Pharmacokinetics of Cajaninstilbene Acid and Its Main Glucuronide

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Pharmacokinetics of Cajaninstilbene Acid and Its Main Glucuronide Metabolite in Rats Li-Sha Wang, Xue Tao, Rui-Le Pan, Fang-Rui Cao, Li Feng, Yong-Hong Liao, Xin-Min Liu, and Qi Chang* Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, People’s Republic of China ABSTRACT: As a major active stilbene from the leaves of pigeon pea (Cajanus cajan), cajaninstilbene acid (CSA) exerts various pharmacological activities. The present study aimed to investigate the pharmacokinetics of CSA and one of its main metabolites (M1) to explore their fate in the body and provide a pharmacokinetic foundation for their in vivo biological activities and functional food or complementary medicine application. M1 was characterized as CSA-3-O-glucuronide using the multiple reaction monitoring−information-dependent acquisition−enhanced product ion technique. After oral and intravenous administration, plasma, urine, and bile were collected and analyzed to estimate pharmacokinetic properties of CSA and M1 and to explore the main excretion route. The oral bioavailability of CSA was estimated to be 44.36%. This study first reported that CSA is mainly metabolized to CSA-3-O-glucuronide via the first-pass effect to limit its oral bioavailability and excreted predominantly through the biliary route, while the enterohepatic circulation, extravascular distribution, and renal reabsorption characteristics of CSA might delay its elimination. KEYWORDS: cajaninstilbene acid, pharmacokinetics, bioavailability, glucuronide metabolite

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INTRODUCTION Pigeon pea [Cajanus cajan (L.) Millsp.], belonging to the family Leguminosae, is a well-known perennial grain legume crop that is widely distributed in the semi-arid tropics and subtropics,1 and it occupies a major position in human diets as a protein source.2 In addition to its nutritional value, pigeon pea is also used as an integral part of traditional folk medicine in India, China, and other developing countries.3 Several parts of pigeon pea, especially its leaves, are known to prevent and cure certain human ailments. Various medicinal values have been confirmed for pigeon pea leaves, such as the treatment of trauma, burn infection, bedsores, and aphthous ulcers.4 The extracts of pigeon pea leaves exhibit therapeutic effects on inflammation,5 diabetes,6 ovariectomy-induced bone loss,7 hypercholesterolemia,8 and Staphylococcus aureus infections.9 The bioactive constituents in pigeon pea leaves are stilbenes and flavonoids, such as cajaninstilbene acid (CSA), longistylins A and C, luteolin, and apigenin.10−12 Among them, CSA, 3hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid, has been recognized as one of the fundamental active components of pigeon pea leaves (Figure 1). Pharmacological studies have

revealed that CSA possesses many bioactivities, including antioxidant properties,13 antibacterial activity against Grampositive bacteria,10 therapeutic potential against breast cancer,14 cytoprotective effects,15 neuroprotective activity in PC12 cells through the inhibition of oxidative stress and the endoplasmic reticulum (ER) stress-mediated pathway,16 relaxation in renal arteries via partial inhibition of calcium entry, 17 antiinflammatory effects,18 and analgesic effects and the inhibition of capillary permeability.4 On the basis of these pharmacological activities, CSA could be a novel functional food or complementary medicine candidate for human health in the future. However, most of the pharmacological properties of CSA were elucidated on cell lines in vitro. Therefore, it is urgent to focus on the pharmacokinetic and bioavailability studies of CSA to provide evidence of in vivo biological activities. Only one publication has reported the pharmacokinetics of CSA in female Kunming rats after oral administration thus far.19 To our knowledge, the metabolism, oral bioavailability, and excretion of CSA have not been clarified. The primary objective of this study was to investigate the metabolism, absorption, and excretion of CSA to explore its fate in the body and provide the pharmacokinetic foundation for its in vivo biological activities and functional food or complementary medicine application. Received: Revised: Accepted: Published:

Figure 1. Chemical structures of CSA and 4-methoxycinnamic acid (IS). © 2017 American Chemical Society

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February 17, 2017 May 5, 2017 May 9, 2017 May 9, 2017 DOI: 10.1021/acs.jafc.7b00743 J. Agric. Food Chem. 2017, 65, 4066−4073

Article

Journal of Agricultural and Food Chemistry

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chromatography−ultraviolet−tandem mass spectrometry (LC−UV− MS/MS) system for metabolite identification. LC−UV−MS/MS Analysis. A metabolite identification analysis was conducted using a QTRAP 4500 mass spectrometer (AB SCIEX, Framingham, MA, U.S.A.) connected to an Agilent 1260 Infinity liquid chromatography system (Agilent Technologies, Santa Clara, CA, U.S.A.) and a 1260 Infinity diode array detector. The chromatographic conditions were the same as mentioned in the HPLC Assay section, and the eluent was finally introduced into the mass spectrometer via an electrospray ionization (ESI) ion source operating in negative ion polarity mode. Nitrogen was used as the nebulizing, curtain, and collision gases. The optimal source parameters, including ion spray voltage, temperature, curtain gas, gas 1, and gas 2, were established at −4500 V, 450 °C, and 25, 40, and 50 psi, respectively. The declustering potential (DP), entrance potential (EP), collision energy (CE), and collision exit potential (CXP) of authentic CSA were optimized using a syringe infusion pump as −60, −10, −40, and −15 V, respectively. The intense precursor-to-product ion transitions (Q1 mass/Q3 mass) of CSA were m/z 337.1/293.1, 337.1/223.1, and 337.1/235.1. Multiple reaction monitoring−information-dependent acquisition− enhanced product ion (MRM−IDA−EPI) measurements were performed to identify the metabolites of CSA. MRM channels of the predicted metabolites were generated by Analyst software based on the most common phase I and II biotransformation pathways and the three original MRM transitions of CSA with a dwelling time of 40 ms for each transition. The IDA threshold was set at 1000 counts per second for the MRM experiment, above which the EPI scan would be triggered. The EPI spectra were obtained over a range from m/z 100 to 800 at a scan rate of 1000 Da/s. The same DP, EP, and CXP values optimized for CSA were used for the MRM transitions of the predicted metabolites. The CE values were set at −40 V with a collision energy spread of 15 V. Dynamic fill time was used to prevent ions from overfilling the ion trap. The UV spectral data were collected from 190.00 to 400.00 nm. Data acquisition and analysis were performed with the Analyst 1.6.2 software (AB SCIEX, Framingham, MA, U.S.A.). Quantitative Determination of CSA and M1. Sample Preparation. An aliquot of 100 μL of plasma sample after oral and iv dose was spiked with 20 μL of IS solution (1000 ng/mL) and then vortex-mixed with methanol (400 μL). After centrifugation at 13800g for 10 min at 4 °C, the supernatant was transferred to a glass tube and evaporated to dryness at 40 °C using a vacuum concentrator (Labconco, Kansas, City, MO, U.S.A.). The residue was reconstituted in 100 μL of 80% methanol aqueous solution by vortex mixing for 1 min. Then, the mixture was centrifuged at 13800g for 10 min at 4 °C, and 15 μL of the supernatant was injected into HPLC for analysis. The bile sample (100 μL; 20 μL of bile at 0−4 h after iv dosing was added to 80 μL of blank bile) diluted with an equal volume of 80% methanol aqueous solution and urine sample (100 μL) were spiked with IS solution (20 μL, 1000 ng/mL for bile sample and 500 ng/mL for urine sample) and vortex-mixed. After centrifugation at 13800g for 10 min at 4 °C, 15 μL of the supernatant was injected into the HPLC system for analysis. The extent of urinary and biliary excretion was calculated on a molar basis by dividing the amount of CSA excreted in biological fluids by the amount of dose administered. HPLC Assay. Chromatographic separation was achieved on an Alltima HP C18 column (100 × 2.1 mm, 3 μm, Grace, Columbia, MD, U.S.A.) at 35 °C using a Waters HPLC system (Waters, Milford, MA, U.S.A.) equipped with an e2695 Separations Module and a 2489 UV/ visible detector.21 The mobile phase comprised acetonitrile (A) and water (B), both containing 0.5% formic acid with subsequent gradient elution at a flow rate of 0.4 mL/min: 0−4 min, 20% A; 4−17 min, 20− 45% A; 17−27 min; 45−80% A; 27−30 min; 80% A; 30−30.1 min; 80−20% A; and 30.1−38 min, 20% A. The wavelength of UV detection was set at 320 nm. The CSA and M1 concentrations were calculated using a CSA standard curve because of their similar molar extinction coefficients.22 Pharmacokinetic Data Analysis. The plasma concentration versus time profile of CSA obtained from each individual rat was submitted to

MATERIALS AND METHODS

Chemicals and Reagents. CSA [98.0% purity by high-performance liquid chromatography (HPLC)] (Figure 1) was separated from the leaves of C. cajan in our laboratory.20 As an internal standard (IS), 4-methoxycinnamic acid [≥98.0%, gas chromatography (GC) grade] (Figure 1) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Acetonitrile, methanol, and ethyl acetate of HPLC grade were obtained from Fisher Co., Ltd. (Waltham, MA, U.S.A.). Formic acid and other reagents of analytical grade were purchased from Beijing Chemical Reagent Company (Beijing, China). Milli-Q (Millford, MA, U.S.A.) water was used for all experiments. Animals. Healthy male Sprague Dawley rats weighing 240 ± 20 g were used and obtained from Vital River Experimental Animal Co., Ltd. (Beijing, China). Before the experiment, the rats had free access to a standard rodent diet and water under controlled temperature, humidity, and light. Before drug administration, the rats were surgically implanted with a polyethylene catheter (0.50 mm inner diameter, 1.00 mm outer diameter, Portex, Ltd., Hythe, U.K.) under anesthesia by a single intraperitoneal injection of chloral hydrate at 350 mg/kg. The catheter was threaded into the right jugular vein and fixed by sutures at the nape. To ensure patency, the catheter was flushed with heparinized saline (20 units/mL). For bile collection, another polyethylene catheter (0.28 mm inner diameter, 0.61 mm outer diameter, Portex, Ltd., Hythe, U.K.) was also surgically cannulated with the bile fistulas of the rat under anesthesia. After the surgery, the rats were allowed to recover for 24 h in metabolism cages individually and fasted overnight prior to drug dosing. All animal procedures were conducted in accordance with the protocols approved by the Animal Ethical Committee at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. Drug Administration and Sample Collection. The CSA dosing drug was freshly prepared at the concentration of 1 mg/mL in 10% (w/v) Solutol HS 15 (BASF, Ludwigshafen, Germany) in saline. For Plasma Collection. Two groups of rats (n = 6 for each group) implanted with only a jugular vein catheter received a single oral (10 mg/kg) or intravenous (iv, 2 mg/kg, through the jugular catheter) dose of CSA. Blood was collected according to the following schedule: 0 (pre-dose), 5, 10, 15, and 30 min and 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, and 84 h post-dose. Blood samples (200 μL) were withdrawn from the jugular catheter into heparinized tubes and immediately centrifuged at 1500g for 10 min at 4 °C. The separated plasma samples (100 μL) were stored at −20 °C until the assay. After drug administration and each blood collection, 200 μL of heparinized saline (20 units/mL) was immediately injected back into the body to flush the catheter for patency. Two other jugular-vein-cannulated rats received a single oral (10 mg/kg) dose of CSA. All blood was collected at 30 min post-dose and immediately centrifuged. This separated plasma was mixed and stored at −20 °C until the identification assay. For Urine and Bile Collection. Another group of rats (n = 5) implanted with jugular vein and bile-duct catheters received CSA by iv injection through the jugular vein catheter at 2 mg/kg. Urine and bile samples were collected before dosing and at the following time intervals: 0−4, 4−8, 8−12, and 12−24 h post-dose. At the end of each collection, the metabolism cages were rinsed with distilled water. The final volume of the diluted urine sample was adjusted to 15 mL. A bile sample was collected from the cannula into a polypropylene tube and diluted with water to make up the volume of 6 mL. All samples were stored at −20 °C until analysis. Identification of CSA Main Metabolite (M1). Sample Preparation. An aliquot of 2 mL of plasma (at 30 min), urine (over 0−4 h), or bile (over 0−4 h) sample was vortex-mixed with methanol (8 mL). After centrifugation at 13800g for 10 min at 4 °C, the supernatant was transferred to a glass tube and evaporated to dryness at 40 °C using a vacuum concentrator. The residue was reconstituted in 200 μL of 80% methanol aqueous solution by vortex mixing for 1 min. Then, the mixture was centrifuged at 18800g for 15 min at 4 °C, and 15 μL of the supernatant was injected into a liquid 4067

DOI: 10.1021/acs.jafc.7b00743 J. Agric. Food Chem. 2017, 65, 4066−4073

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Journal of Agricultural and Food Chemistry a non-compartmental model using WinNonlin software (Pharsight Corporation, Mountain View, CA, U.S.A., version 6.1). The following pharmacokinetic parameters were calculated: the initial plasma concentration (C0) for the iv dose, the maximum plasma concentration (Cmax) and the time to Cmax (Tmax) for the oral dose, the terminal elimination half-life (t1/2,λz), the area under the plasma concentration versus time curve from zero to the last sampling time (AUC0−t) and to infinity (AUC0−∞), the total body clearance (CL), the mean residence time (MRT), the volume of distribution (Vd,λz), and the volume of distribution at steady state (Vss). The absolute oral bioavailability (F) of CSA was calculated by the equation F = (AUC0−∞,oral × doseiv)/ (AUC0−∞,iv × doseoral) × 100%. All data are presented as the mean ± standard deviation (SD).

are identical to those of the authentic CSA (Figure 2). Taken together, these data showed that M1 was identified as CSA-3O-glucuronide. M1 was abundant in the three types of samples (plasma, urine, and bile) (bottom plots in Figure 3), especially in plasma. Thus, the present study focused on the pharmacokinetics of CSA and M1. Furthermore, many other unique peaks, not just M1, were clearly observed in the urine and bile samples after oral administration by comparing the blank and CSA-dosed samples. They could also be identified as CSA metabolites and require a further detailed study for the metabolism pathway of CSA. HPLC Method Development and Optimization. To attain satisfactory sensitivity in the HPLC assay, the UV spectra of CSA were monitored and showed two absorption peaks at approximately 259 and 303 nm. The wavelength at 259 nm, the maximum efficacy absorption, was originally selected for the analysis. However, the unstable chromatographic baseline at this wavelength sacrificed the sensitivity of CSA. Hence, the UV results obtained at the wavelengths of 259, 303, 320, and 340 nm were rigorously reviewed. Despite a certain decrease in the peak intensity of CSA as the wavelengths increased, the peak intensity of CSA at 320 nm at low concentrations (29.58−59.16 nM) was superior to that of others because of the stable baseline and a relatively high intensity. Considering that both CSA and M1 could also produce a relatively strong signal at 320 nm, this wavelength was selected for quantification. In this study, the critical effect of the composition and pH of the mobile phase on separation was examined to optimize the chromatographic conditions. Acetonitrile was chosen in the organic phase as a result of its low viscosity, which reduced the back pressure and achieved a slightly better peak shape. Because CSA, M1, and IS are weak acid compounds, the impact of adding 0.0, 0.1, 0.2, 0.4, and 0.5% formic acid to the mobile phase was evaluated. The addition of 0.5% formic acid to the mobile phase resulted in a good resolution of these compounds as well as satisfactory peak symmetry and shape. To achieve resolution of CSA ad M1, a slow gradient elution was performed (Figure 3). In the final developed condition, CSA, M1, and IS in the biological samples after CSA dosing had better resolution and could be completely separated within 38 min on a reversed-phase HPLC column. Plasma Sample Preparation. Different solvents for plasma sample preparation were tested and compared, including methanol for protein precipitation and ethyl acetate for liquid−liquid extraction. The absolute extraction recoveries of CSA (4437.00 nM) and IS from plasma treated by the two solvents were very similar, 88.41 and 83.48% for methanol and 88.07 and 82.24% for ethyl acetate, respectively, However, for M1, the extraction efficiencies were quite different using the two solvents, which were compared by peak area in the same plasma sample obtained from a rat after oral administration of CSA. After extraction with ethyl acetate or methanol, the organic layer or supernatant was collected and evaporated to dryness and then reconstituted in the same volume (100 μL) of 80% methanol aqueous solution. The peak area of M1 extracted with methanol was larger than that with ethyl acetate. This might be caused by the higher solubility of glucuronide metabolites in a polar solvent than in a nonpolar solvent. The quantitation of M1 should be underestimated if ethyl acetate was used. Considering the extraction efficiency of both CSA and M1, methanol was finally chosen as the extraction solvent.

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RESULTS AND DISCUSSION Identification of M1. CSA could be detected in the plasma, urine, and bile samples of rats after dosing; however, it existed in the body mainly as M1 rather than as its parent form. The chemical structure of M1 was identified using the MRM−IDA− EPI technique as described below. CSA (at 25.0 min) had a precursor mass of m/z 337.1 [M − H]−. Its product ion spectrum (Figure 2A) yielded a diagnostic

Figure 2. MS/MS product ion mass spectra of (A) CSA and (B) M1 by performing the MRM−IDA−EPI analysis of the plasma sample of a rat after oral administration of CSA.

fragment ion at m/z 293.1 resulting from the loss of a carboxyl group. Other typical fragment ions were at m/z 223.1, 235.1, 238.1, and 277.1. This is in accordance with the fragment pathway of CSA reported in an earlier study.23 M1 displayed a [M − H]− ion at m/z 513.2 and a retention time of approximately 16.7 min, which was 176 Da higher than that of CSA (Figure 2B). Its MS2 product ion spectrum showed a dominant fragment ion at m/z 337.1 resulting from the neutral loss of 176 Da, suggesting that M1 was a glucuronide conjugate of CSA. A fragment ion at m/z 469.1 was obtained by carboxyl group removal from the benzene ring of M1, indicating that glucuronide was conjugated with the 3-hydroxyl group of CSA. This was further confirmed by the observation of the fragment ions at m/z 223.1, 235.1, 238.1, 277.1, and 293.1 of M1, which 4068

DOI: 10.1021/acs.jafc.7b00743 J. Agric. Food Chem. 2017, 65, 4066−4073

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Figure 3. Representative HPLC chromatograms of the (A) plasma, (B) urine, and (C) bile samples: (top plots) blank biological samples, (middle plots) blank biological samples spiked with CSA at 2958 nM and IS, and (bottom plots) plasma obtained from a rat at 30 min after oral administration of CSA (10 mg/kg) spiked with IS and urine and bile obtained from a rat over 0−4 h post-iv dosing of CSA (2 mg/kg) spiked with IS.

Table 1. Intra- and Interday Precision and Accuracy, Extraction Recovery of the HPLC Method for the Determination of CSA in Rat Plasma, and the Stability of CSA in Prepared Plasma at 4 °Ca intraday

a

interday (3 days)

stability (% of initial concentrations)

concentration (nM)

precision (RSD, %)

accuracy (%)

precision (RSD, %)

accuracy (%)

103.53 1035.30 4437.00 44370.00

4.02 1.53 0.58 0.40

99.56 98.15 99.54 98.70

5.39 2.91 1.90 1.05

110.67 98.26 98.97 100.69

recovery (%) 81.70 87.84 88.41 88.57

± ± ± ±

3.67 0.58 0.49 0.98

12 h 98.24 101.93 99.57 100.05

± ± ± ±

24 h 4.60 1.44 0.52 0.25

100.26 99.63 100.34 100.91

± ± ± ±

48 h 3.69 1.59 0.81 0.42

100.14 100.54 99.98 99.48

± ± ± ±

2.85 1.75 0.73 0.33

Data are expressed as the mean ± SD (n = 5).

87.84 ± 0.58, 88.41 ± 0.49, and 88.57 ± 0.98%, respectively. The method showed good reproducibility with intraday (n = 5) and interday (n = 5, for 3 days) precision less than 5.39% and accuracy ranging from 98.15 to 110.67%. The LOD (the concentration at a signal-to-noise ratio of 3) and LOQ (the concentration at a signal-to-noise ratio of 10, with precision less than 20% and accuracy between 80 and 120%) were 29.58 and 59.16 nM, respectively. CSA remained stable in the prepared samples for 48 h at 4 °C. In addition, the absolute extraction recovery of the IS samples was found to be 83.48 ± 1.98%, and IS was stable in the prepared samples for 48 h at 4 °C, with the stability of 103.37 ± 1.06%. Therefore, the established HPLC method has good sensitivity and reproducibility for the determination of CSA, and it can successfully investigate the pharmacokinetic behaviors of CSA. Pharmacokinetics of CSA and M1 in Plasma. The mean plasma concentration versus time profiles and the principal pharmacokinetic parameters of CSA after oral and iv administration are described in Figure 4 and Table 2. After iv dosing, the plasma concentration of CSA rapidly declined and

Method Validation. Before the pharmacokinetic study, the established HPLC method for the quantitation of CSA concentrations in the biological samples was validated in terms of specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), inter- and intraday accuracy and precision, extraction recovery, and stability. Representative chromatograms of the plasma, urine, and bile samples are shown in Figure 3. The analytical method was specific and selective. All chromatograms were free from any interference at the retention times of CSA, M1, and IS, and they were eluted completely and appeared without peak tailing. The calibration curves of peak area ratios of CSA/IS versus CSA concentrations ranging from 59.16 to 2958.00 nM and from 2958.00 to 147 900.00 nM in plasma samples and from 59.16 to 5916.00 nM in urine and bile samples all had an excellent linearity, with correlation coefficients greater than 0.999. Table 1 shows the results of inter- and intraday precision and accuracy, recovery, and stability of the quality control samples consisting of CSA at 103.53, 1035.30, 4437.00, and 44 370.00 nM in blank plasma (n = 5). The absolute extraction recoveries were 81.70 ± 3.67, 4069

DOI: 10.1021/acs.jafc.7b00743 J. Agric. Food Chem. 2017, 65, 4066−4073

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Journal of Agricultural and Food Chemistry

Figure 4. (Top plots) Plasma concentration versus time profiles of CSA and M1 in rats after (A) iv (2 mg/kg) and (B) oral (10 mg/kg) administration of CSA, with each point representing the mean ± SD (n = 6) and (bottom plots) plasma concentration versus time profile of (C) CSA in an individual rat after iv administration of CSA (2 mg/kg) and (D) M1 in an individual rat after oral administration of CSA (10 mg/kg).

Table 2. Pharmacokinetic Parameters of CSA and M1 in Rats after Oral (10 mg/kg) and iv (2 mg/kg) Administration of CSAa oral dose parameter Tmax,1 (h) Cmax,1 (μM) Tmax,2 (h) Cmax,2 (μM) C0 (μM) t1/2,λz (h) AUC0−t (μM h) AUC0−∞ (μM h) Vd,λz/F (L/kg) Vss (L/kg) CL/F (L h−1 kg−1) MRT0−t (h) MRT0−∞ (h) F (%) a

iv dose

CSA 0.35 2.77 9.20 0.52

± ± ± ±

0.14 1.38 2.68 0.23

12.90 10.07 10.69 54.64

± ± ± ±

8.99 5.83 6.10 20.02

M1 1.10 83.23 16.67 53.41

± ± ± ±

CSA 0.55 27.84 8.16 10.20

15.40 ± 4.06 1980.47 ± 554.81 2069.35 ± 653.16

3.54 ± 1.75 12.98 ± 7.58 16.03 ± 7.92 44.36

22.83 ± 8.01 25.55 ± 10.82

M1 0.46 3.80 13.60 2.72

41.74 3.04 4.72 4.82 5.34 0.84 1.26 0.44 0.69

± ± ± ± ± ± ± ± ±

13.12 1.23 0.83 0.88 1.85 0.44 0.23 0.29 0.40

± ± ± ±

0.10 0.56 9.53 0.94

15.05 ± 5.78 78.25 ± 23.51 81.83 ± 26.87

20.95 ± 5.61 24.11 ± 7.44

All data are expressed as the mean ± SD (n = 6).

then remained at a relatively low level, with a Vd of 5.34 L/kg, t1/2,λz of 3.04 h, and CL of 1.26 L h−1 kg−1. As the plasma concentration of CSA decreased, M1 appeared rapidly in the plasma at 27.6 min to reach Cmax of 3.80 μM and was eliminated slowly with a t1/2,λz of 15.05 h, nearly 5-fold longer than that of CSA. Although interanimal variability limits any conclusion from the mean concentration versus time profile of CSA after iv administration (Figure 4A), a secondary absorption peak was observed in the CSA concentration versus time profiles from four of the six tested rats, typically at 4−6 h (see Figure 4C for a representative individual profile). Furthermore, in the elimination phase, a second peak plasma

level of M1 evidently occurred at approximately 6−24 h (Figure 4A). These facts were interpreted as strong evidence for enterohepatic circulation.24 Once M1 was secreted into the bile (see below) and entered the intestine, it could be reabsorbed; otherwise, microbial and intestinal β-glucuronidases hydrolyze M1, rendering free CSA sufficiently lipophilic for reabsorption. The repeated circulation process resulted in a long elimination period of CSA and its glucuronide. Vss (0.84 L/kg) of CSA was substantially greater than the total blood volume (approximately 0.0312 L/kg) of rats,25 and t1/2, λz was relatively long, indicating that CSA had an extensive affinity with the tissues rather than plasma protein and was 4070

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Journal of Agricultural and Food Chemistry Table 3. Urinary and Biliary Excretion of CSA and M1 in Rats after iv Administrationa bile (% of dose)

a

time (h)

CSA

0−4 4−8 8−12 12−24 total

0.440 ± 0.104 0.020 ± 0.005 ndb ndb 0.460 ± 0.101

M1 19.403 2.631 1.581 1.297 24.912

± ± ± ± ±

urine (% of dose) sum

3.339 0.807 0.531 0.659 3.624

19.843 2.651 1.581 1.297 25.372

± ± ± ± ±

3.327 0.803 0.531 0.659 3.619

CSA

M1

sum

0.068 ± 0.004 0.031 ± 0.002 ndb ndb 0.099 ± 0.006

0.683 ± 0.112 0.217 ± 0.137 0.133 ± 0.069 ndb 1.032 ± 0.296

0.751 ± 0.109 0.247 ± 0.136 0.133 ± 0.069 ndb 1.131 ± 0.293

total (% of dose) 20.594 2.898 1.714 1.297 26.503

± ± ± ± ±

3.378 0.910 0.584 0.659 3.791

Data represent the mean ± SD (n = 5). bnd indicated not detected.

readily distributed into the extravascular system.26 This is in agreement with the study of Hua et al.,19 where CSA underwent a rapid and wide distribution in tissues and organs throughout the whole body, especially in the small intestine, liver, and kidneys. After oral administration, CSA was absorbed rapidly into the circulation to reach Cmax (2.77 μM) at approximately 21 min and eliminated slowly with a t1/2,λz of 12.90 h, CL/F of 3.54 L h−1 kg−1, and relatively large Vd/F of 54.64 L/kg. Meanwhile, M1 was extensively formed to achieve Cmax (83.23 μM), which was nearly 30-fold higher than that of CSA, at a longer Tmax of approximately 1.10 h. M1 was eliminated slowly with a t1/2,λz of approximately 15.40 h, which was similar to that of CSA. The M1 plasma concentration was higher than the CSA concentration in the absorption, distribution, and elimination phases. As depicted in Figure 4B, it was apparent that CSA exhibited another Cmax (0.52 μM) at approximately 9.20 h, which could be due to enterohepatic circulation.27 This evidence alone was not as convincing as that provided by the observation for iv administration because the differential rates of absorption along the gastrointestinal tract after oral dosing could also produce such a phenomenon. M1 exhibited another Cmax (53.41 μM) as well, at 8−24 h in all six tested rats (Figure 4D), although the secondary absorption peak could not be observed from the mean concentration versus time profile of M1 after oral administration. The absolute oral bioavailability of CSA was estimated as 44.36% of the dose. The plasma exposure ratio between M1 and CSA (AUC0−t,M1/AUC0−t,CSA) after oral dosing was 196.67, which was approximately 12-fold higher than that (16.58) after iv injection. This difference between the two dosing routes was strong evidence for a highly efficient first-pass effect of CSA in the gut and/or the liver, whereby after absorption in the gastrointestinal tract, much CSA was converted to M1 before it went into the circulation. Therefore, more CSA was metabolized to M1 following oral administration, leading to a higher plasma exposure of M1.28 The total dose-normalized AUC0−t of CSA and M1 from oral administration was 4.8-fold higher than that from iv dosing, which suggested that the absorption of CSA from the gut was not the main barrier limiting its oral exposure. In summary, M1 formation via firstpass metabolism had attenuated the oral bioavailability of CSA. Considering the importance of M1 in CSA bioavailability, the evident drug−drug interaction of M1 with other drugs interfering with UDP-glucuronosyltransferase activities requires much attention.29 Furthermore, the sample collection period (84 h) was more than 3 times the elimination half-lives of CSA and M1, which was in accordance with the principles of the United States Food and Drug Administration.30 For both administration routes, the AUC0−t covered at least 93% of AUC0−∞, indicating that the duration of sample collection was appropriate and the method

was reliable for estimating the pharmacokinetic parameters of CSA and M1.31 Urinary and Biliary Excretion of CSA and M1. To understand the main excretion route of CSA and M1 from the body, its urinary and biliary recoveries after iv dosing were determined and the results are displayed in Table 3. CSA was mainly excreted as M1 from bile with approximately 19.403, 2.631, 1.581, and 1.297% of the CSA dose over the periods of 0−4, 4−8, 8−12, and 12−24 h post-dose, respectively. However, a lower amount of administered CSA was recovered in urine as M1 with 0.683, 0.217, and 0.133% over 0−4, 4−8, and 8−12 h post-dose, respectively. In its parent form, CSA was excreted from bile with approximately 0.440 and 0.020% of the dose and from urine with approximately 0.068 and 0.031% of the dose over the periods of 0−4 and 4−8 h post-dose, respectively. After 8 h post-dose, CSA could not be detected in either urine or bile. The accumulative recovery (25.372% of dose) of CSA in combination with M1 in bile was nearly 22.4-fold higher than that in urine (1.131%), signifying that CSA was excreted principally via the biliary route in rats in its parent and glucuronide metabolite forms, which might cause enterohepatic circulation. This result is consistent with the double-peak phenomenon mentioned above. Only 0.559% of the CSA dose was excreted from bile and urine in its parent form. Nevertheless, M1 excreted from both urine and bile accounted for 25.944% of the CSA dose. In addition, several other CSA metabolites with evidently large peaks in bile samples post-dose (Figure 3C) covered more than half of dose in total (not described precisely in this paper). This finding indicates that CSA was extensively transferred to its metabolites, especially the glucuronide metabolite, in the systemic circulation predominantly by liver metabolism and mainly excreted as its metabolites.32 Therefore, drugs inhibiting or inducing CSA metabolic enzymes might change CSA metabolism and the level of its parent form in the circulation when used in combination, which may finally affect its biological activities. The renal clearance [CLr; CLr = CL × fe (fraction excreted unchanged in urine) = 1.26 × 0.099% = 0.001 247 L h−1 kg−1]33 of CSA was substantially smaller than the glomerular filtration rate (approximately 0.3 L h−1 kg−1) of rats,25 indicating that CSA underwent extensive renal reabsorption and might or might not be secreted in the renal tubule.34 Thus, the urine flow rate and/or pH might affect the CLr of CSA. The renal reabsorption contributed to the low urinary recovery and long elimination half-life of CSA, especially after oral administration (12.90 h). In summary, this study first reported that CSA is mainly metabolized to CSA-3-O-glucuronide via the first-pass effect to limit its oral bioavailability and excreted predominantly through the biliary route, whereas the enterohepatic circulation, 4071

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Journal of Agricultural and Food Chemistry

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extravascular distribution, and renal reabsorption characteristics of CSA might delay its elimination.

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: 86-10-57833224. E-mail: [email protected]. cn. ORCID

Qi Chang: 0000-0001-9546-1827 Funding

This study was supported by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-1-012). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED CSA, cajaninstilbene acid; M1, CSA-3-O-glucuronide; iv, intravenous; IS, internal standard; DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision exit potential; MRM−IDA−EPI, multiple reaction monitoring− information-dependent acquisition−enhanced product ion; C0, initial plasma concentration for the iv dose; Cmax, maximum plasma concentration; Tmax, time to Cmax for the oral dose; t1/2,λz, terminal elimination half-life; AUC0−t, area under the plasma concentration versus time curve from zero to last sampling time; AUC0−∞, area under the plasma concentration versus time curve from zero to infinity; CL, total body clearance; CLr, renal clearance; MRT, mean residence time; Vd,λz, volume of distribution; Vss, volume of distribution at steady state; F, absolute oral bioavailability

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