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Herb–drug interaction of Evodia rutaecarpa extract on the pharmacokinetics of theophylline in rats
Journal of Ethnopharmacology 102 (2005) 440–445
Herb–drug interaction of Evodia rutaecarpa extract on the pharmacokinetics of theophylline in rats Woan-Ching Jan a,b , Lie-Chwen Lin c , Chieh-Fu-Chen c , Tung-Hu Tsai c,d,∗ a
c
Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan b Mackay Medicine, Nursing and Management College, Taipei, Taiwan National Research Institute of Chinese Medicine, 155-1 Li-Nong Street Section 2, Taipei 112, Taiwan d Institute of Traditional Medicine, National Yang-Ming University, Taipei 112, Taiwan Received 22 June 2004; received in revised form 21 March 2005; accepted 6 July 2005 Available online 15 August 2005
Abstract The extract of Evodia rutaecarpa fruit and its preparation were used for the treatment of gastrointestinal disorders and headache. To assess the possible herb–drug interaction, the ethanol extract of Evodia rutaecarpa fruit (1 and 2 g/kg/day, p.o.) and the herbal preparation Wu-ChuYu-Tang (1 and 5 g/kg/day) were given to rats daily for three consecutive days and on the fourth day theophylline was administered (2 mg/kg, i.v.). Theophylline concentration in blood was measured by a microdialysis coupled to a liquid chromatographic system. Pharmacokinetic data were calculated by noncompartmental model. The results indicate that the theophylline level was significantly decreased by the pretreatment with the extract of Evodia rutaecarpa and herbal preparation Wu-Chu-Yu-Tang with dose-related manner. It is suggested that the herb–drug interaction may occur through the induction of the metabolism of theophylline. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Evodia rutaecarpa; Microdialysis; Pharmacokinetics; Theophylline; Traditional Chinese medicine
1. Introduction Theophylline is a potent bronchodilator that has been widely used in the treatment of acute asthma. Almost entirely 90% of theophylline is metabolized in the human liver by the cytochrome P450 (CYP) with its members CYP1A2 and CYP2E1 (Ogilvie, 1978). It was also reported (Teunissen et al., 1985) that theophylline was metabolized to 1,3dimethyluric acid (1,3-DMU) via CYP1A2 and CYP2E1 and to 1-methylxanthine via CYP1A2 which was further metabolized to 1-methyluric acid (1-MU) via xanthine oxidase in rats. Hence, it could be expected that the pharmacokinetic parameters of theophylline could be changed in the pretreatment of herbal medicine which affects the activity of CYP1A2 and CYP2E1. Theophylline has been charac∗ Corresponding author. Tel.: +886 2 2820 1999x8091; fax: +886 2 2826 4276. E-mail address: [email protected] (T.-H. Tsai).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.07.002
terized by a narrow therapeutic index with the therapeutic concentration ranges of 5–20 g/ml. Therefore, drug–drug or herb–drug interaction may sensitively affect the therapeutics of theophylline. The dried unripened fruits of Evodia rutaecarpa (Chinese name: Wu-Chu-Yu) are a traditional Chinese herbal medicine that has been used as a remedy for gastrointestinal disorders, headache, amenorrhea, and postpartum hemorrhage for a long time (Liao et al., 1981; Sheu, 1999). Recent reports indicate that Evodia rutaecarpa is a potent inducer of CYP1A (Ueng et al., 2001, 2002b). Rutaecarpine, an active ingredient, was originally isolated from Evodia rutaecarpa (Asahina and Kashiwaki, 1915) and total synthesized (Chavan and Sivappa, 2004) which possesses antihypertensive (Chiou et al., 1994), anti-platelet (Sheu et al., 1998) and antithrombotic (Sheu et al., 2000) activities. In addition, rutaecarpine has been identified as a potent inhibitor of CYP1A2 in both mouse and human liver microsomes (Ueng et al., 2002a). WuChu-Yu-Tang (Goshuyu-to in Japanese Kampo medicine) is a
W.-C. Jan et al. / Journal of Ethnopharmacology 102 (2005) 440–445
traditional Chinese preparation for the treatment of migraine and vomiting which contains four herbs with Evodia rutaecarpa, Ginseng Radix, Zingiber Rhizoma, Zizyphi Fructus (Kano et al., 1991). Microdialysis is a biological fluid sampling technique with a principle of passive diffusion processes through semi-permeable membrane. The mass transport through the membrane is governed by diffusion and forced by concentration gradient. Microdialysis sampling from blood vessels is applied to investigate the protein-unbound sample with no blood loss. Due to the constriction of blood volume, pharmacokinetic studies are often limited by the temporal resolution for a small experimental animal. With minimum disturbance of physiological function of the body, microdialysis compensate the disadvantage of blood withdrawing and increase the temporal resolution. Few published data are available concerning the Evodia rutaecarpa related herb–drug interaction on the perspective of pharmacokinetics (Ueng and Wang, 2003). The purpose of this paper is to explore the effect of Evodia rutaecarpa and the herbal preparation Wu-Chu-Yu-Tang pretreatment on the pharmacokinetics of theophylline to rats with a microdialysis application.
441
tain starch:concentrate herbal decoction (3.8:5.2, w/w). The concentrate herbal decoction was prepared from 7.5 g Evodia Fructus, 4.5 g Ginseng Radix, 9 g Zingiber Rhizoma and 6 g Zizyphi Fructus and the extraction yield of the decoction was 19.26%. Chromatographic solvents were obtained from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA). Triple deionized water from Millipore (Bedford, MA, USA) was utilized for all preparations. 2.2. Liquid chromatography HPLC was performed with a chromatographic pump (BAS PM-80, West Lafayette, IN, USA), a Rheodyne Model 7125 injector equipped with a 10 l sampling loop and an ultraviolet detector (Varian, Walnut Creek, CA, USA). Separation was achieved by a Phenomenex LUNA microbore PhenylHexyl column (150 mm × 1 mm i.d.; 5 m, Torrance, CA, USA) (Tsai and Liu, 2004). The mobile phase consisted of acetonitrile–methanol–10 mM monosodium phosphate (pH 3.0) (10:20:70, v/v), with a flow rate of 0.05 ml/min, and the wavelength was 270 nm. Output data from the detector were integrated using an EZChrom chromatographic data system (Scientific Software, San Roman, CA, USA). 2.3. Animals
2. Materials and methods 2.1. Chemicals and reagents Theophylline was purchased from Sigma Chemicals (St. Louis, MO, USA). Rutaecarpine (purity 99.5%, by HPLC, Fig. 1) was isolated from Evodia rutaecarpa (Juss) Benth (Rutaceae) (Lin et al., 1991). Dried fruit of Evodia rutaecarpa (600 g) was collected at Lishan, Taichung, Taiwan. A voucher specimen (No. 700191) has been deposited in the National Research Institute of Chinese Medicine, Taipei, Taiwan. Fruits were powdered using a crushing machine (Yu Chi Machinery Co., Taiwan). The powder was immersed in deionized water–ethanol (1:1, v/v) overnight and then shaken at 50 ◦ C, 200 rpm for 1 h. The mixture was filtered through filter paper (Advantac #1, Tokyo, Japan). The filtrate was concentrated using a rotary vacuum evaporator. Extracts were lyophilized (169 g; yield 28.17%) and then stored at room temperature. The herbal extract preparation, Wu-Chu-YuTang was purchased from Sheng Chang Pharmaceutical Co., Taipei, Taiwan. Nine gram of Wu-Chu-Yu-Tang extract con-
Fig. 1. Chemical structure of rutaecarpine.
The institutional animal experimentation committee of the National Research Institute of Chinese Medicine reviewed and approved all experimental protocols involving animals. Male, specific pathogen-free Sprague–Dawley rats were obtained from the Laboratory Animal Center of the National Yang-Ming University, Taipei. The animals had free access to food (Laboratory rodent diet #5P14, PMI Feeds Inc., Richmond, IN, USA) and water until 18 h prior to being supplied for experiments, at which time only food was removed. The rats were initially anaesthetized with urethane 1 g/ml and ␣-chloralose 0.1 g/ml (1 ml/kg, i.p.), and remained anaesthetized throughout the experimental period. The femoral vein was exposed for further drug administration. The rats’ body temperature was maintained at 37 ◦ C with a heating pad during the experiment. 2.4. Microdialysis experiment Blood microdialysis system was comprised of a CMA/100 microinjection pump (CMA, Stockholm, Sweden) and microdialysis probes. The dialysis probe for blood (10 mm in length) was made of silica capillary in a concentric design (Tsai et al., 1999). Their tips were covered by dialysis membrane (Spectrum Lab., 200 m inner diameter with a cut-off at nominal molecular weight of 13,000, Laguna Hills, CA, USA) and all unions were cemented with epoxy. At least 24 h was allowed for the epoxy to dry. The blood microdialysis probe was located within the jugular vein/right atrium and then perfused with anticoagulant dextrose (ACD) solution (citric acid 3.5 mM; sodium citrate 7.5 mM; dextrose
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W.-C. Jan et al. / Journal of Ethnopharmacology 102 (2005) 440–445
13.6 mM) at a flow rate of 2 l/min employing the microinjection pump (Tsai, 2001). A retrograde calibration technique was utilized during in vivo recovery. The microdialysis probe was inserted into the rats’ jugular vein under anesthesia. Following a stabilization period of 2 h post-probe implantation, the perfusate (Cperf ) and dialysate (Cdiai ) concentrations of theophylline were determined by HPLC. ACD solution containing theophylline was perfused through the probe at a constant flow rate (2 l/min) employing the infusion pump (CMA/100). The in vivo relative recovery (Rdial ) of theophylline across the microdialysis probe was calculated by the following equation: Rdial = (Cperf − Cdial )/Cperf . Theophylline microdialysate concentrations (Cm ) were converted to unbound concentration (Cu ) as follows: Cu = Cm /Rdial (Tsai, 2003). The microdialysate recovery and concentration calculations were performed according to our previous reports (Tsai et al., 2001). 2.5. Liquid chromatography–tandem mass spectrometry (LC–MS–MS) for the identification of rutaecarpine from herbal extract and herbal preparation LC–MS–MS analysis was performed using a Waters 2690 with a 996 photodiode array (PDA) detector together with an automatic liquid chromatographic sampler and an autoinjection system hyphenated to a Micromass Quattro Ultima tandem quadrupole mass spectrometry (Micromass, Manchester, UK) equipped with an electrospray ionization (ESI) source. The separation was achieved using a reversed-phase C18 column (150 mm × 4.6 mm i.d.) (Agilent, USA). The solvent delivery system was kept constant at 1 ml/min and it obeyed a linear gradient elution according to the following profile: 0–10 min, 80–20% water, 20–80% methanol. The volume of injection was 10 l. For operation in MS–MS mode, a mass spectrometer with an orthogonal Z-spray electrospray interface (ESI) was used. The infusion experiment was performed using a Mode 22 multiple syringe pump (Harvard, Holliston, MA, USA). For rutaecarpine assay, the ESI parameters were set as follows: capillary voltage, 3 kV for positive mode; source temperature, 80 ◦ C; desolvation temperature, 300 ◦ C; cone gas flow, 95 l/h; and desolvation gas flow, 440 l/h. The cone voltage of m/z 288 was 70 V and the collision voltages were 30 eV. All LC–MS–MS data were processed by the MassLynx Version 4.0 NT Quattro data acquisition software. 2.6. Drug administration The drug was subsequently administered according to the following study design. Six animals were used in each group. For the extract of Evodia rutaecarpa extract and Wu-Chu-Yu-Tang pretreated group, rats were treated with Evodia rutaecarpa extract 1 or 2 g/kg/day and Wu-Chu-YuTang extract 1 or 5 g/kg/day, respectively, by gastrogavage
for three consecutive days. Both extracts of Evodia rutaecarpa and Wu-Chu-Yu-Tang were dissolved in corn oil for administration (Ueng et al., 2001). On the fourth day, the rats were administered theophylline 2 mg/kg via femoral vein. The rats for control group, same amount of corn oil was given orally for three consecutive days and on the fourth day theophylline 2 mg/kg was injected via the femoral vein into the rats. Outflow dialysates from blood was collected in a fraction collector (CMA/140) every 10 min. These dialysate samples were measured by HPLC during the same experimental day. 2.7. Pharmacokinetics Pharmacokinetic calculations were performed on each individual animal’s data utilizing the pharmacokinetic calculation software WinNonlin Standard Edition Version 1.1 (Scientific Consulting Inc., Apex, NC, USA) by a noncompartmental method (Gabrielsson and Weiner, 1994). Formation rate constants were calculated from the extrapolated formation slope determined by the residual method. The AUCs from time zero to time infinity (AUC0–inf ) were calculated by the trapezoidal rule and extrapolated to time infinity by the addition of AUCt–inf . The clearance (Cl) was calculated as follows: Cl = dose/AUC. 2.8. Statistical analysis The statistical analysis was performed with SPSS Version 10.0 (SPSS Inc. Chicago, IL, USA). One-way ANOVA was used for the comparison between the control group (theophylline alone), Evodia rutaecarpa extract and Wu-Chu-Yu-Tang extract pretreated groups. All statistical tests were performed at the two-sided 5% level of significance.
3. Results The validated chromatographic method of theophylline has been reported in our previous studies. In vivo recovery of theophylline in blood (1 g/ml) was 0.77 ± 0.01 (mean ± S.E.M., n = 6) (Tsai and Liu, 2004). The contents of rutaecarpine in the Evodia rutaecarpa extract and herbal preparation Wu-Chu-Yu-Tang were 5.59 ± 0.39 and 0.30 ± 0.002 mg/g, respectively, which were identified by liquid chromatography coupled to tandem mass spectrometry (Fig. 2). The results show that the concentration of theophylline in blood was significantly decreased in the pretreatment of Evodia rutaecarpa extract and the herbal preparation Wu-Chu-Yu-Tang in the daily dosages of 1 and 2 g/kg/day, and 1 and 5 g/kg/day for three consecutive days, respectively (Fig. 3). The declining slope of the theophylline concentration curve in the pretreated group of Evodia rutaecarpa extract was sharper then the group of herbal preparation Wu-Chu-Yu-Tang which may relate to
W.-C. Jan et al. / Journal of Ethnopharmacology 102 (2005) 440–445
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Fig. 2. (A) The full scan of rutaecarpine for positive mode of electrospray ionization (ESI) source with m/z 288. (B) Daughter ion scan of rutaecarpine with m/z 288 and 271.
the contents of rutaecarpine in the herbal extract and herbal preparation. The pharmacokinetic parameters of theophylline indicate that the rats pretreatment with Evodia rutaecarpa extract in the daily dosages of 1 and 2 g/kg/day for three consecutive days resulted in 72.9 and 72.8% decreases the AUC of theophylline, respectively. The elimination half-life of theophylline was dose-relatively decreased but the total clearance was increased in the pre-treatment of Evodia rutaecarpa extract (Table 1).
The rat pretreatment with Wu-Chu-Yu-Tang in the daily dosages of 1 and 5 g/kg/day for three consecutive days resulted in 26.3 and 59.7% decreases the AUC of theophylline, respectively. The elimination half-life of theophylline was decreased at higher dose (5 g/kg/day) but there was no significant difference at lower dose (1 g/kg/day) pretreatment with the herbal preparation. The total clearance of theophylline was dose-relatively increased in the pretreatment of the herbal preparation. The contents of rutaecarpine in the herbal preparation Wu-Chu-Yu-Tang was less than that
Table 1 Pharmacokinetic parameters of the control group, with theophylline administration (2 mg/kg, i.v.) alone; the Evodia rutaecarpa extract (1 or 2 g/kg/day, p.o. for 3 days) and Wu-Chu-Yu-Tang extract (1 or 5 g/kg/day, p.o. for 3 days) pretreated groups, on the fourth day theophylline 2 mg/kg was injected via femoral vein injection Parameters
AUC (min g/ml)
Theophylline (2 mg/kg) +Evodia rutaecarpa (1 g/kg) +Evodia rutaecarpa (2 g/kg) +Wu-Chu-Yu-Tang (1 g/kg) +Wu-Chu-Yu-Tang (5 g/kg)
204.1 55.3 55.4 150.4 82.2
Data are expressed as mean ± S.E.M. (n = 6). * p < 0.05 vs. theophylline alone group.
± ± ± ± ±
18.2 5.9* 4.7* 12.7* 6.2*
Cl (ml/kg/min)
t1/2 (min)
10.2 ± 1.0 36.6 ± 2.9* 37.6 ± 3.5* 13.8 ± 1.2* 25.1 ± 2.1*
108 23 16 99 63
± ± ± ± ±
14 7* 3* 8 4*
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showed that rutaecarpine-treatment increased the protein levels of CYP1A1 and CYP1A2 in the liver, whereas hepatic level of CYP3A-immunoreacted protein was not affected by rutaecarpine (Ueng et al., 2001). In summary, these results indicate that Evodia rutaecarpa extract and the herbal preparation Wu-Chu-Yu-Tang act as a selective and potent inducer of the CYP1A enzymes responsible for oxidative biotransformation of chemicals such as theophylline. This evidence provides a fundamental explanation for the herb–drug interactions experienced in clinical practice. Acknowledgements This study was supported in part by research grants: VGH92-377-4D, VGH93-377-4D, VGH94-366-3 from the Veterans General Hospital, Taipei; and NSC94-2113-M-077001, NSC94-2320-B-077-002 from the National Science Council, Taiwan. References Fig. 3. Mean unbound theophylline concentration–time curves in rat blood after theophylline (2 mg/kg, i.v.) administration for control group, and the pretreated groups of which Evodia rutaecarpa extract (1 and 2 g/kg/day) and the herbal preparation Wu-Chu-Yu-Tang (1 and 5 g/kg/day) were given orally for three consecutive days and on the fourth day theophylline (2 mg/kg, i.v.) was injected via femoral vein (n = 6). Data are presented as mean ± S.E.M.
in the herbal extract of Evodia rutaecarpa. This result may lead to diminish the CYP1A activity for the theophylline metabolism.
4. Discussion Bachmann et al. (1993) provided the evidence that theophylline is metabolized principally by CYP1A in rats. In the pretreatment with the CYP1A inducer, beta-naphthoflavone (BNF), increased theophylline clearance 4.5-fold (p < 0.001), and the CYP1A inhibitor, alpha-naphthoflavone, significantly attenuated the BNF effect. However, the substrates of CYP2E, CYP2D, CYP3A and CYP4A have also been investigated and the result is that it has no significant effect on theophylline clearance. While the powerful CYP3A inducer clotrimazole did not increase theophylline clearance, troleandomycin, an inhibitor of CYP3A, decreased theophylline clearance by about 25% (p < 0.002). Our data are consistent with Ueng et al. (2002a) in an in vivo study of rutaecarpine in which the mice were administered with rutaecarpine (50 mg/kg/day for 3 days) by gastrogavage resulted in marked increases of hepatic microsomal benzo(a)pyrene hydroxylase (CYP1A), 7-ethoxycoumarin O-deethylase (CYP1A), 7-ethoxyresorufin O-deethylase (CYP1A), and 7-methoxyresorufin O-demethylase (CYP1A) activities. Immunoblot analysis of microsomal proteins
Asahina, Y., Kashiwaki, K., 1915. Chemical constituents of the fruits of Evodia rutaecarpa. Journal of Pharmaceutical Society Japan 35, 1293. Bachmann, K., Sanyal, G., Potter, J., Schiavone, R., Loch, J., 1993. In vivo evidence that theophylline is metabolized principally by CYP1A in rats. Pharmacology 47, 1–7. Chavan, S.P., Sivappa, R., 2004. A facile total synthesis of rutaecarpine. Tetrahedron Letter 45, 997–999. Chiou, W.F., Chou, C.J., Liao, J.F., Sham, A.Y.C., Chen, C.F., 1994. The mechanism of the vasodilator effect of rutaecarpine, and alkaloid isolated form Evodia rutaecarpa. European Journal of Pharmacology 257, 59–66. Gabrielsson, J., Weiner, D., 1994. Non-compartmental analysis. In: Pharmacokinetic and Pharmacodynamic Data Analysis Concepts and Applications. Swedish Pharm. Press, Stockholm, pp. 621–626. Kano, Y., Zong, Q., Komatsu, K., 1991. Pharmacological properties of Galenical preparation. XIV. Body temperature retaining effect of the Chinese traditional medicine “Goshuyu-to” and component crude drugs. Chemical Pharmacological Bulletin (Tokyo) 39, 690–692. Liao, J.F., Chen, C.F., Chow, S.Y., 1981. Pharmacological studies of Chinese herbs. 9. Pharmacological effects of Evodiae fructus. Journal of Formosan Medical Association 79, 30–38. Lin, L.C., Chou, C.J., Chen, K.T., Chen, C.F., 1991. Flavonoids from Evodiae fructus. Journal of Chinese Medicine 2, 94–100. Ogilvie, R.I., 1978. Clinical pharmacokinetics of theophylline. Clinical Pharmacology 3, 267–293. Sheu, J.R., 1999. Pharmacological effects of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa. Cardiovascular Drug Review 17, 237–245. Sheu, J.R., Hung, W.C., Wu, C.H., Lee, Y.M., Yen, M.H., 2000. Antithrombotic effect of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, on platelet plug formation in in vivo experiments. British Journal of Haematology 110, 110–115. Sheu, J.R., Kan, Y.C., Hung, W.C., Su, C.H., Lin, C.H., Lee, Y.M., Yen, M.H., 1998. The antiplatelet activity of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, is mediated through inhibition of phospholipase C. Thrombosis Research 92, 53–64. Teunissen, M.W., Brorens, I.O., Geerlings, J.M., Breimer, D.D., 1985. Dose-dependent elimination of theophylline in rats. Xenobiotica 15, 165–171.
W.-C. Jan et al. / Journal of Ethnopharmacology 102 (2005) 440–445 Tsai, T.H., Chen, Y.F., Chen, I.F., Chen, C.F., 1999. Measurement of unbound caffeic acid in rat blood by on-line microdialysis coupled with liquid chromatography and its application to pharmacokinetic studies. Journal of Chromatography B 729, 119–125. Tsai, T.H., 2001. Pharmacokinetics of pefloxacin and its interaction with cyclosporin A, a P-glycoprotein modulator, in rat blood, brain and bile, using simultaneous microdialysis. British Journal Pharmacology 132, 1310–1316. Tsai, T.H., Lee, C.H., Yeh, P.H., 2001. Effect of cyclosporine, a Pglycoprotein modulator, on the disposition and biliary excretion of camptothecin using microdialysis. British Journal of Pharmacology 134, 1245–1252. Tsai, T.H., 2003. Assaying protein unbound drugs using microdialysis techniques. Journal of Chromatography B 797, 161–173. Tsai, T.H., Liu, M.C., 2004. Determination and brain distribution of theophylline in rat blood and brain using microdialysis and micro-
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bore liquid chromatography. Journal of Chromatography A 1032, 97– 101. Ueng, Y.F., Wang, J.J., Lin, L.C., Park, S.S., Chen, C.F., 2001. Induction of cytochrome P450-dependent monooxygenase in mouse liver and kidney by rutaecarpine, an alkaloid of the herbal drug Evodia rutaecarpa. Life Sciences 70, 207–217. Ueng, Y.F., Jan, W.C., Lin, L.C., Chen, T.L., Guengerich, F.P., Chen, C.F., 2002a. The alkaloid rutaecarpine is a selective inhibitor of cytochrome P450 1A in mouse and human liver microsomes. Drug Metabolism Disposition 30, 349–353. Ueng, Y.F., Ko, H.C., Chen, C.F., Wang, J.J., Chen, K.T., 2002b. Modulation of drug-metabolizing enzymes by extracts of a herbal medicine Evodia rutaecarpa in C57BL/6J mice. Life Sciences 71, 1267–1277. Ueng, Y.F., Wang, S.Y., 2003. The herbal preparation Wu-Chu-Yu-Tang enhance the excretion of the CYP1A2 substrate caffeine in mice. Journal of Chinese Medicine 14, 175–181.
Herb–drug interaction of Evodia rutaecarpa extract on the pharmacokinetics of theophylline in rats Woan-Ching Jan a,b , Lie-Chwen Lin c , Chieh-Fu-Chen c , Tung-Hu Tsai c,d,∗ a
c
Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan b Mackay Medicine, Nursing and Management College, Taipei, Taiwan National Research Institute of Chinese Medicine, 155-1 Li-Nong Street Section 2, Taipei 112, Taiwan d Institute of Traditional Medicine, National Yang-Ming University, Taipei 112, Taiwan Received 22 June 2004; received in revised form 21 March 2005; accepted 6 July 2005 Available online 15 August 2005
Abstract The extract of Evodia rutaecarpa fruit and its preparation were used for the treatment of gastrointestinal disorders and headache. To assess the possible herb–drug interaction, the ethanol extract of Evodia rutaecarpa fruit (1 and 2 g/kg/day, p.o.) and the herbal preparation Wu-ChuYu-Tang (1 and 5 g/kg/day) were given to rats daily for three consecutive days and on the fourth day theophylline was administered (2 mg/kg, i.v.). Theophylline concentration in blood was measured by a microdialysis coupled to a liquid chromatographic system. Pharmacokinetic data were calculated by noncompartmental model. The results indicate that the theophylline level was significantly decreased by the pretreatment with the extract of Evodia rutaecarpa and herbal preparation Wu-Chu-Yu-Tang with dose-related manner. It is suggested that the herb–drug interaction may occur through the induction of the metabolism of theophylline. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Evodia rutaecarpa; Microdialysis; Pharmacokinetics; Theophylline; Traditional Chinese medicine
1. Introduction Theophylline is a potent bronchodilator that has been widely used in the treatment of acute asthma. Almost entirely 90% of theophylline is metabolized in the human liver by the cytochrome P450 (CYP) with its members CYP1A2 and CYP2E1 (Ogilvie, 1978). It was also reported (Teunissen et al., 1985) that theophylline was metabolized to 1,3dimethyluric acid (1,3-DMU) via CYP1A2 and CYP2E1 and to 1-methylxanthine via CYP1A2 which was further metabolized to 1-methyluric acid (1-MU) via xanthine oxidase in rats. Hence, it could be expected that the pharmacokinetic parameters of theophylline could be changed in the pretreatment of herbal medicine which affects the activity of CYP1A2 and CYP2E1. Theophylline has been charac∗ Corresponding author. Tel.: +886 2 2820 1999x8091; fax: +886 2 2826 4276. E-mail address: [email protected] (T.-H. Tsai).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.07.002
terized by a narrow therapeutic index with the therapeutic concentration ranges of 5–20 g/ml. Therefore, drug–drug or herb–drug interaction may sensitively affect the therapeutics of theophylline. The dried unripened fruits of Evodia rutaecarpa (Chinese name: Wu-Chu-Yu) are a traditional Chinese herbal medicine that has been used as a remedy for gastrointestinal disorders, headache, amenorrhea, and postpartum hemorrhage for a long time (Liao et al., 1981; Sheu, 1999). Recent reports indicate that Evodia rutaecarpa is a potent inducer of CYP1A (Ueng et al., 2001, 2002b). Rutaecarpine, an active ingredient, was originally isolated from Evodia rutaecarpa (Asahina and Kashiwaki, 1915) and total synthesized (Chavan and Sivappa, 2004) which possesses antihypertensive (Chiou et al., 1994), anti-platelet (Sheu et al., 1998) and antithrombotic (Sheu et al., 2000) activities. In addition, rutaecarpine has been identified as a potent inhibitor of CYP1A2 in both mouse and human liver microsomes (Ueng et al., 2002a). WuChu-Yu-Tang (Goshuyu-to in Japanese Kampo medicine) is a
W.-C. Jan et al. / Journal of Ethnopharmacology 102 (2005) 440–445
traditional Chinese preparation for the treatment of migraine and vomiting which contains four herbs with Evodia rutaecarpa, Ginseng Radix, Zingiber Rhizoma, Zizyphi Fructus (Kano et al., 1991). Microdialysis is a biological fluid sampling technique with a principle of passive diffusion processes through semi-permeable membrane. The mass transport through the membrane is governed by diffusion and forced by concentration gradient. Microdialysis sampling from blood vessels is applied to investigate the protein-unbound sample with no blood loss. Due to the constriction of blood volume, pharmacokinetic studies are often limited by the temporal resolution for a small experimental animal. With minimum disturbance of physiological function of the body, microdialysis compensate the disadvantage of blood withdrawing and increase the temporal resolution. Few published data are available concerning the Evodia rutaecarpa related herb–drug interaction on the perspective of pharmacokinetics (Ueng and Wang, 2003). The purpose of this paper is to explore the effect of Evodia rutaecarpa and the herbal preparation Wu-Chu-Yu-Tang pretreatment on the pharmacokinetics of theophylline to rats with a microdialysis application.
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tain starch:concentrate herbal decoction (3.8:5.2, w/w). The concentrate herbal decoction was prepared from 7.5 g Evodia Fructus, 4.5 g Ginseng Radix, 9 g Zingiber Rhizoma and 6 g Zizyphi Fructus and the extraction yield of the decoction was 19.26%. Chromatographic solvents were obtained from Mallinckrodt Baker Inc. (Phillipsburg, NJ, USA). Triple deionized water from Millipore (Bedford, MA, USA) was utilized for all preparations. 2.2. Liquid chromatography HPLC was performed with a chromatographic pump (BAS PM-80, West Lafayette, IN, USA), a Rheodyne Model 7125 injector equipped with a 10 l sampling loop and an ultraviolet detector (Varian, Walnut Creek, CA, USA). Separation was achieved by a Phenomenex LUNA microbore PhenylHexyl column (150 mm × 1 mm i.d.; 5 m, Torrance, CA, USA) (Tsai and Liu, 2004). The mobile phase consisted of acetonitrile–methanol–10 mM monosodium phosphate (pH 3.0) (10:20:70, v/v), with a flow rate of 0.05 ml/min, and the wavelength was 270 nm. Output data from the detector were integrated using an EZChrom chromatographic data system (Scientific Software, San Roman, CA, USA). 2.3. Animals
2. Materials and methods 2.1. Chemicals and reagents Theophylline was purchased from Sigma Chemicals (St. Louis, MO, USA). Rutaecarpine (purity 99.5%, by HPLC, Fig. 1) was isolated from Evodia rutaecarpa (Juss) Benth (Rutaceae) (Lin et al., 1991). Dried fruit of Evodia rutaecarpa (600 g) was collected at Lishan, Taichung, Taiwan. A voucher specimen (No. 700191) has been deposited in the National Research Institute of Chinese Medicine, Taipei, Taiwan. Fruits were powdered using a crushing machine (Yu Chi Machinery Co., Taiwan). The powder was immersed in deionized water–ethanol (1:1, v/v) overnight and then shaken at 50 ◦ C, 200 rpm for 1 h. The mixture was filtered through filter paper (Advantac #1, Tokyo, Japan). The filtrate was concentrated using a rotary vacuum evaporator. Extracts were lyophilized (169 g; yield 28.17%) and then stored at room temperature. The herbal extract preparation, Wu-Chu-YuTang was purchased from Sheng Chang Pharmaceutical Co., Taipei, Taiwan. Nine gram of Wu-Chu-Yu-Tang extract con-
Fig. 1. Chemical structure of rutaecarpine.
The institutional animal experimentation committee of the National Research Institute of Chinese Medicine reviewed and approved all experimental protocols involving animals. Male, specific pathogen-free Sprague–Dawley rats were obtained from the Laboratory Animal Center of the National Yang-Ming University, Taipei. The animals had free access to food (Laboratory rodent diet #5P14, PMI Feeds Inc., Richmond, IN, USA) and water until 18 h prior to being supplied for experiments, at which time only food was removed. The rats were initially anaesthetized with urethane 1 g/ml and ␣-chloralose 0.1 g/ml (1 ml/kg, i.p.), and remained anaesthetized throughout the experimental period. The femoral vein was exposed for further drug administration. The rats’ body temperature was maintained at 37 ◦ C with a heating pad during the experiment. 2.4. Microdialysis experiment Blood microdialysis system was comprised of a CMA/100 microinjection pump (CMA, Stockholm, Sweden) and microdialysis probes. The dialysis probe for blood (10 mm in length) was made of silica capillary in a concentric design (Tsai et al., 1999). Their tips were covered by dialysis membrane (Spectrum Lab., 200 m inner diameter with a cut-off at nominal molecular weight of 13,000, Laguna Hills, CA, USA) and all unions were cemented with epoxy. At least 24 h was allowed for the epoxy to dry. The blood microdialysis probe was located within the jugular vein/right atrium and then perfused with anticoagulant dextrose (ACD) solution (citric acid 3.5 mM; sodium citrate 7.5 mM; dextrose
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13.6 mM) at a flow rate of 2 l/min employing the microinjection pump (Tsai, 2001). A retrograde calibration technique was utilized during in vivo recovery. The microdialysis probe was inserted into the rats’ jugular vein under anesthesia. Following a stabilization period of 2 h post-probe implantation, the perfusate (Cperf ) and dialysate (Cdiai ) concentrations of theophylline were determined by HPLC. ACD solution containing theophylline was perfused through the probe at a constant flow rate (2 l/min) employing the infusion pump (CMA/100). The in vivo relative recovery (Rdial ) of theophylline across the microdialysis probe was calculated by the following equation: Rdial = (Cperf − Cdial )/Cperf . Theophylline microdialysate concentrations (Cm ) were converted to unbound concentration (Cu ) as follows: Cu = Cm /Rdial (Tsai, 2003). The microdialysate recovery and concentration calculations were performed according to our previous reports (Tsai et al., 2001). 2.5. Liquid chromatography–tandem mass spectrometry (LC–MS–MS) for the identification of rutaecarpine from herbal extract and herbal preparation LC–MS–MS analysis was performed using a Waters 2690 with a 996 photodiode array (PDA) detector together with an automatic liquid chromatographic sampler and an autoinjection system hyphenated to a Micromass Quattro Ultima tandem quadrupole mass spectrometry (Micromass, Manchester, UK) equipped with an electrospray ionization (ESI) source. The separation was achieved using a reversed-phase C18 column (150 mm × 4.6 mm i.d.) (Agilent, USA). The solvent delivery system was kept constant at 1 ml/min and it obeyed a linear gradient elution according to the following profile: 0–10 min, 80–20% water, 20–80% methanol. The volume of injection was 10 l. For operation in MS–MS mode, a mass spectrometer with an orthogonal Z-spray electrospray interface (ESI) was used. The infusion experiment was performed using a Mode 22 multiple syringe pump (Harvard, Holliston, MA, USA). For rutaecarpine assay, the ESI parameters were set as follows: capillary voltage, 3 kV for positive mode; source temperature, 80 ◦ C; desolvation temperature, 300 ◦ C; cone gas flow, 95 l/h; and desolvation gas flow, 440 l/h. The cone voltage of m/z 288 was 70 V and the collision voltages were 30 eV. All LC–MS–MS data were processed by the MassLynx Version 4.0 NT Quattro data acquisition software. 2.6. Drug administration The drug was subsequently administered according to the following study design. Six animals were used in each group. For the extract of Evodia rutaecarpa extract and Wu-Chu-Yu-Tang pretreated group, rats were treated with Evodia rutaecarpa extract 1 or 2 g/kg/day and Wu-Chu-YuTang extract 1 or 5 g/kg/day, respectively, by gastrogavage
for three consecutive days. Both extracts of Evodia rutaecarpa and Wu-Chu-Yu-Tang were dissolved in corn oil for administration (Ueng et al., 2001). On the fourth day, the rats were administered theophylline 2 mg/kg via femoral vein. The rats for control group, same amount of corn oil was given orally for three consecutive days and on the fourth day theophylline 2 mg/kg was injected via the femoral vein into the rats. Outflow dialysates from blood was collected in a fraction collector (CMA/140) every 10 min. These dialysate samples were measured by HPLC during the same experimental day. 2.7. Pharmacokinetics Pharmacokinetic calculations were performed on each individual animal’s data utilizing the pharmacokinetic calculation software WinNonlin Standard Edition Version 1.1 (Scientific Consulting Inc., Apex, NC, USA) by a noncompartmental method (Gabrielsson and Weiner, 1994). Formation rate constants were calculated from the extrapolated formation slope determined by the residual method. The AUCs from time zero to time infinity (AUC0–inf ) were calculated by the trapezoidal rule and extrapolated to time infinity by the addition of AUCt–inf . The clearance (Cl) was calculated as follows: Cl = dose/AUC. 2.8. Statistical analysis The statistical analysis was performed with SPSS Version 10.0 (SPSS Inc. Chicago, IL, USA). One-way ANOVA was used for the comparison between the control group (theophylline alone), Evodia rutaecarpa extract and Wu-Chu-Yu-Tang extract pretreated groups. All statistical tests were performed at the two-sided 5% level of significance.
3. Results The validated chromatographic method of theophylline has been reported in our previous studies. In vivo recovery of theophylline in blood (1 g/ml) was 0.77 ± 0.01 (mean ± S.E.M., n = 6) (Tsai and Liu, 2004). The contents of rutaecarpine in the Evodia rutaecarpa extract and herbal preparation Wu-Chu-Yu-Tang were 5.59 ± 0.39 and 0.30 ± 0.002 mg/g, respectively, which were identified by liquid chromatography coupled to tandem mass spectrometry (Fig. 2). The results show that the concentration of theophylline in blood was significantly decreased in the pretreatment of Evodia rutaecarpa extract and the herbal preparation Wu-Chu-Yu-Tang in the daily dosages of 1 and 2 g/kg/day, and 1 and 5 g/kg/day for three consecutive days, respectively (Fig. 3). The declining slope of the theophylline concentration curve in the pretreated group of Evodia rutaecarpa extract was sharper then the group of herbal preparation Wu-Chu-Yu-Tang which may relate to
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Fig. 2. (A) The full scan of rutaecarpine for positive mode of electrospray ionization (ESI) source with m/z 288. (B) Daughter ion scan of rutaecarpine with m/z 288 and 271.
the contents of rutaecarpine in the herbal extract and herbal preparation. The pharmacokinetic parameters of theophylline indicate that the rats pretreatment with Evodia rutaecarpa extract in the daily dosages of 1 and 2 g/kg/day for three consecutive days resulted in 72.9 and 72.8% decreases the AUC of theophylline, respectively. The elimination half-life of theophylline was dose-relatively decreased but the total clearance was increased in the pre-treatment of Evodia rutaecarpa extract (Table 1).
The rat pretreatment with Wu-Chu-Yu-Tang in the daily dosages of 1 and 5 g/kg/day for three consecutive days resulted in 26.3 and 59.7% decreases the AUC of theophylline, respectively. The elimination half-life of theophylline was decreased at higher dose (5 g/kg/day) but there was no significant difference at lower dose (1 g/kg/day) pretreatment with the herbal preparation. The total clearance of theophylline was dose-relatively increased in the pretreatment of the herbal preparation. The contents of rutaecarpine in the herbal preparation Wu-Chu-Yu-Tang was less than that
Table 1 Pharmacokinetic parameters of the control group, with theophylline administration (2 mg/kg, i.v.) alone; the Evodia rutaecarpa extract (1 or 2 g/kg/day, p.o. for 3 days) and Wu-Chu-Yu-Tang extract (1 or 5 g/kg/day, p.o. for 3 days) pretreated groups, on the fourth day theophylline 2 mg/kg was injected via femoral vein injection Parameters
AUC (min g/ml)
Theophylline (2 mg/kg) +Evodia rutaecarpa (1 g/kg) +Evodia rutaecarpa (2 g/kg) +Wu-Chu-Yu-Tang (1 g/kg) +Wu-Chu-Yu-Tang (5 g/kg)
204.1 55.3 55.4 150.4 82.2
Data are expressed as mean ± S.E.M. (n = 6). * p < 0.05 vs. theophylline alone group.
± ± ± ± ±
18.2 5.9* 4.7* 12.7* 6.2*
Cl (ml/kg/min)
t1/2 (min)
10.2 ± 1.0 36.6 ± 2.9* 37.6 ± 3.5* 13.8 ± 1.2* 25.1 ± 2.1*
108 23 16 99 63
± ± ± ± ±
14 7* 3* 8 4*
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showed that rutaecarpine-treatment increased the protein levels of CYP1A1 and CYP1A2 in the liver, whereas hepatic level of CYP3A-immunoreacted protein was not affected by rutaecarpine (Ueng et al., 2001). In summary, these results indicate that Evodia rutaecarpa extract and the herbal preparation Wu-Chu-Yu-Tang act as a selective and potent inducer of the CYP1A enzymes responsible for oxidative biotransformation of chemicals such as theophylline. This evidence provides a fundamental explanation for the herb–drug interactions experienced in clinical practice. Acknowledgements This study was supported in part by research grants: VGH92-377-4D, VGH93-377-4D, VGH94-366-3 from the Veterans General Hospital, Taipei; and NSC94-2113-M-077001, NSC94-2320-B-077-002 from the National Science Council, Taiwan. References Fig. 3. Mean unbound theophylline concentration–time curves in rat blood after theophylline (2 mg/kg, i.v.) administration for control group, and the pretreated groups of which Evodia rutaecarpa extract (1 and 2 g/kg/day) and the herbal preparation Wu-Chu-Yu-Tang (1 and 5 g/kg/day) were given orally for three consecutive days and on the fourth day theophylline (2 mg/kg, i.v.) was injected via femoral vein (n = 6). Data are presented as mean ± S.E.M.
in the herbal extract of Evodia rutaecarpa. This result may lead to diminish the CYP1A activity for the theophylline metabolism.
4. Discussion Bachmann et al. (1993) provided the evidence that theophylline is metabolized principally by CYP1A in rats. In the pretreatment with the CYP1A inducer, beta-naphthoflavone (BNF), increased theophylline clearance 4.5-fold (p < 0.001), and the CYP1A inhibitor, alpha-naphthoflavone, significantly attenuated the BNF effect. However, the substrates of CYP2E, CYP2D, CYP3A and CYP4A have also been investigated and the result is that it has no significant effect on theophylline clearance. While the powerful CYP3A inducer clotrimazole did not increase theophylline clearance, troleandomycin, an inhibitor of CYP3A, decreased theophylline clearance by about 25% (p < 0.002). Our data are consistent with Ueng et al. (2002a) in an in vivo study of rutaecarpine in which the mice were administered with rutaecarpine (50 mg/kg/day for 3 days) by gastrogavage resulted in marked increases of hepatic microsomal benzo(a)pyrene hydroxylase (CYP1A), 7-ethoxycoumarin O-deethylase (CYP1A), 7-ethoxyresorufin O-deethylase (CYP1A), and 7-methoxyresorufin O-demethylase (CYP1A) activities. Immunoblot analysis of microsomal proteins
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