursor-product ion reaction monitoring
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ESI (+) 2.7 46 28 150 550 1000 m/z 385.2 → 160.0
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Determination of protein-unbound rhynchiphylline brain distribution 8.6 g; KCl 0.3 g; CaCl2 0.33 g in 10 00 mL H2O; pH 7.0). The flow rates of the ACD and Ringer’s solution were set at 2.0 μL/min by a microinjection. The dialysates were collected every 15 min for 6 h and preserved at 20 °C in a refrigerator.
Method validation The proposed method was validated according to the US Food and Drug Administration guidelines. A stock solution of rhynchophylline was prepared in methanol at a concentration of 1 mg/mL. The working standard solution was serially diluted by the mobile phase (35% MeOH). The calibration standards were prepared by adding different concentrations of working standards (5 μL) and blank blood or brain dialysates (45 μL) to give 5, 10, 25, 50, 100, 250 and 500 ng/mL solutions of rhynchophylline. The calibration curves were constructed by least-squares linear regression of the peak-area ratios vs the concentrations of rhynchophylline. The lower limit of quantification (LLOQ) was defined as the lowest concentration of the calibration range having an accuracy within ±20% and a precision within 20%. The intra-day precision and accuracy of the analytical method were assessed by three concentrations (1, 10 and 100 ng/mL) of quality control samples in six replicates. The inter-day precision was determined across three concentrations on six different days, and the mean concentrations and the coefficient of variation were calculated. A study of the matrix effect was conducted to evaluate selectivity, by comparing the peak areas of the analytes in those samples spiked with the analyte. Three concentrations of the analytes, each in six replicates, were studied. When the peak area ratio (A/B × 100)% of the analytes and the internal standard solution was between 80 and 120%, the matrix effect could be considered as negligible. The freeze–thaw stability was assessed over three freeze–thaw cycles.
Recovery of microdialysates The recovery of the microdialysates was estimated by in vivo assay. Three different concentrations (0.1, 1 and 10 μg/mL) of rhynchophylline were dissolved in ACD and Ringer’s solution for blood and brain probes, respectively. After balancing for 2 h, the ACD and Ringer’s solution containing rhynchophylline was perfused through the microdialysis probes. The perfusion flow rate was constant at 2 μL/min. The in vivo recovery (R) of rhynchophylline was calculated as R = (Cperf – Cdial)/Cdial, where Cperf is the concentration of the perfusate and Cdial the concentration of the collected dialysate.
Figure 1. Chemical structures of rhynchophylline. Molecular weight = 384.
Figure 2. Product ion spectrum of protonated rhynchophylline.
[C9H8N2O]+. Electron ionization mass spectrometry revealed that rhynchophylline underwent loss of hydroxyl, methyl or methoxyl groups from the molecular ion, and cleavage of the spiran ring C, in agreement with a previous report (Saxton, 1965). The quantification of rhynchophylline was performed by montoring the m/z 385.2 → 160.0 transition. The mobile phases were water (A) and methanol (B), both of which contained 0.1% formic acid. An isocratic elution, A:B = 65:35 for 4 min, was chosen to achieve a short run time. The retention time of rhynchophylline with this system was 1.74 min.
Animal preparation The ethanolic extract of U. rhynchophylla (5 g) was dissolved in 100 mL of double-distilled water with PEG 400 (1:1), and sonicated (Branson 2510, Danbury, CT, USA) for 10 min. The final concentration was 50 mg/mL. The drug solution was given to rats via gastric gavage in the group for oral administration (dose 1000 mg/kg, equal to 1.21 mg/kg rhynchophylline, n = 5). For the other groups, rhynchophylline was dissolved in PEG 400 and intravenously administered at three different concentrations (1 or 10 mg/kg, i.v.). The microdialysis sample was stored at 20°C before analysis. WinNonlin Standard Edition version 1.1 software (Pharsight Corp., Mountain View, CA, USA) was used to calculate each individual set of data by a noncompartmental model.
Results and discussion Method development
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Selectivity. The specificity of this method was evaluated by analyzing individual blank microdialysis samples from six different rats. No significant interference from endogenous components was found at the retention times of rhynchophylline. Figure 3 shows UPLC/MS/MS chromatograms of blank microdialysate, blank microdialysate spiked with rhynchophylline, and a microdialysate sample collected 15 min after the i.v. administration of rhynchophylline. Linearity and LLOQ. The linearity was evaluated on six separate occasions with two sets of calibration curves per occasion. The calibration curves indicated that the linearity was good over the concentration range 0.5–1000 ng/mL for rhynchophylline. Typical linear regression equations for the calibration curves were y = 4266.7x + 2754.8 (r = 0.9991), with relative standard deviation (RSD) of the slope 5.6% and RSD of the intercept 1.5% for ACD, and y = 4759x + 5957.2 (r = 0.9957), with RSD of the slope 0.9% and RSD of the intercept 3.5% for Ringer’s solution. The LLOQ of the assay was 0.5 ng/mL for both ACD and Ringer’s solution, for which the signal-to-noise ratios were
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The chemical structure of rhynchophylline is shown in Fig. 1. The optimized ESI conditions for rhynchophylline are shown in Table 1, and the major peak detected for rhynchophylline in positive ion mode was the [M + H]+ ion at m/z 385.2. Fragmentation of m/z 385.2 produced a major product ion at m/z 160 (Fig. 2), which was assigned to the oxindole structure
Method validation
C.-J. Lee et al.
Figure 3. UPLC/MS/MS chromatograms of rhynchophylline: (A) a blank rat plasma sample; (B) blank plasma spiked with rhynchophylline standard solution (100 ng/mL); (C) rat blood sample (72.8 ng/mL) collected at 15 min after rhynchophylline (1 mg/kg i.v.) administration; and (D) rat sample brain (88.7 ng/mL) collected at 15 min after rhynchophylline (1 mg/kg i.v.) administration.
both >10. These data demonstrated that this method was sensitive enough for the pharmacokinetic study of rhynchophylline in vivo. Accuracy and precision. The intra-day and inter-day accuracy and precision for rhynchophylline in different perfusates are summarized in Table 2. All the results for the samples tested were within the acceptable criterion of ±20%, and the matrix effect ranged from 114 to 121% (Table 3), which indicates a stable matrix effect for rhynchophylline. Pharmacokinetic applications. The validated method was successfully applied to the pharmacokinetic study of rhynchophylline. The concentrations of rhynchophylline in the rat plasma and brain were monitored by the validated UPLC/MS/MS method after rhynchophylline adiministration. After oral administration of U.
rhynchophylla extract at 1 g/kg, rhynchophylline was undetectable in plasma or brain samples, which may be due to the low rhynchophylline content (1.21 ± 0.01 mg/g) in the herbal extract. In previous research, Cai et al. (2013) gave a single high dosage of rhynchophylline 15 mg/kg, and Wang et al. (2010) gave a single dosage of 37.5 mg/kg, so that the concentrations of rhynchophylline and its metabolites could be measured in plasma. The mean blood and brain concentration–time profiles after rhynchophylline administration (1 or 10 mg/kg, i.v.) dosedependently are shown in Fig. 4. The plasma concentrations appeared to decline after either a low dose or after a high dose with rapid distribution and elimination. The data demonstrated that rhynchonphylline easily penetrates the blood–brain barrier, and can be detected at the first microdialysis collection time point within 15 min of administration. Comparing the rhynchophylline
Table 2. Precision and accuracy for analysis of rhynchophylline in different dialysate Spiked concentration (ng/mL)
Intra-day (n = 6) Observed concentration (ng/mL)
ACD (blood)
Ringer’s solution (brain)
1 10 100 1 10 100
0.85 ± 0.03 10.03 ± 0.12 99.48 ± 0.96 0.89 ± 0.04 9.92 ± 0.14 99.28 ± 1.22
Precision (RSD, %) 2.7 1.24 0.96 4.3 1.37 1.22
Inter-day (n = 6) Accuracy (RE, %)
Observed concentration (ng/mL)
84.27 100.29 99.48 89.41 99.18 99.28
0.81 ± 0.08 10.38 ± 0.91 99.17 ± 1.02 0.92 ± 0.05 10.05 ± 0.23 99.18 ± 2.24
Precision (RSD, %) 7.74 9.07 1.02 4.82 2.34 2.24
Accuracy (RE, %) 80.75 103.79 99.17 92.17 100.52 99.18
ACD, Anticoagulant citrate dextrose.
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Mean extraction recovery (n = 6) Mean (%) RSD (%) 6.3 2.6 0.8 3.3 4.4 0.7
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114.13 ± 15.62 15.59 ± 7.87 1.64 ± 0.26 489.92 ± 70.80 92.08 ± 14.22 15.90 108.87 ± 44.85 732.90 ± 220.68 81.66 ± 9.27 160.66 ± 33.15 28.09 ± 1.14
10 mg/kg
37.13 ± 8.34 76.42 ± 21.38 10.31 ± 2.91 125.36 ± 19.04 16.88 ± 3.56
1 mg/kg
Blood Parameters
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levels in blood and in brain after rhynchophylline administration (10 mg/kg, i.v.), the AUC (area under the curve) of rhynchophylline in blood was 81.66 ± 9.27 min μg/mL, which was higher than the AUC of rhynchophylline in the brain (14.01 ± 3.13 min μg/mL) (Table 4). It is suggested that rhynchophylline may contribute to the permeability of the blood–brain barrier. The AUCbrain/blood ratio of 17.15% was calculated from the results of the rhynchophylline administration (10 mg/kg, i.v.) (Table 4).
Table 4. Pharmacokinetic parameters of blood and brain after rhynchophylline administration (1 and 10 mg/kg, i.v.)
Figure 4. Blood (A) and brain (B) drug concentration–time curves of rhynchophylline after i.v. administration of rhynchophylline in rats.
t1/2 (min) C0 (ng/mL) AUC (min μg/mL) Cl (mL/min/kg) Vss (L/kg) Brain–blood ratio (%)
121.07 121.30 114.06 113.53 126.78 115.04
1 mg/kg
Ringer’s solution (Brain)
1 10 100 1 10 100
Brain
ACD (blood)
Data are expressed as the mean ± standard deviation (n = 6). AUC, area under the plasma concentration versus the time curve; t1/2, half-life; Tmax, time to reach the drug concentration in plasma; C0, maximal drug concentration in plasma; Vss, volume of distribution at steady state.
Spiked concentration (ng/mL)
10 mg/kg
Table 3. Precision, accuracy, matrix effect, and recovery for analysis of rhynchophylline in rat plasma
146.01 ± 15.68 337.16 ± 183.69 14.01 ± 3.13 609.25 ± 125.30 82.64 ± 12.06 17.15
Determination of protein-unbound rhynchiphylline brain distribution
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Conclusion We have developed and validated a combined UPLC/MS/MS microdialysis sampling method to concurrently monitor the concentrations of rhynchophylline in rat plasma and brain. This is the first time that rhynchophylline has been monitored in the brain, and it provided the first evidence that rhynchophylline can cross the blood–brain barrier.
Acknowledgments This study was supported by National Science Council grants (NSC102-2113-M-010-001-MY3), post-doctoral training grant (NSC 100-2811-M-010-005 and NSC 101-2811-M-010-003) and TCH 10202; 10102-62-084 from Taipei City Hospital, Taipei, Taiwan.
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