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Influences of Fructus evodiae pretreatment on the pharmacokinetics of Rhizoma coptidis alkaloids
Journal of Ethnopharmacology 137 (2011) 1395–1401
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Influences of Fructus evodiae pretreatment on the pharmacokinetics of Rhizoma coptidis alkaloids Bing-Liang Ma a , Meng-Kan Yao a , Xiang-Hui Han a , Yue-Ming Ma a,∗ , Jia-Sheng Wu a , Chang-Hong Wang b a b
Laboratory of Pharmacokinetics, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China
a r t i c l e
i n f o
Article history: Received 13 October 2010 Received in revised form 29 July 2011 Accepted 2 August 2011 Available online 9 August 2011 Keywords: Rhizoma coptidis Fructus evodiae Berberine Herb–herb interactions UGT1A1 Pharmacokinetics
a b s t r a c t Ethnopharmacological relevance: Rhizoma coptidis is a traditional Chinese medicine with pharmacological properties. It is usually prescribed with Fructus evodiae as traditional Chinese medicine (TCM) formulas. Here we report the influences of Fructus evodiae on the pharmacokinetics of the Rhizoma coptidis alkaloids and propose possible mechanisms. Materials and methods: Pharmacokinetic experiments were performed in rats. In vitro absorption experiments were performed in everted rat gut sacs, while in vitro metabolism experiments and determination of hepatic UDP-glucuronosyltransferase (UGT) 1A1 mRNA expression were performed in rat liver microsomes. Results: Pretreatment with Fructus evodiae extract for two weeks decreased the systemic exposure of the Rhizoma coptidis alkaloids. This effect was not due to inhibition of absorption or enhanced hepatic phase I metabolism of the Rhizoma coptidis alkaloids. However, Fructus evodiae pretreatment enhanced both the activity and expression of hepatic UGT1A1. Conclusions: The results showed that Fructus evodiae pretreatment decreased the systemic exposure of the Rhizoma coptidis alkaloids by inducing hepatic UGT1A1. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Rhizoma coptidis (Huang lian) is the dried rhizomes of several medicinal plants of the family Ranunculaceae such as Coptis chinensis Franch, which is a perennial stemless herb. It is a commonly used traditional Chinese medicine (TCM) with antibacterial (Choi et al., 2007), antifungal (Seneviratne et al., 2008), antiviral (Kim et al., 2008a), anti-dysentery (Xu et al., 2004), antioxidant (Yokozawa et al., 2005), anti-inflammatory (Kim et al., 2008b), and anticancer (Liu et al., 2009a) activities. It yields alkaloids such as berberine, coptisine, jatrorrhizine, and palmatine (Chen et al., 2008) (Fig. 1). Berberine is the primary active constituent. Rhizoma coptidis is usually prescribed with Fructus evodiae (Wu zhu yu) as TCM formulas such as Zuojin Wan in eastern Asia and overseas Chinese communities, which show therapeutic actions on gastric (Zhao et al., 2009) and intestinal diseases (Luo et al., 2010). Fructus evodiae is the dried fruits of Evodia rutaecarpa (Juss.) Benth and related varieties, which are shrubs or small trees belonging to the family Rutaceae. Evodiamine and rutaecarpine are active constituents in Fructus evodiae (Yang et al., 2009) (Fig. 1).
∗ Corresponding author. Tel.: +86 21 51322386; fax: +86 21 51322386. E-mail address: mayueming [email protected] (Y.-M. Ma). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.08.002
Several studies on interactions between Rhizoma coptidis and Fructus evodiae were reported (Jia et al., 2009). Pharmaceutically, the dissolution rate of the Rhizoma coptidis alkaloids was decreased by Fructus evodiae when these two TCMs were extracted together (Jia et al., 2009). Pharmacologically, Fructus evodiae showed antagonistic effects on Rhizoma coptidis (Zhao et al., 2010). Pharmacokinetically, the absorption of berberine in Rhizoma coptidis–Fructus evodiae drug-pair was improved when compared to that in Rhizoma coptidis (Sun et al., 2008), while the systemic exposure of berberine in Rhizoma coptidis was reduced in the presence of Fructus evodiae (Jia et al., 2009). However, the reduced systemic exposure of berberine might be caused by the decreased dissolution rate of berberine since both extracts were oral administered together. Furthermore, only single administration was studied in above researches. The influence of multiple administration of Fructus evodiae was still unclear, which might significantly differ from single administration and show more relevance to clinical practices. In brief, the influences of Fructus evodiae on the pharmacokinetics of the Rhizoma coptidis alkaloids and underlying mechanisms needed further studies. Fructus evodiae and its major constituents or metabolites influence the pharmacokinetics of drugs like caffeine (Tsai et al., 2005), theophylline (Jan et al., 2005), and acetaminophen (Lee et al., 2007), possibly by modulating hepatic drug-metabolizing enzymes (Ueng
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Fig. 1. Structures of the Rhizoma coptidis alkaloids (berberine, coptisine, palmatine, and jatrorrhizine) and the Fructus evodiae alkaloids (evodiamine and rutaecarpine).
et al., 2001, 2002a,b,c; Lee et al., 2004; Iwata et al., 2005). Berberine is the main active constituent in Rhizoma coptidis, and it is eliminated mainly by hepatic metabolism (Yang et al., 2010). Therefore, we focused on studying whether multiple administration of Fructus evodiae could influence the pharmacokinetics of the Rhizoma coptidis alkaloids by influencing their metabolism in this study.
2. Materials and methods 2.1. Materials Both Rhizoma coptidis (Coptis chinensis Franch) and Fructus evodiae [Evodia rutaecarpa (Juss.) Benth], which were produced according to The preparing standardization of the crude Traditional Chinese Medicine of Shanghai (2008 edition), were purchased from the GMP certificated manufacturer Shanghai Kang Qiao herbal pieces Co. Ltd. The authentication of this herb was performed by Prof. Zhi-Li Zhao of the Department of Botany, Shanghai University of Traditional Chinese Medicine. The authentication was performed by comparison with appropriate voucher specimens at the herbaria and by analyzing both physical and chemical properties according to The Pharmacopoeia of People’s Republic of China (2010 edition). All of the alkaloid standards (except coptisine) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
Coptisine standard was purchased from the Wako Pure Chemical Ind. Ltd. (Japan). Demethyleneberberine standard, with purity greater than 99%, was obtained from Dr. Hong-Feng Luo, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. A BCA protein assay kit was obtained from the Shanghai Usen Biological Technology Co. (Shanghai, China). Deaminotriphosphopyridine nucleotide (NADPH), uridine-5 -diphosphoglucuronic acid (UDPGA), saccharic acid-1,4-lactone, alamethicin, ethidium bromide, and agarose were products of the Sigma Chemical Co. (USA). Trizol reagent was obtained from the Invitrogen Corporation (USA). The Revert Aid First Strand cDNA Synthesis Kit was purchased from Mathematical Biosciences Institute (USA). Acetonitrile and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany). The pure water used in this study was produced using the Milli-Q system (Millipore, Bedford, MA, USA). All other materials were of analytical grade or better.
2.2. Standardized preparation and quality control of extracts Both extracts were prepared by standardized operating procedures according to traditional method. In brief, Rhizoma coptidis or Fructus evodiae was immersed in water (TCM:water at 1:10, w/w) for 1 h and then boiled for 1.5 h for the first decocting. The second decocting was performed like the first. After filtration, the two decoctions were mixed and evaporated to dryness under reduced
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pressure at 60 ◦ C (yield at 37.1% or 31.6% for Rhizoma coptidis or Fructus evodiae, respectively). The active compounds were determined by HPLC for quality control of both extracts. The mass percent of berberine, coptisine, palmatine, and jateorhizine in the extract of Rhizoma coptidis was 15.5%, 4.2%, 1.9%, and 3.2%, respectively. The mass percent of evodiamine and rutaecarpine in the extract of Fructus evodiae was 0.012% and 0.009%, respectively. 2.3. Animals Grade II Sprague–Dawley rats weighing 200–220 g were purchased from the Shanghai Slac Laboratory Animal Co. (Shanghai, China). The rats were housed in an air conditioned room at 22–24 ◦ C under a 12 h dark/light cycle and given food and water ad libitum. The rats were fasted for 12 h before experiments. All animal experiments were performed in accordance with the guidelines of the National Research Council. 2.4. Influence of Fructus evodiae on the pharmacokinetics of the Rhizoma coptidis alkaloids in rats Twenty rats (male: female = 1:1) were randomly divided into four groups according to body weight. The rats received 0.32 g/kg of the oral extract of Fructus evodiae or the same volume of saline daily for two weeks. After 12 h of fasting, rats were administered 0.56 or 2.23 g/kg of the oral extract of Rhizoma coptidis. Venous blood samples (about 0.3 mL) were collected in heparinized 1.5 mL tubes by eye puncture at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18 (or 21), and 24 h after Rhizoma coptidis ingestion. Blood samples were centrifuged at 12,000 rpm for 10 min immediately upon collection, and the plasma collected and stored at −80 ◦ C for later analysis using LC/MS. 2.5. Influence of Fructus evodiae on the absorption of the Rhizoma coptidis alkaloids across everted rat gut sacs Rats received the oral extract of Fructus evodiae (0.08, 0.16, or 0.32 g/kg) or an equivalent volume of saline daily for two weeks. Absorption of the Rhizoma coptidis alkaloids across everted rat sacs was evaluated as described (Veau et al., 2001) with modifications. Briefly, after laparotomy, the ileum was taken 5 cm above the caecum, washed with isotonic saline, and everted. A 12 cm segment was cut off and ligated at one end. Everted gut sacs were filled on the serosal side (artificial inside) with 1 mL blank Krebs–Ringer buffer (containing 118 mM NaCl, 25 mM NaHCO3 , 1.2 mM MgSO4 , 2.5 mM CaCl2 , 11 mM glucose, 1.2 mM KH2 PO4 , and 4.7 mM KCl, pH 6.8). The other end was then tightly ligated to create a gut sac which was immediately incubated in a Magnus bath containing 19 mL oxygenated Krebs–Ringer buffer at 37 ◦ C. After 5 min, 1 mL Rhizoma coptidis solution (1 mg/mL in Krebs–Ringer buffer) was added to the bath. Aliquots of the solution (200 L) in the serosal side were taken every 15 min to 60 min. The same volume of fresh Krebs–Ringer buffer was resupplied every time. After incubation was terminated, the lengths of the sacs were measured. The samples were analyzed using LC/MS/MS. 2.6. Influence of Fructus evodiae on the metabolism of berberine in rat liver microsomes Rats were treated as described in the absorption experiment. Livers were removed and immediately stored at −80 ◦ C. Liver microsomes were prepared as described (Hill, 2003) except that the microsomes were resuspended and homogenized in ice-cold sucrose solution (0.25 M). Protein concentration was determined using the bicinchoninic acid (BCA) method. The protein concentration was adjusted to 10 mg/mL with the sucrose solution and 0.5 mL aliquots were dispensed into labeled tubes and stored at −80 ◦ C.
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Both phase I and phase II microsomal incubations were performed as described (Liu et al., 2009b) with modifications. Briefly, reaction conditions were optimized to ensure the linearity of the reactions. Finally, for the phase I microsomal incubation, the reactions were conducted in a medium containing 100 mM potassium phosphate buffer (pH 7.4) with 1 mM NADPH, 0.5 mg microsomes, and 10 M berberine. After a preincubation for 5 min, the reaction was initiated by the addition of the NADPH solution. After incubation at 37 ◦ C for 15 min, the reaction was terminated by adding an equal volume of cold methanol with internal standard. For the phase II microsomal incubation, the reactions were conducted in a medium containing 0.5 mg microsomes, 1 mM UDPGA, 10 mM MgCl2 , 5 mM saccharic acid-1,4-lactone, 50 mM potassium phosphate buffer (pH 7.4), and 10 M demethyleneberberine. The microsomes were pretreated with 25 g/mL alamethicin on ice for 15 min. After further preincubation for 5 min at 37 ◦ C, the reactions were started by adding the UDPGA solution. After 2 min, the reaction was stopped by addition of an equal volume of cold methanol with internal standard. After centrifugation at 12,000 rpm for 15 min at 4 ◦ C, samples were injected into a Waters HPLC system for analysis.
2.7. Quantitative analysis in various biological samples The LC/MS analysis was performed using a Shimadzu LC-10AD HPLC series liquid chromatograph and a Shimadzu LC/MS-2010A single quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface and a Q-array Octapole-Quadrupole mass analyzer (Shimadzu, Japan). The samples were precipitated with three volumes of methanol. Tetrahydropalmatine was used as the internal standard. The supernatant was evaporated to dryness under a stream of nitrogen. The samples were eluted through an XTerra RP 18 analytical column (5 m, 3.9 × 150 mm) at 40 ◦ C with a gradient of solution A (0.1% formic acid and 2 mM ammonium formate) and solution B (methanol) (0 min, 85:15; 3 min, 70:30; 10 min, 70:30; 11 min, 20:80; 12 min, 85:15; 15 min, 85:15) at a flow rate of 0.8 mL/min. A post-column split was used to maintain a flow rate of 0.2 mL/min into the mass spectrometer source. The ESI source was set to positive ion mode. Quantification was obtained by using the selected ion monitoring (SIM) mode of the m/z 320.1 for coptisine, 336.1 for berberine, 338.1 for jateorrhizine, 352.1 for palmatine, and 356.3 for tetrahydropalmatine. The LC/MS/MS system was comprised of an Agilent 1200 series HPLC and a Quattro premier triple-quadrupole mass spectrometer (AB, USA) equipped with an ESI source. The samples were precipitated with three volumes of acetonitrile with phenacetin as internal standard. The supernatant was then mixed with an equal volume of pure water, and then 10 L samples were injected into the LC/MS/MS system. The samples were eluted through the XTerra RP 18 analytical column at 40 ◦ C with the aqueous phase (0.08%, v/v formic acid in 4 mM ammonium acetate) and the acetonitrile phase (48:52) at a flow rate of 0.6 mL/min. The ESI source was set to positive ion mode. Data acquisition was performed in the multiple reaction monitoring (MRM) mode of the selective mass transition for each compound. The transitions of the protonated precursor ions to the selected product ions were m/z 338.10 → m/z 322.90 for jatrorhizine, m/z 320.20 → m/z 292.00 for coptisine, m/z 336.10 → m/z 292.00 for berberine, m/z 352.00 → m/z 335.90 for palmatine, and m/z 180.00 → m/z 110.00 for phenacetin. The integrated HPLC system (Waters, USA) was equipped with a 2695 separation module, a 2487 dual absorbance detector and an Empower2 chemstation. Separation and determination were performed using the XTerra RP 18 analytical column at 35 ◦ C with = 346 nm. The samples were eluted through the column with a gradient of water–formic acid–triethylamine (100:0.1:0.2,
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v/v/v) and methanol (0 min, 75:25; 15 min, 60:40; 15.5 min, 75:25; 20 min, 75:25) at a flow rate of 0.5 mL/min. 2.8. Influence of Fructus evodiae on the mRNA expression of UGT1A1 in rat livers Rats were treated and liver samples collected as described in the metabolism experiment. Approximately 100 mg of frozen liver tissue was pulverized in liquid nitrogen, and total RNA was extracted using a Trizol reagent according to the manufacturer’s instruction. RNA was quantified and purity was monitored by spectrophotometry (Beckman Coulter, USA) at 260 and 280 nm. The RNA samples were stored at −80 ◦ C until analysis. Reverse transcriptase reactions were performed using a Revert Aid First Strand cDNA Synthesis Kit according to the manufacturer’s instruction. The UGT1A1-specific sense and antisense primers were 5 -TGGTGTGCCGGAGCTCATGTTCG-3 and 5 -ACTCCGCCCAAGTTCCACAAAAGCA-3 , respectively. The polymerase chain reaction (PCR) reactions began with 3 min at 95 ◦ C, followed by 35 cycles (30 s at 95 ◦ C, 30 s at 55 ◦ C, 45 s at 72 ◦ C), and finally by 5 min at 72 ◦ C. PCR products were electrophoresed on an ethidium bromide-stained 1.5% agarose gel, and the DNA fragments obtained were quantified using a gel image analysis system (Tanon2500, Shanghai). All reverse transcriptase reactions were controlled by using specific oligonucleotides for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 2.9. Statistical analysis Results were expressed as mean ± S.D. To determine pharmacokinetic parameters, data were processed by non-compartmental analysis using the DAS 2.0 package (Chinese Pharmacological Society). Maximum plasma concentration (Cmax ) and time to maximum plasma concentration (Tmax ) were obtained directly from the observed concentration versus time data. Analysis of variance was performed to compare multiple treatment means. P < 0.05 was considered significant, P < 0.01 was considered highly significant.
Fig. 2. Effects of Fructus evodiae pretreatment on the pharmacokinetics of berberine in rats receiving the oral Rhizoma coptidis extract (mean ± S.D, n = 5). Rats received a single 0.56 g/kg (A) or 2.23 g/kg (B) dose of Rhizoma coptidis oral extract following pretreatment with Fructus evodiae (0.32 g/kg daily for two weeks) or saline. Venous blood samples were collected and the berberine content was determined using LC/MS/MS. RC: Rhizoma coptidis; FE: Fructus evodiae.
3. Results 3.1. Influence of Fructus evodiae on the pharmacokinetics of the Rhizoma coptidis alkaloids in rats Multiple peaks in the plasma concentration of berberine were observed (Fig. 2). Berberine reached its first (C1max ), second (C2max ), and third (C3max ) peak concentrations about 1, 5–6, and 16–18 h post dosing. The pharmacokinetic parameters of berberine are summarized in Table 1. The results showed that two weeks pretreatment with the oral extract of Fructus evodiae significantly (P = 0.029) decreased the systemic exposure of berberine as determined by the area under concentration–time curve from 0 to 24 h (AUC0–24 h ). The C3max was significantly decreased (P = 0.008) by Fructus evodiae pretreatment. The mean plasma concentration–time curves of coptisine, palmatine, and jateorrhizine mirrored the temporal kinetics of the major alkaloid berberine, but the AUC0–24 h values were lower. The AUC0–24 h of coptisine was 27% that of berberine that of palmatine was 23% of the berberine AUC0–24 h , and the AUC0–24 h of jateorrhizine was only 13% of the berberine AUC0–24 h . 3.2. Influence of Fructus evodiae on the absorption of the Rhizoma coptidis alkaloids in rat everted gut sacs The mean absorption–time curves of the Rhizoma coptidis alkaloids are shown in Fig. 3. The results revealed that pretreatment with 0.08 g/kg of Fructus evodiae daily for two weeks significantly
increased the absorption of the Rhizoma coptidis alkaloids (P = 0.031, 0.008, 0.024, and 0.015 for berberine, coptisine, palmatine, and jatrorrhizine, respectively). However, both 0.16 and 0.32 g/kg of Fructus evodiae had no significant effect on the in vitro absorption of the Rhizoma coptidis alkaloids. 3.3. Influence of Fructus evodiae on berberine metabolism in rat liver microsomes Two major phase I metabolites, M1 and M2 (demethyleneberberine), with fragment ions at m/z 322 and m/z 324 according to an LC/MS/MS identification, were formed during incubation of berberine with rat liver microsomes in the presence of NADPH. Incubation of M2 standard in the rat liver microsomes in the presence of UDPGA led to formation of glucuronide M3, with fragment ions at m/z 500 according to the LC/MS/MS identification. We could not obtain sufficient standards of these metabolites, so semiquantitative concentrations of M1 were deduced according to the accompanying calibration curve of berberine; the semiquantitative concentration of M3 was calculated according to the accompanying calibration curve of M2. As shown in Fig. 4, pretreatment with 0.32 g/kg of Fructus evodiae for two weeks significantly decreased the phase I (P = 0.016) metabolism of berberine to M1. On the other hand, both 0.16 g/kg and 0.32 g/kg of Fructus evodiae significantly increased the phase II metabolism of M2 to M3 (P = 0.021 for 0.16 g/kg, P = 0.027 for 0.32 g/kg).
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Table 1 Pharmacokinetic parameters of berberine in rats received the oral extract of Rhizoma coptidis with or without the pretreatment with Fructus evodiae (mean ± SD, n = 5). Parameters
RC (2.23 g/kg) Control
AUC0–24 h (ng h/mL) MRT0–24 h (h) T1max (h) C1max (ng/mL) T2max (h) C2max (ng/mL) T3max (h) C3max (ng/mL)
410.1 11.4 1.1 50.5 5.2 23.5 16.8 32.6
± ± ± ± ± ± ± ±
RC (0.56 g/kg) FE (0.32 g/kg)
309.0 2.9 0.6 72.2 1.8 20.1 5.0 28.8
214.8 9.8 1.8 37.9 5.6 14.3 15.6 9.0
± ± ± ± ± ± ± ±
172.5 2.6 0.4 67.7 0.9 8.2 5.4 5.2
Control 282.7 13.0 0.3 3.9 6.4 3.8 18 38.1
± ± ± ± ± ± ± ±
FE (0.32 g/kg) 370.3 2.4 0.1 2.3 1.7 3.6 5.6 55.5
61.7 11.2 1.1 12.3 4.8 3.6 15.6 3.9
± ± ± ± ± ± ± ±
10.3 1.4 0.9 18.9 1.8 3.6 4.9 0.7
RC: Rhizoma coptidis; FE: Fructus evodiae. AUC, area under concentration–time curve; Cmax , maximum plasma concentration; MRT, mean residence time; Tmax , time to reach maximum plasma concentration.
Fig. 3. Effects of Fructus evodiae pretreatment on the absorption of the Rhizoma coptidis alkaloids in rat everted gut sacs (mean ± S.D, n = 6). After pretreatment of Fructus evodiae (0.08, 0.16, or 0.32 g/kg for two weeks), the rats were killed and everted gut sacs were prepared. Absorption of the Rhizoma coptidis alkaloids in the rat everted gut sacs was measured. Concentrations of berberine were determined using LC/MS/MS. FE: Fructus evodiae; L, M, and H: 0.08, 0.16, or 0.32 g/kg respectively; Ber: berberine; Cop: coptisine; Jat: jatrorrhizine; Pal: palmatine.
3.4. Influence of Fructus evodiae on the mRNA expression of UGT1A1 in rat livers Pretreatment with the three dosages of Fructus evodiae (0.08, 0.16, 0.32 g/kg) for two weeks significantly increased the hepatic mRNA expression of UGT1A1 in the rats (0.08 g/kg: P = 0.049, 0.16 g/kg: P = 0.018, 0.32 g/kg: P = 0.045; Fig. 5). 4. Discussion and conclusions Traditional Chinese medicines are usually prescribed as multiherb formulas. The combination of several herbs is believed to increase curative effects and to reduce side effects. Both synergy and antagonistic effects between TCMs components may be derived from herb–herb interactions. Similar to drug–drug interactions, herb–herb interactions may occur at pharmacokinetic level through modulation of absorption, metabolism, or elimination of active components. Studies on herb–herb interactions, especially herb–herb pharmacokinetic interactions, will aid in the elucidation of both synergistic and antagonistic interactions between herbs in
TCMs. Indeed, such data will help to modernization clinical applications of TCMs. Therapeutically, Rhizoma coptidis and Fructus evodiae are extracted and oral administered together. However, in this situation, interactions between these two TCMs are very complicated. Interactions also happen pharmaceutically and pharmacologically besides pharmacokinetically (Jia et al., 2009; Zhao et al., 2010). Therefore, in this study, to eliminate the influence of pharmaceutical interactions, we adopted the subsequent treatment methodological approach, which is not matching the therapeutical use but could also offer useful hints for further studies. The results showed that the pretreatment with 0.32 g/kg of the Fructus evodiae extract decreased the systemic exposure of berberine. In addition, C3max was significantly decreased. The pharmacokinetic interactions were due to enhancement of phase II glucuronidation by Fructus evodiae based on following results. Firstly, pretreatment with 0.32 g/kg of Fructus evodiae extract had no significant effect on the in vitro absorption of the Rhizoma coptidis alkaloids. Secondly, the in vitro metabolism experiments excluded the possibility that the effect of Fructus evodiae was
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Fig. 4. Effects of Fructus evodiae pretreatment on phase I metabolism of berberine and phase II metabolism of demethyleneberberine (M2) in rat liver microsomes (mean ± S.D, n = 6). After the Fructus evodiae pretreatment (0.08, 0.16, or 0.32 g/kg for two weeks), the rats were killed and liver microsomes were prepared. Phase I and phase II metabolism was studied in rat liver microsomes and analyzed using HPLC. M1, M2, and M3: metabolites at m/z 322, 324 (demethyleneberberine), and 500 respectively; L, M, and H: 0.08, 0.16, and 0.32 g/kg, respectively.
Fig. 5. Effect of Fructus evodiae pretreatment on UGT1A1 mRNA expression in rat liver (mean ± S.D, n = 3). Rats were treated with either saline or Fructus evodiae (0.08, 0.16, or 0.32 g/kg) for two weeks. Hepatic UGT1A1 mRNA expression was determined (A), and values expressed relative to the appropriate control group (B). C: Control; L, M, and H: 0.08, 0.16, and 0.32 g/kg, respectively. *P < 0.05 compared with control.
due to induction of phase I hepatic drug-metabolizing enzymes. Actually, the phase I metabolism of berberine to M1 was inhibited by the pretreatment with 0.32 g/kg of Fructus evodiae. Finally, both the in vitro metabolism and PCR experiments confirmed that phase II glucuronidation was enhanced by 0 .32 g/kg of the Fructus evodiae extract. Multiple cytochrome P450 isoenzymes (CYPs) were involved in the phase I metabolism of berberine, including CYP3A1/2, CYP2B, and CYP2D1 (Liu et al., 2009b). Pretreatment with Fructus evodiae was reported to increase the activities of CYP1A1, CYP1A2, and CYP2B in C57BL/6J mice (Ueng et al., 2002c). In addition, pretreatment of rutaecarpine, one of the major alkaloids of Fructus evodiae, reportedly increased protein levels of both CYP1A1 and CYP1A2 in
liver without altering hepatic protein levels of CYP3A (Ueng et al., 2001). To our knowledge, this is the first time that an inhibitory effect of Fructus evodiae on phase I metabolism of berberine has been reported. Phase II glucuronidation metabolism was demonstrated to be one of the major elimination pathway for the Rhizoma coptidis alkaloids (Yang et al., 2010), indicating that phase II glucuronidation metabolism was crucial to systemic exposure of the Rhizoma coptidis alkaloids. In the in vitro phase II metabolism experiments, we only tested incubation of the M2 standard since M1 standard was not available in sufficient quantities. It was reported, however, that both M1 and M2 were mainly catalyzed by UGT1A1 (Liu et al., 2009b), suggesting that the incubation of the M2 standard reflects the metabolism capability of UGT1A1 in rat liver microsomes. Our results suggested differential effects of Fructus evodiae on phase I and phase II hepatic drug metabolism enzymes, and that the enhancement of phase II was more important than any change in phase I in the systemic exposure of the Rhizoma coptidis alkaloids. After oral administration of the Rhizoma coptidis extract, the Rhizoma coptidis alkaloids were widely distributed into various tissues, including liver, heart, kidney, lung, and brain (Ma et al., 2010). Tissue concentrations of the Rhizoma coptidis alkaloids increase nonlinearly with higher doses and finally cause acute toxicity (Ma et al., 2010). The median lethal dose (LD50 ) of the oral Rhizoma coptidis extract was 2.95 g/kg in mice (Ma et al., 2010). Traditionally, Fructus evodiae is prescribed with Rhizoma coptidis at certain ratio. For example, the ratio of Fructus evodiae to Rhizoma coptidis is 1:6 (calculated by the weight of the dried herbal pieces) in Zuojin Wan. Therefore, for toxic dosage of Rhizoma coptidis, the dosage of the combined Fructus evodiae should be higher than 0.3 g/kg. In this situation, Fructus evodiae would influence the final in vivo activities of Rhizoma coptidis by influencing the pharmacokinetics of the Rhizoma coptidis alkaloids. In fact, although the detailed mechanism involved remained to be studied, the oral Fructus evodiae extract pretreatment decreased the acute toxicity of Rhizoma coptidis in mice (data not shown). However, for conventional clinical application, our results did not suggest that the oral Fructus evodiae would reduce the curative effects of the combined Rhizoma coptidis based on pharmacokinetic influences. The clinical conventional dosage of Fructus evodiae extract is 0.47–1.42 g per adult in China. Thus, the equivalent surface area dosage of rat is about 0.05–0.14 g/kg. In this study, 0.08 g/kg of the oral Fructus evodiae pretreatment increased the absorption of the Rhizoma coptidis alkaloids. This result was consistent with what was reported (Sun et al., 2008; Wang et al., 2009). All the Rhizoma coptidis alkaloids are substrates of p-glycoprotein (P-gp) (Zhang et al., 2011), a well known drug efflux pump, and the extract of Fructus evodiae reportedly showed significant inhibitory effects on P-gp (Yoshida et al., 2005). Hence, Fructus evodiae might increase the absorption of the Rhizoma coptidis alkaloids by inhibiting the intestinal P-gp. Furthermore, although 0.08 g/kg of the oral Fructus evodiae pretreatment showed weak induction on the expression of UGT1A1, it did not show significant effects on both phase I and phase II metabolism of berberine. Our results indicated that different dosages of Fructus evodiae resulted in different pharmacokinetic influences on the Rhizoma coptidis alkaloids. In contrast to pure compound drugs used in Western medicine, TCMs consist of a multitude of constituents (some still not described) that differ in terms of herb content and quality. Thus, different dosages of TCM result in different dosages of the active constituents and significantly different outcomes. The detailed mechanism involved, however, has yet to be elucidated. In conclusion, the results showed that Fructus evodiae pretreatment decreased the systemic exposure of the Rhizoma coptidis alkaloids by inducing hepatic UGT1A1.
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Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was financially supported by the Construction Program for Innovative Research Team in Shanghai Institutions of Higher Education, the National Natural Science Foundation of China (No. 30873231), the Shanghai Leading Academic Discipline Project (No. J50303), and the key projects of PhD Programs Foundation of Shanghai (2011). References Chen, J., Zhao, H., Wang, X., Lee, F.S., Yang, H., Zheng, L., 2008. Analysis of major alkaloids in Rhizoma coptidis by capillary electrophoresis–electrospray-time of flight mass spectrometry with different background electrolytes. Electrophoresis 29, 2135–2147. Choi, U.K., Kim, M.H., Lee, N.H., 2007. Optimization of antibacterial activity by gold-thread (Coptidis rhizoma Franch) against Streptococcus mutans using evolutionary operation-factorial design technique. Journal of Microbiology and Biotechnology 17, 1880–1884. Hill, J.R., 2003. In vitro drug metabolism using liver microsomes. In: Enna, S.J. (Ed.), Current Protocols in Pharmacology. John Wiley and Sons, Massachusetts, pp. 7.8.1–7.8.11. Iwata, H., Tezuka, Y., Kadota, S., Hiratsuka, A., Watabe, T., 2005. Mechanismbased inactivation of human liver microsomal CYP3A4 by rutaecarpine and limonin from Evodia fruit extract. Drug Metabolism and Pharmacokinetics 20, 34–45. Jan, W.C., Lin, L.C., Chieh Fu, C., Tsai, T.H., 2005. Herb–drug interaction of Evodia rutaecarpa extract on the pharmacokinetics of theophylline in rats. Journal of Ethnopharmacology 102, 440–445. Jia, X., Jiang, J., Chen, B., Chen, Y., Hao, K., Cai, B., Wu, H., Yang, G., 2009. Progress in research of processing of Fructus Evodiae–Rhizoma Coptidis and research thoughts and methods of its processing mechanism. China Journal of Chinese Materia Medica 34, 1314–1317. Kim, H.Y., Shin, H.S., Park, H., Kim, Y.C., Yun, Y.G., Park, S., Shin, H.J., Kim, K., 2008a. In vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, Cimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, and Phellodendron cortex. Journal of Clinical Virology 41, 122–128. Kim, K.S, Rhee, H.I., Park, E.K., Jung, K., Jeon, H.J., Kim, J.H., Yoo, H., Han, C.K., Cho, Y.B., Ryu, C.J., Yang, H., Yoo, M.C., 2008b. Anti-inflammatory effects of Radix Gentianae Macrophyllae (Qinjiao), Rhizoma Coptidis (Huanglian) and Citri Unshiu Pericarpium (Wenzhou migan) in animal models. Chinese Medical Journal 3, 10. Lee, S.K., Bista, S.R., Jeong, H., Kim, D.H., Kang, M.J., Jahng, Y., Jeong, T.C., 2007. The effects of rutaecarpine on the pharmacokinetics of acetaminophen in rats. Archives of Pharmacal Research 30, 1629–1634. Lee, S.K., Kim, N.H., Lee, J., Kim, D.H., Lee, E.S., Choi, H.G., Chang, H.W., Jahng, Y., Jeong, T.C., 2004. Induction of cytochrome P450s by rutaecarpine and metabolism of rutaecarpine by cytochrome P450s. Planta Medica 70, 753–757. Liu, J., He, C., Zhou, K., Wang, J., Kang, J.X., 2009a. Coptis extracts enhance the anticancer effect of estrogen receptor antagonists on human breast cancer cells. Biochemical and Biophysical Research Communications 378, 174–178. Liu, Y., Hao, H., Xie, H., Lv, H., Liu, C., Wang, G., 2009b. Oxidative demethylenation and subsequent glucuronidation are the major metabolic pathways of berberine in rats. Journal of Pharmaceutical Sciences 98, 4391–4401.
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Luo, Y., Zhao, H., Liu, Z., Ju, D., He, X., Xiao, C., Zhong, G., Chen, S., Yang, D., Chan, A.S., Lu, A., 2010. Comparison of the enteric mucosal immunomodulatory activity of combinations of Coptis chinensis Franch. rhizomes and Evodia rutaecarpa (Juss.) Benth. fruits in mice with dextran sulphate sodium-induced ulcerative colitis. Planta Medica 76, 766–772. Ma, B.L., Ma, Y.M., Shi, R., Wang, T.M., Zhang, N., Wang, C.H., Yang, Y., 2010. Identification of the toxic constituents in Rhizoma Coptidis. Journal of Ethnopharmacology 128, 357–364. Seneviratne, C.J., Wong, R.W., Samaranayake, L.P., 2008. Potent anti-microbial activity of traditional Chinese medicine herbs against Candida species. Mycoses 51, 30–34. Sun, W.-J., Zhang, Y.-J., Yao, N., Jiang, H., Wang, Q., 2008. Influence on rat intestinal absorption of huanglian alkalis after match-pair of huanglian-wuzhuyu. China Journal of Chinese Materia Medica 33, 2614–2616. Tsai, T.H., Chang, C.H., Lin, L.C., 2005. Effects of Evodia rutaecarpa and rutaecarpine on the pharmacokinetics of caffeine in rats. Planta Medica 71, 640–645. Ueng, Y.F., Don, M.J., Peng, H.C., Wang, S.Y., Wang, J.J., Chen, C.F., 2002a. Effects of Wu-chu-yu-tang and its component herbs on drug-metabolizing enzymes. The Japanese Journal of Pharmcology 89, 267–273. Ueng, Y.F., Jan, W.C., Lin, L.C., Chen, T.L., Guengerich, F.P., Chen, C.F., 2002b. The alkaloid rutaecarpine is a selective inhibitor of cytochrome P450 1A in mouse and human liver microsomes. Drug Metabolism and Disposition 30, 349–353. Ueng, Y.F., Ko, H.C., Chen, C.F., Wang, J.J., Chen, K.T., 2002c. Modulation of drugmetabolizing enzymes by extracts of an herbal medicine Evodia rutaecarpa in C57BL/6J mice. Life Sciences 71, 1267–1277. 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. Veau, C., Leroy, C., Banide, H., Auchere, D., Tardivel, S., Farinotti, R., Lacour, B., 2001. Effect of chronic renal failure on the expression and function of rat intestinal P-glycoprotein in drug excretion. Nephrology Dialysis Transplantation 16, 1607–1614. Wang, J., Yuan, Z.-M., Zhang, Z.-Q., 2009. Pharmacokinetic studied on berberine hydrochloride in Rhizoma Coptidis–Fructus Evodiae drug-pair in rat. Chinese Journal of Pharmaceutical Analysis 29, 755–759. Xu, J.-T., Wang, L.-Q., Xu, B., 2004. Research development of Coptis Chinensis. Acta Academiae Medicinae Sinicae 26, 705–707. Yang, X.W., Teng, J., Wang, Y., 2009. Thepermeability and efflux of alkaloids of Fructus evodiae in the Caco-2 model. Phytotherapy Research 23, 56–60. Yang, Y., Kang, N., Xia, H., Li, J., Chen, L., Qiu, F., 2010. Metabolites of protoberberine alkaloids in human urine following oral administration of Coptidis Rhizoma decoction. Planta Medica 76, 1859–1863. Yokozawa, T., Satoh, A., Cho, E.J., Kashiwada, Y., Ikeshiro, Y., 2005. Protective role of Coptidis Rhizoma alkaloids against peroxynitrite-induced damage to renal tubular epithelial cells. Journal of Pharmacy and Pharmacology 57, 367–374. Yoshida, N., Takagi, A., Kitazawa, H., Kawakami, J., Adachi, I., 2005. Inhibition of P-glycoprotein-mediated transport by extracts of and monoterpenoids contained in Zanthoxyli fructus. Toxicology and Applied Pharmacology 209, 167–173. Zhang, X., Qiu, F., Jiang, J., Gao, C., Tan, Y., 2011. Intestinal absorption mechanisms of berberine, palmatine, jateorhizine, and coptisine: involvement of P-glycoprotein. Xenobiotica 41, 290–296. Zhao, F.R., Mao, H.P., Zhang, H., Hu, L.M., Wang, H., Wang, Y.F., Yanagihara, N., Gao, X.M., 2010. Antagonistic effects of two herbs in Zuojin Wan, a traditional Chinese medicine formula, on catecholamine secretion in bovine adrenal medullary cells. Phytomedicine 17, 659–668. Zhao, Y.L., Shi, W.L., Shan, L.M., Wang, J.B., Zhao, H.P., Xiao, X.H., 2009. Differences in effects of Zuojin Pills and its similar formulas on Wei cold model in rats. Chinese Journal of Integrative Medicine 15, 293–298.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Influences of Fructus evodiae pretreatment on the pharmacokinetics of Rhizoma coptidis alkaloids Bing-Liang Ma a , Meng-Kan Yao a , Xiang-Hui Han a , Yue-Ming Ma a,∗ , Jia-Sheng Wu a , Chang-Hong Wang b a b
Laboratory of Pharmacokinetics, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China
a r t i c l e
i n f o
Article history: Received 13 October 2010 Received in revised form 29 July 2011 Accepted 2 August 2011 Available online 9 August 2011 Keywords: Rhizoma coptidis Fructus evodiae Berberine Herb–herb interactions UGT1A1 Pharmacokinetics
a b s t r a c t Ethnopharmacological relevance: Rhizoma coptidis is a traditional Chinese medicine with pharmacological properties. It is usually prescribed with Fructus evodiae as traditional Chinese medicine (TCM) formulas. Here we report the influences of Fructus evodiae on the pharmacokinetics of the Rhizoma coptidis alkaloids and propose possible mechanisms. Materials and methods: Pharmacokinetic experiments were performed in rats. In vitro absorption experiments were performed in everted rat gut sacs, while in vitro metabolism experiments and determination of hepatic UDP-glucuronosyltransferase (UGT) 1A1 mRNA expression were performed in rat liver microsomes. Results: Pretreatment with Fructus evodiae extract for two weeks decreased the systemic exposure of the Rhizoma coptidis alkaloids. This effect was not due to inhibition of absorption or enhanced hepatic phase I metabolism of the Rhizoma coptidis alkaloids. However, Fructus evodiae pretreatment enhanced both the activity and expression of hepatic UGT1A1. Conclusions: The results showed that Fructus evodiae pretreatment decreased the systemic exposure of the Rhizoma coptidis alkaloids by inducing hepatic UGT1A1. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Rhizoma coptidis (Huang lian) is the dried rhizomes of several medicinal plants of the family Ranunculaceae such as Coptis chinensis Franch, which is a perennial stemless herb. It is a commonly used traditional Chinese medicine (TCM) with antibacterial (Choi et al., 2007), antifungal (Seneviratne et al., 2008), antiviral (Kim et al., 2008a), anti-dysentery (Xu et al., 2004), antioxidant (Yokozawa et al., 2005), anti-inflammatory (Kim et al., 2008b), and anticancer (Liu et al., 2009a) activities. It yields alkaloids such as berberine, coptisine, jatrorrhizine, and palmatine (Chen et al., 2008) (Fig. 1). Berberine is the primary active constituent. Rhizoma coptidis is usually prescribed with Fructus evodiae (Wu zhu yu) as TCM formulas such as Zuojin Wan in eastern Asia and overseas Chinese communities, which show therapeutic actions on gastric (Zhao et al., 2009) and intestinal diseases (Luo et al., 2010). Fructus evodiae is the dried fruits of Evodia rutaecarpa (Juss.) Benth and related varieties, which are shrubs or small trees belonging to the family Rutaceae. Evodiamine and rutaecarpine are active constituents in Fructus evodiae (Yang et al., 2009) (Fig. 1).
∗ Corresponding author. Tel.: +86 21 51322386; fax: +86 21 51322386. E-mail address: mayueming [email protected] (Y.-M. Ma). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.08.002
Several studies on interactions between Rhizoma coptidis and Fructus evodiae were reported (Jia et al., 2009). Pharmaceutically, the dissolution rate of the Rhizoma coptidis alkaloids was decreased by Fructus evodiae when these two TCMs were extracted together (Jia et al., 2009). Pharmacologically, Fructus evodiae showed antagonistic effects on Rhizoma coptidis (Zhao et al., 2010). Pharmacokinetically, the absorption of berberine in Rhizoma coptidis–Fructus evodiae drug-pair was improved when compared to that in Rhizoma coptidis (Sun et al., 2008), while the systemic exposure of berberine in Rhizoma coptidis was reduced in the presence of Fructus evodiae (Jia et al., 2009). However, the reduced systemic exposure of berberine might be caused by the decreased dissolution rate of berberine since both extracts were oral administered together. Furthermore, only single administration was studied in above researches. The influence of multiple administration of Fructus evodiae was still unclear, which might significantly differ from single administration and show more relevance to clinical practices. In brief, the influences of Fructus evodiae on the pharmacokinetics of the Rhizoma coptidis alkaloids and underlying mechanisms needed further studies. Fructus evodiae and its major constituents or metabolites influence the pharmacokinetics of drugs like caffeine (Tsai et al., 2005), theophylline (Jan et al., 2005), and acetaminophen (Lee et al., 2007), possibly by modulating hepatic drug-metabolizing enzymes (Ueng
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Fig. 1. Structures of the Rhizoma coptidis alkaloids (berberine, coptisine, palmatine, and jatrorrhizine) and the Fructus evodiae alkaloids (evodiamine and rutaecarpine).
et al., 2001, 2002a,b,c; Lee et al., 2004; Iwata et al., 2005). Berberine is the main active constituent in Rhizoma coptidis, and it is eliminated mainly by hepatic metabolism (Yang et al., 2010). Therefore, we focused on studying whether multiple administration of Fructus evodiae could influence the pharmacokinetics of the Rhizoma coptidis alkaloids by influencing their metabolism in this study.
2. Materials and methods 2.1. Materials Both Rhizoma coptidis (Coptis chinensis Franch) and Fructus evodiae [Evodia rutaecarpa (Juss.) Benth], which were produced according to The preparing standardization of the crude Traditional Chinese Medicine of Shanghai (2008 edition), were purchased from the GMP certificated manufacturer Shanghai Kang Qiao herbal pieces Co. Ltd. The authentication of this herb was performed by Prof. Zhi-Li Zhao of the Department of Botany, Shanghai University of Traditional Chinese Medicine. The authentication was performed by comparison with appropriate voucher specimens at the herbaria and by analyzing both physical and chemical properties according to The Pharmacopoeia of People’s Republic of China (2010 edition). All of the alkaloid standards (except coptisine) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
Coptisine standard was purchased from the Wako Pure Chemical Ind. Ltd. (Japan). Demethyleneberberine standard, with purity greater than 99%, was obtained from Dr. Hong-Feng Luo, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. A BCA protein assay kit was obtained from the Shanghai Usen Biological Technology Co. (Shanghai, China). Deaminotriphosphopyridine nucleotide (NADPH), uridine-5 -diphosphoglucuronic acid (UDPGA), saccharic acid-1,4-lactone, alamethicin, ethidium bromide, and agarose were products of the Sigma Chemical Co. (USA). Trizol reagent was obtained from the Invitrogen Corporation (USA). The Revert Aid First Strand cDNA Synthesis Kit was purchased from Mathematical Biosciences Institute (USA). Acetonitrile and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany). The pure water used in this study was produced using the Milli-Q system (Millipore, Bedford, MA, USA). All other materials were of analytical grade or better.
2.2. Standardized preparation and quality control of extracts Both extracts were prepared by standardized operating procedures according to traditional method. In brief, Rhizoma coptidis or Fructus evodiae was immersed in water (TCM:water at 1:10, w/w) for 1 h and then boiled for 1.5 h for the first decocting. The second decocting was performed like the first. After filtration, the two decoctions were mixed and evaporated to dryness under reduced
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pressure at 60 ◦ C (yield at 37.1% or 31.6% for Rhizoma coptidis or Fructus evodiae, respectively). The active compounds were determined by HPLC for quality control of both extracts. The mass percent of berberine, coptisine, palmatine, and jateorhizine in the extract of Rhizoma coptidis was 15.5%, 4.2%, 1.9%, and 3.2%, respectively. The mass percent of evodiamine and rutaecarpine in the extract of Fructus evodiae was 0.012% and 0.009%, respectively. 2.3. Animals Grade II Sprague–Dawley rats weighing 200–220 g were purchased from the Shanghai Slac Laboratory Animal Co. (Shanghai, China). The rats were housed in an air conditioned room at 22–24 ◦ C under a 12 h dark/light cycle and given food and water ad libitum. The rats were fasted for 12 h before experiments. All animal experiments were performed in accordance with the guidelines of the National Research Council. 2.4. Influence of Fructus evodiae on the pharmacokinetics of the Rhizoma coptidis alkaloids in rats Twenty rats (male: female = 1:1) were randomly divided into four groups according to body weight. The rats received 0.32 g/kg of the oral extract of Fructus evodiae or the same volume of saline daily for two weeks. After 12 h of fasting, rats were administered 0.56 or 2.23 g/kg of the oral extract of Rhizoma coptidis. Venous blood samples (about 0.3 mL) were collected in heparinized 1.5 mL tubes by eye puncture at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18 (or 21), and 24 h after Rhizoma coptidis ingestion. Blood samples were centrifuged at 12,000 rpm for 10 min immediately upon collection, and the plasma collected and stored at −80 ◦ C for later analysis using LC/MS. 2.5. Influence of Fructus evodiae on the absorption of the Rhizoma coptidis alkaloids across everted rat gut sacs Rats received the oral extract of Fructus evodiae (0.08, 0.16, or 0.32 g/kg) or an equivalent volume of saline daily for two weeks. Absorption of the Rhizoma coptidis alkaloids across everted rat sacs was evaluated as described (Veau et al., 2001) with modifications. Briefly, after laparotomy, the ileum was taken 5 cm above the caecum, washed with isotonic saline, and everted. A 12 cm segment was cut off and ligated at one end. Everted gut sacs were filled on the serosal side (artificial inside) with 1 mL blank Krebs–Ringer buffer (containing 118 mM NaCl, 25 mM NaHCO3 , 1.2 mM MgSO4 , 2.5 mM CaCl2 , 11 mM glucose, 1.2 mM KH2 PO4 , and 4.7 mM KCl, pH 6.8). The other end was then tightly ligated to create a gut sac which was immediately incubated in a Magnus bath containing 19 mL oxygenated Krebs–Ringer buffer at 37 ◦ C. After 5 min, 1 mL Rhizoma coptidis solution (1 mg/mL in Krebs–Ringer buffer) was added to the bath. Aliquots of the solution (200 L) in the serosal side were taken every 15 min to 60 min. The same volume of fresh Krebs–Ringer buffer was resupplied every time. After incubation was terminated, the lengths of the sacs were measured. The samples were analyzed using LC/MS/MS. 2.6. Influence of Fructus evodiae on the metabolism of berberine in rat liver microsomes Rats were treated as described in the absorption experiment. Livers were removed and immediately stored at −80 ◦ C. Liver microsomes were prepared as described (Hill, 2003) except that the microsomes were resuspended and homogenized in ice-cold sucrose solution (0.25 M). Protein concentration was determined using the bicinchoninic acid (BCA) method. The protein concentration was adjusted to 10 mg/mL with the sucrose solution and 0.5 mL aliquots were dispensed into labeled tubes and stored at −80 ◦ C.
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Both phase I and phase II microsomal incubations were performed as described (Liu et al., 2009b) with modifications. Briefly, reaction conditions were optimized to ensure the linearity of the reactions. Finally, for the phase I microsomal incubation, the reactions were conducted in a medium containing 100 mM potassium phosphate buffer (pH 7.4) with 1 mM NADPH, 0.5 mg microsomes, and 10 M berberine. After a preincubation for 5 min, the reaction was initiated by the addition of the NADPH solution. After incubation at 37 ◦ C for 15 min, the reaction was terminated by adding an equal volume of cold methanol with internal standard. For the phase II microsomal incubation, the reactions were conducted in a medium containing 0.5 mg microsomes, 1 mM UDPGA, 10 mM MgCl2 , 5 mM saccharic acid-1,4-lactone, 50 mM potassium phosphate buffer (pH 7.4), and 10 M demethyleneberberine. The microsomes were pretreated with 25 g/mL alamethicin on ice for 15 min. After further preincubation for 5 min at 37 ◦ C, the reactions were started by adding the UDPGA solution. After 2 min, the reaction was stopped by addition of an equal volume of cold methanol with internal standard. After centrifugation at 12,000 rpm for 15 min at 4 ◦ C, samples were injected into a Waters HPLC system for analysis.
2.7. Quantitative analysis in various biological samples The LC/MS analysis was performed using a Shimadzu LC-10AD HPLC series liquid chromatograph and a Shimadzu LC/MS-2010A single quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface and a Q-array Octapole-Quadrupole mass analyzer (Shimadzu, Japan). The samples were precipitated with three volumes of methanol. Tetrahydropalmatine was used as the internal standard. The supernatant was evaporated to dryness under a stream of nitrogen. The samples were eluted through an XTerra RP 18 analytical column (5 m, 3.9 × 150 mm) at 40 ◦ C with a gradient of solution A (0.1% formic acid and 2 mM ammonium formate) and solution B (methanol) (0 min, 85:15; 3 min, 70:30; 10 min, 70:30; 11 min, 20:80; 12 min, 85:15; 15 min, 85:15) at a flow rate of 0.8 mL/min. A post-column split was used to maintain a flow rate of 0.2 mL/min into the mass spectrometer source. The ESI source was set to positive ion mode. Quantification was obtained by using the selected ion monitoring (SIM) mode of the m/z 320.1 for coptisine, 336.1 for berberine, 338.1 for jateorrhizine, 352.1 for palmatine, and 356.3 for tetrahydropalmatine. The LC/MS/MS system was comprised of an Agilent 1200 series HPLC and a Quattro premier triple-quadrupole mass spectrometer (AB, USA) equipped with an ESI source. The samples were precipitated with three volumes of acetonitrile with phenacetin as internal standard. The supernatant was then mixed with an equal volume of pure water, and then 10 L samples were injected into the LC/MS/MS system. The samples were eluted through the XTerra RP 18 analytical column at 40 ◦ C with the aqueous phase (0.08%, v/v formic acid in 4 mM ammonium acetate) and the acetonitrile phase (48:52) at a flow rate of 0.6 mL/min. The ESI source was set to positive ion mode. Data acquisition was performed in the multiple reaction monitoring (MRM) mode of the selective mass transition for each compound. The transitions of the protonated precursor ions to the selected product ions were m/z 338.10 → m/z 322.90 for jatrorhizine, m/z 320.20 → m/z 292.00 for coptisine, m/z 336.10 → m/z 292.00 for berberine, m/z 352.00 → m/z 335.90 for palmatine, and m/z 180.00 → m/z 110.00 for phenacetin. The integrated HPLC system (Waters, USA) was equipped with a 2695 separation module, a 2487 dual absorbance detector and an Empower2 chemstation. Separation and determination were performed using the XTerra RP 18 analytical column at 35 ◦ C with = 346 nm. The samples were eluted through the column with a gradient of water–formic acid–triethylamine (100:0.1:0.2,
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v/v/v) and methanol (0 min, 75:25; 15 min, 60:40; 15.5 min, 75:25; 20 min, 75:25) at a flow rate of 0.5 mL/min. 2.8. Influence of Fructus evodiae on the mRNA expression of UGT1A1 in rat livers Rats were treated and liver samples collected as described in the metabolism experiment. Approximately 100 mg of frozen liver tissue was pulverized in liquid nitrogen, and total RNA was extracted using a Trizol reagent according to the manufacturer’s instruction. RNA was quantified and purity was monitored by spectrophotometry (Beckman Coulter, USA) at 260 and 280 nm. The RNA samples were stored at −80 ◦ C until analysis. Reverse transcriptase reactions were performed using a Revert Aid First Strand cDNA Synthesis Kit according to the manufacturer’s instruction. The UGT1A1-specific sense and antisense primers were 5 -TGGTGTGCCGGAGCTCATGTTCG-3 and 5 -ACTCCGCCCAAGTTCCACAAAAGCA-3 , respectively. The polymerase chain reaction (PCR) reactions began with 3 min at 95 ◦ C, followed by 35 cycles (30 s at 95 ◦ C, 30 s at 55 ◦ C, 45 s at 72 ◦ C), and finally by 5 min at 72 ◦ C. PCR products were electrophoresed on an ethidium bromide-stained 1.5% agarose gel, and the DNA fragments obtained were quantified using a gel image analysis system (Tanon2500, Shanghai). All reverse transcriptase reactions were controlled by using specific oligonucleotides for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 2.9. Statistical analysis Results were expressed as mean ± S.D. To determine pharmacokinetic parameters, data were processed by non-compartmental analysis using the DAS 2.0 package (Chinese Pharmacological Society). Maximum plasma concentration (Cmax ) and time to maximum plasma concentration (Tmax ) were obtained directly from the observed concentration versus time data. Analysis of variance was performed to compare multiple treatment means. P < 0.05 was considered significant, P < 0.01 was considered highly significant.
Fig. 2. Effects of Fructus evodiae pretreatment on the pharmacokinetics of berberine in rats receiving the oral Rhizoma coptidis extract (mean ± S.D, n = 5). Rats received a single 0.56 g/kg (A) or 2.23 g/kg (B) dose of Rhizoma coptidis oral extract following pretreatment with Fructus evodiae (0.32 g/kg daily for two weeks) or saline. Venous blood samples were collected and the berberine content was determined using LC/MS/MS. RC: Rhizoma coptidis; FE: Fructus evodiae.
3. Results 3.1. Influence of Fructus evodiae on the pharmacokinetics of the Rhizoma coptidis alkaloids in rats Multiple peaks in the plasma concentration of berberine were observed (Fig. 2). Berberine reached its first (C1max ), second (C2max ), and third (C3max ) peak concentrations about 1, 5–6, and 16–18 h post dosing. The pharmacokinetic parameters of berberine are summarized in Table 1. The results showed that two weeks pretreatment with the oral extract of Fructus evodiae significantly (P = 0.029) decreased the systemic exposure of berberine as determined by the area under concentration–time curve from 0 to 24 h (AUC0–24 h ). The C3max was significantly decreased (P = 0.008) by Fructus evodiae pretreatment. The mean plasma concentration–time curves of coptisine, palmatine, and jateorrhizine mirrored the temporal kinetics of the major alkaloid berberine, but the AUC0–24 h values were lower. The AUC0–24 h of coptisine was 27% that of berberine that of palmatine was 23% of the berberine AUC0–24 h , and the AUC0–24 h of jateorrhizine was only 13% of the berberine AUC0–24 h . 3.2. Influence of Fructus evodiae on the absorption of the Rhizoma coptidis alkaloids in rat everted gut sacs The mean absorption–time curves of the Rhizoma coptidis alkaloids are shown in Fig. 3. The results revealed that pretreatment with 0.08 g/kg of Fructus evodiae daily for two weeks significantly
increased the absorption of the Rhizoma coptidis alkaloids (P = 0.031, 0.008, 0.024, and 0.015 for berberine, coptisine, palmatine, and jatrorrhizine, respectively). However, both 0.16 and 0.32 g/kg of Fructus evodiae had no significant effect on the in vitro absorption of the Rhizoma coptidis alkaloids. 3.3. Influence of Fructus evodiae on berberine metabolism in rat liver microsomes Two major phase I metabolites, M1 and M2 (demethyleneberberine), with fragment ions at m/z 322 and m/z 324 according to an LC/MS/MS identification, were formed during incubation of berberine with rat liver microsomes in the presence of NADPH. Incubation of M2 standard in the rat liver microsomes in the presence of UDPGA led to formation of glucuronide M3, with fragment ions at m/z 500 according to the LC/MS/MS identification. We could not obtain sufficient standards of these metabolites, so semiquantitative concentrations of M1 were deduced according to the accompanying calibration curve of berberine; the semiquantitative concentration of M3 was calculated according to the accompanying calibration curve of M2. As shown in Fig. 4, pretreatment with 0.32 g/kg of Fructus evodiae for two weeks significantly decreased the phase I (P = 0.016) metabolism of berberine to M1. On the other hand, both 0.16 g/kg and 0.32 g/kg of Fructus evodiae significantly increased the phase II metabolism of M2 to M3 (P = 0.021 for 0.16 g/kg, P = 0.027 for 0.32 g/kg).
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Table 1 Pharmacokinetic parameters of berberine in rats received the oral extract of Rhizoma coptidis with or without the pretreatment with Fructus evodiae (mean ± SD, n = 5). Parameters
RC (2.23 g/kg) Control
AUC0–24 h (ng h/mL) MRT0–24 h (h) T1max (h) C1max (ng/mL) T2max (h) C2max (ng/mL) T3max (h) C3max (ng/mL)
410.1 11.4 1.1 50.5 5.2 23.5 16.8 32.6
± ± ± ± ± ± ± ±
RC (0.56 g/kg) FE (0.32 g/kg)
309.0 2.9 0.6 72.2 1.8 20.1 5.0 28.8
214.8 9.8 1.8 37.9 5.6 14.3 15.6 9.0
± ± ± ± ± ± ± ±
172.5 2.6 0.4 67.7 0.9 8.2 5.4 5.2
Control 282.7 13.0 0.3 3.9 6.4 3.8 18 38.1
± ± ± ± ± ± ± ±
FE (0.32 g/kg) 370.3 2.4 0.1 2.3 1.7 3.6 5.6 55.5
61.7 11.2 1.1 12.3 4.8 3.6 15.6 3.9
± ± ± ± ± ± ± ±
10.3 1.4 0.9 18.9 1.8 3.6 4.9 0.7
RC: Rhizoma coptidis; FE: Fructus evodiae. AUC, area under concentration–time curve; Cmax , maximum plasma concentration; MRT, mean residence time; Tmax , time to reach maximum plasma concentration.
Fig. 3. Effects of Fructus evodiae pretreatment on the absorption of the Rhizoma coptidis alkaloids in rat everted gut sacs (mean ± S.D, n = 6). After pretreatment of Fructus evodiae (0.08, 0.16, or 0.32 g/kg for two weeks), the rats were killed and everted gut sacs were prepared. Absorption of the Rhizoma coptidis alkaloids in the rat everted gut sacs was measured. Concentrations of berberine were determined using LC/MS/MS. FE: Fructus evodiae; L, M, and H: 0.08, 0.16, or 0.32 g/kg respectively; Ber: berberine; Cop: coptisine; Jat: jatrorrhizine; Pal: palmatine.
3.4. Influence of Fructus evodiae on the mRNA expression of UGT1A1 in rat livers Pretreatment with the three dosages of Fructus evodiae (0.08, 0.16, 0.32 g/kg) for two weeks significantly increased the hepatic mRNA expression of UGT1A1 in the rats (0.08 g/kg: P = 0.049, 0.16 g/kg: P = 0.018, 0.32 g/kg: P = 0.045; Fig. 5). 4. Discussion and conclusions Traditional Chinese medicines are usually prescribed as multiherb formulas. The combination of several herbs is believed to increase curative effects and to reduce side effects. Both synergy and antagonistic effects between TCMs components may be derived from herb–herb interactions. Similar to drug–drug interactions, herb–herb interactions may occur at pharmacokinetic level through modulation of absorption, metabolism, or elimination of active components. Studies on herb–herb interactions, especially herb–herb pharmacokinetic interactions, will aid in the elucidation of both synergistic and antagonistic interactions between herbs in
TCMs. Indeed, such data will help to modernization clinical applications of TCMs. Therapeutically, Rhizoma coptidis and Fructus evodiae are extracted and oral administered together. However, in this situation, interactions between these two TCMs are very complicated. Interactions also happen pharmaceutically and pharmacologically besides pharmacokinetically (Jia et al., 2009; Zhao et al., 2010). Therefore, in this study, to eliminate the influence of pharmaceutical interactions, we adopted the subsequent treatment methodological approach, which is not matching the therapeutical use but could also offer useful hints for further studies. The results showed that the pretreatment with 0.32 g/kg of the Fructus evodiae extract decreased the systemic exposure of berberine. In addition, C3max was significantly decreased. The pharmacokinetic interactions were due to enhancement of phase II glucuronidation by Fructus evodiae based on following results. Firstly, pretreatment with 0.32 g/kg of Fructus evodiae extract had no significant effect on the in vitro absorption of the Rhizoma coptidis alkaloids. Secondly, the in vitro metabolism experiments excluded the possibility that the effect of Fructus evodiae was
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Fig. 4. Effects of Fructus evodiae pretreatment on phase I metabolism of berberine and phase II metabolism of demethyleneberberine (M2) in rat liver microsomes (mean ± S.D, n = 6). After the Fructus evodiae pretreatment (0.08, 0.16, or 0.32 g/kg for two weeks), the rats were killed and liver microsomes were prepared. Phase I and phase II metabolism was studied in rat liver microsomes and analyzed using HPLC. M1, M2, and M3: metabolites at m/z 322, 324 (demethyleneberberine), and 500 respectively; L, M, and H: 0.08, 0.16, and 0.32 g/kg, respectively.
Fig. 5. Effect of Fructus evodiae pretreatment on UGT1A1 mRNA expression in rat liver (mean ± S.D, n = 3). Rats were treated with either saline or Fructus evodiae (0.08, 0.16, or 0.32 g/kg) for two weeks. Hepatic UGT1A1 mRNA expression was determined (A), and values expressed relative to the appropriate control group (B). C: Control; L, M, and H: 0.08, 0.16, and 0.32 g/kg, respectively. *P < 0.05 compared with control.
due to induction of phase I hepatic drug-metabolizing enzymes. Actually, the phase I metabolism of berberine to M1 was inhibited by the pretreatment with 0.32 g/kg of Fructus evodiae. Finally, both the in vitro metabolism and PCR experiments confirmed that phase II glucuronidation was enhanced by 0 .32 g/kg of the Fructus evodiae extract. Multiple cytochrome P450 isoenzymes (CYPs) were involved in the phase I metabolism of berberine, including CYP3A1/2, CYP2B, and CYP2D1 (Liu et al., 2009b). Pretreatment with Fructus evodiae was reported to increase the activities of CYP1A1, CYP1A2, and CYP2B in C57BL/6J mice (Ueng et al., 2002c). In addition, pretreatment of rutaecarpine, one of the major alkaloids of Fructus evodiae, reportedly increased protein levels of both CYP1A1 and CYP1A2 in
liver without altering hepatic protein levels of CYP3A (Ueng et al., 2001). To our knowledge, this is the first time that an inhibitory effect of Fructus evodiae on phase I metabolism of berberine has been reported. Phase II glucuronidation metabolism was demonstrated to be one of the major elimination pathway for the Rhizoma coptidis alkaloids (Yang et al., 2010), indicating that phase II glucuronidation metabolism was crucial to systemic exposure of the Rhizoma coptidis alkaloids. In the in vitro phase II metabolism experiments, we only tested incubation of the M2 standard since M1 standard was not available in sufficient quantities. It was reported, however, that both M1 and M2 were mainly catalyzed by UGT1A1 (Liu et al., 2009b), suggesting that the incubation of the M2 standard reflects the metabolism capability of UGT1A1 in rat liver microsomes. Our results suggested differential effects of Fructus evodiae on phase I and phase II hepatic drug metabolism enzymes, and that the enhancement of phase II was more important than any change in phase I in the systemic exposure of the Rhizoma coptidis alkaloids. After oral administration of the Rhizoma coptidis extract, the Rhizoma coptidis alkaloids were widely distributed into various tissues, including liver, heart, kidney, lung, and brain (Ma et al., 2010). Tissue concentrations of the Rhizoma coptidis alkaloids increase nonlinearly with higher doses and finally cause acute toxicity (Ma et al., 2010). The median lethal dose (LD50 ) of the oral Rhizoma coptidis extract was 2.95 g/kg in mice (Ma et al., 2010). Traditionally, Fructus evodiae is prescribed with Rhizoma coptidis at certain ratio. For example, the ratio of Fructus evodiae to Rhizoma coptidis is 1:6 (calculated by the weight of the dried herbal pieces) in Zuojin Wan. Therefore, for toxic dosage of Rhizoma coptidis, the dosage of the combined Fructus evodiae should be higher than 0.3 g/kg. In this situation, Fructus evodiae would influence the final in vivo activities of Rhizoma coptidis by influencing the pharmacokinetics of the Rhizoma coptidis alkaloids. In fact, although the detailed mechanism involved remained to be studied, the oral Fructus evodiae extract pretreatment decreased the acute toxicity of Rhizoma coptidis in mice (data not shown). However, for conventional clinical application, our results did not suggest that the oral Fructus evodiae would reduce the curative effects of the combined Rhizoma coptidis based on pharmacokinetic influences. The clinical conventional dosage of Fructus evodiae extract is 0.47–1.42 g per adult in China. Thus, the equivalent surface area dosage of rat is about 0.05–0.14 g/kg. In this study, 0.08 g/kg of the oral Fructus evodiae pretreatment increased the absorption of the Rhizoma coptidis alkaloids. This result was consistent with what was reported (Sun et al., 2008; Wang et al., 2009). All the Rhizoma coptidis alkaloids are substrates of p-glycoprotein (P-gp) (Zhang et al., 2011), a well known drug efflux pump, and the extract of Fructus evodiae reportedly showed significant inhibitory effects on P-gp (Yoshida et al., 2005). Hence, Fructus evodiae might increase the absorption of the Rhizoma coptidis alkaloids by inhibiting the intestinal P-gp. Furthermore, although 0.08 g/kg of the oral Fructus evodiae pretreatment showed weak induction on the expression of UGT1A1, it did not show significant effects on both phase I and phase II metabolism of berberine. Our results indicated that different dosages of Fructus evodiae resulted in different pharmacokinetic influences on the Rhizoma coptidis alkaloids. In contrast to pure compound drugs used in Western medicine, TCMs consist of a multitude of constituents (some still not described) that differ in terms of herb content and quality. Thus, different dosages of TCM result in different dosages of the active constituents and significantly different outcomes. The detailed mechanism involved, however, has yet to be elucidated. In conclusion, the results showed that Fructus evodiae pretreatment decreased the systemic exposure of the Rhizoma coptidis alkaloids by inducing hepatic UGT1A1.
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