Baicalin (purity, >90.0%) was supplied form Tokyo Chemical Industry. Baicalein, caffeine, paraxanthine, theobromine, theophylline, ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, resorufin, p-nitrophenol, erythromycin, glucose-6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Sigma (St. Louis, MO, USA). The reduced form of β-nicotinamide adenine dinucleotide phosphate (β-NADPH) was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan), and methanol and acetonitrile were HPLC-grade from J.T. Baker (Central Valley, PA, USA). All other chemicals were of analytical grade and used as received.

Male Sprague-Dawley (SD) rats (7 weeks, 240–270 g) were obtained from Samtako Bio Korea, and randomized and housed three per cage. The animal room was maintained at a temperature of 22 ± 2°C, relative humidity of 50 ± 10% with 10–20 air changes/h, and light intensity of 150–300 Lux with a 12-hr light/dark cycle. This study was approved by the Yeungnam University Animal Care and Use Committee (approved No., 2014-008). All animals used in this study were cared in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Rats were pretreated with either 3-methylcholanthrene (CYP1A inducer), phenobarbital sodium (CYP2B inducer), dexamethasone (CYP3A inducer) or acetone (CYP2E1 inducer). Dexamethasone and 3-methylcholanthrene suspended in corn-oil were intraperitoneally given to rats at a dose of 50 mg/kg and 40 mg/kg for 3 consecutive days, respectively. Phenobarbital sodium dissolved in saline was also intraperitoneally administered to rats at a dose of 80 mg/kg for 3 days. Acetone was given once only to rats by oral administration at 5 mL/kg. Two days after acetone administration, rats were sacrificed to remove livers. Then, rat liver microsomes were prepared as described previously (Kim et al., 2014). In brief, livers removed from each treatment group were homogenized with four volumes of ice-cold 0.1 M potassium phosphate buffer (pH 7.4). The liver homogenates were centrifuged at 9,000×g for 20 min at 4°C, and the resulting supernatants were centrifuged again at 105,000×g for 60 min at 4°C. Then, the microsomal pel-lets were resuspended in 0.1 M potassium phosphate buffer, pH 7.4, containing 20% glycerol, and aliquots were stored at −70°C until use. The content of protein in rat liver microsomes was determined using bovine serum albumin as a standard (Lowry et al., 1951).

Ethoxyresorufin O-deethylase (EROD) activity was determined, as previously described, with a slight modification (Blank et al., 1987). The reaction mixture (1 mL) consisted of 0.1 M potassium phosphate buffer, pH 7.4, containing 2 mg/mL of bovine serum albumin, 5 mM glucose-6-phosphate, 1 U of glucose-6-phosphate dehydrogenase, 5 μM β-NADPH, and 2.5 μM 7-ethoxyresorufin as a substrate. The formation of resorufin was fluorometrically measured at λEx/λEm of 550/585 nm. Methoxyresorufin O-demethylase (MROD) and benzyloxyresorufin O-debenzylase (BROD) activities were determined by the method of Lubet et al. (1985) with a slight modification. The reactions were conducted under the same condition for EROD, except that the substrates were used 2.0 μM methoxyresorufin and benzyloxyresorufin for MROD and BROD, respectively. p-Nitrophenol hydroxylase (PNPH) activity was determined as described previously (Koop, 1986). The reaction mixture (1 mL) consisted of 0.1 M potassium phosphate buffer, pH 7.4, containing 100 μM p-nitrophenol, 1 mM NADPH, and an enzyme source. The reaction was terminated by adding 0.6 N perchloric acid, and the amount of 4-nitrocatechol formed was measured spectrophotometrically at λmax of 512 nm. Erythromycin N-demethylase (ERDM) activity was determined by measuring the amount of formaldehyde formed, as described previously (Nash, 1953). Erythromycin at 400 μM was used as a substrate. The reaction mixture (1.5 mL) consisted of 0.1 M potassium phosphate buffer, pH 7.4, containing 3 mM glucose-6-phosphate, 1 U glucose-6-phosphate dehydrogenase, 0.8 mM β-NADPH, 7.5 mM semicarbazide, and 5.0 mM MgCl₂. The reaction was terminated by adding 20% trichloroacetic acid, and the amount of formaldehyde formed was measured spectrophotometrically at λmax of 412 nm.

Rats were fasted overnight, and divided into two groups (n=5). Baicalin was suspended in corn oil and vortexed before treatment. Either vehicle (corn oil at 10 ml/kg) or baicalin (200 mg/kg/10 ml corn oil) were administered orally 8 hr prior to the caffeine administration, because oral baicalin reached its maximum concentration in plasma 8 hr after administration (Lu et al., 2007). Caffeine was given to rats by an oral administration at 1 mg/kg. Blood samples (200 μL), collected into heparinized centrifuge tubes, were taken at 5 min before and 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 14 and 24 hr after caffeine administration via subclavian vein. After centrifugation at 10,000×g for 5 min, the plasma was separated. The obtained plasma samples were stored at −70°C until analysis.

Sixty microliter of methanol containing 1 ng/mL methaqua-lone (internal standard, IS) was added to 20 μL of plasma sample. After vortexing for 30 sec, plasma samples were centrifuged at 10,000×g for 10 min. Then, the supernatant was transferred into a vial, and 5 μL of supernatant was injected for analysis.

Plasma concentrations of caffeine and its three metabolites, paraxanthine, theobromine, and theophylline, were determined by a previously reported method with some modifications (Choi et al., 2013). Caffeine and its metabolites were analyzed by using an HPLC (1260 system, Agilent) with mass spectrometry (API-4000, AB SCIEX). Analytes were separated using ZORBAX Bonus-RP column (2.1×150 mm, 5 μm, Agilent). The mobile phase was composed of 0.2% formic acid (A) and methanol (B), and eluted with a gradient condition as follows: initially at 90% mobile phase A; from 90% mobile phase A to 5% mobile phase A from 0.5 to 4 min; 5% mobile phase A holding for 2 min (4–6 min); from 5% mobile phase A to 90% mobile phase A during 6–7 min; and 90% mobile phase A holding for 7 min (7–14 min). The flow rate and temperature were maintained at 0.4 mL/min and 35°C during analysis, respectively. Caffeine, paraxanthine, theobromine, theophylline and IS were detected in the positive ion mode. Mass transitions used in the analysis were m/z 195.2→138.1 for caffeine, m/z 181.1→124.0 for paraxanthine and theophylline, m/z 181.1→138.1 for theobromine, and m/z 251→132.1 for IS. Quantitative analysis for caffeine and its metabolites were performed by multiple reaction monitoring of the precursor ion and the related product ion using the ratio of area under the peak for each sample.

Plasma concentration of baicalin was determined by using same instruments above mentioned. Atlantis T3 column (2.1×150 mm, 3 μm, Waters) was used for separation, and column oven was maintained at 40°C. The mobile phase was composed of 0.1% formic acid (A) and acetonitrile (B), and eluted with a gradient condition as follows: initially at 75% mobile phase A; from 75% mobile phase A to 10% mobile phase A from 0 to 2 min; 10% mobile phase A holding for 6 min (2–8 min); from 10% mobile phase A to 75% mobile phase A from 8 to 10 min; and 75% mobile phase A holding for 8 min (10–18 min). The flow rate was maintained at 0.2 mL/min during analysis. Baicalin was detected in the positive ion mode, and mass transition used in the analysis was m/z 447.1→271.1. The mass transition for IS was the same as in the analytical method for caffeine and its metabolites. Quantitative analysis for baicalin was performed by multiple reaction monitoring of the precursor ion and the related product ion using the ratio of area under the peak for each sample.

Pharmacokinetic parameters of caffeine and its metabolites were obtained from time course plasma concentrations and the peak area ratios of analyte to IS in rats. Standard methods were used to calculate the following pharmacokinetic parameters by using non-compartmental analysis (WinNonlin; version 2.1; Scientific Consulting): maximum observed plasma concentration (C_max), time of maximum observed plasma concentration (T_max), area under the plasma concentration-time curve from the time of dosing extrapolated to infinity (AUC_∞), apparent volume of distribution based on the terminal phase (V_d/F) and terminal half-life (t_1/2). AUC was calculated using the trapezoidal rule-extrapolation method (Chiou, 1978). The pharmacokinetic parameters were expressed as mean ± SD obtained from 5 rats (Noh et al., 2011). The statistical significance of the results was analyzed using Student’s t-test, with p-values of less than 0.05 considered statistically significant.

The half maximal inhibitory concentration (IC₅₀) of baicalin and baicalein on CYP enzymes were shown in Table 1. Baicalin inhibited EROD, MROD and BROD activities in rat liver microsomes with the IC₅₀ values of 24.2, 9.3 and 22.9 μM, respectively. Baicalein also showed inhibitory effects on EROD, MROD and BROD activities. Especially, the inhibition was much more potent on EROD and MROD, when compared to the inhibition by baicalin. There were no inhibitory effects on PNPH and ERDM activities by neither baicalin nor baicalein.

Subsequently, possible drug interaction of baicalin with caffeine was studied in male SD rats, because baicalin and baicalein were inhibitory on CYP 1A activity in rat liver microsomes. Baicalin was administered orally, because baicalin could be metabolized to baicalein, the aglycone form, in the intestine by intestinal microbiota prior to absorption (Kang et al., 2014). Time-concentration profiles of caffeine and its three metabolites in rats pretreated with or without baicalin were depicted in Fig. 2, and the relevant pharmacokinetic parameters of them were presented in Table 2. In control group, caffeine reached to the C_max of 0.35 ± 0.10 mg/L at 0.40 ± 0.14 hr, and estimated AUC_∞ and t_1/2 were 0.87 ± 0.51 mg·hr/L and 1.37 ± 0.94 hr, respectively. The C_max of metabolites were 0.25 ± 0.05 mg/L for paraxanthine, 0.16 ± 0.02 mg/L for theobromine, and 0.10 ± 0.02 mg/L for theophylline, respectively, and the highest concentrations of them were achieved at 2.20–2.80 hr after caffeine administration. The obtained AUC_∞ and t_1/2 of paraxanthine, theobromine and theophylline were 1.13 ± 0.24 mg·hr/L and 1.69 ± 0.63 hr, 1.12 ± 0.32 mg·hr/L and 3.13 ± 1.46 hr, and 0.70 ± 0.20 mg·hr/L and 2.79 ± 1.24 hr, respectively.

In baicalin-pretreated rats, the pharmacokinetic parameters of caffeine and its three metabolites, paraxanthine, theobromine, and theophylline, were comparable with those in the control group, and there were no significant differences of pharmacokinetic parameters between groups, as shown in Table 2. The present results indicated that baicalin might not interact with caffeine in vivo, although baicalin could inhibit some CYP enzyme activities in vitro.

Because baicalin at 200 mg/kg did not show any drug interaction with caffeine, the concentration of plasma baicalin was determined to explain the reason for inconsistency. Based on the dosing schedule for drug interaction study in Fig. 2, time-concentration profile of baicalin from 8 hr after oral administration of rats with 200 mg/kg baicalin were obtained, as depicted in Fig. 3. The plasma concentrations of baicalin were highly variable. The mean maximum concentration of baicalin was calculated to be 3.7 μM, which was below IC₅₀ values for EROD and MROD for baicalin that showed inhibition. The results clearly explained the reason why baicalin might not interact with caffeine in rats, and indicated that baicalin at the dose tested in the present study would be safe in terms of the possible drug interaction with certain drugs that are CYP1A2 and 2E1 substrates.