Abstract
Kaempferol is a well-known flavonoid; however, it lacks extensive pharmacokinetic studies. Phase II metabolic enzymes and efflux transporters play an important role in the disposition of flavonoids. This study aimed to investigate the mechanism by which phase II metabolic enzymes and efflux transporters determine the in vivo exposure of kaempferol. Pharmacokinetic analysis in Sprague–Dawley rats revealed that kaempferol was mostly biotransformed to conjugates, namely, kaempferol-3-glucuronide (K-3-G), kaempferol-7-glucuronide (K-7-G), and kaempferol-7-sulfate, in plasma. K-3-G represented the major metabolite. Compared with that in wild-type mice, pharmacokinetics in knockout FVB mice demonstrated that the absence of multidrug resistance protein 2 (MRP2) and breast cancer resistance protein (BCRP) significantly increased the area under the curve (AUC) of the conjugates. The lack of MRP1 resulted in a much lower AUC of the conjugates. Intestinal perfusion in rats revealed that the glucuronide conjugates were mainly excreted in the small intestine, but K-7-S was mainly excreted in the colon. In Caco-2 monolayers, K-7-G efflux toward the apical (AP) side was significantly higher than K-3-G efflux. In contrast, K-3-G efflux toward the basolateral (BL) side was significantly higher than K-7-G efflux. The BL-to-AP efflux was significantly reduced in the presence of the MRP2 inhibitor LTC4. The AP-to-BL efflux was significantly decreased in the presence of the BL-side MRPs inhibitor MK571. BCRP inhibitor Ko143 decreased the glucuronide conjugate efflux. Therefore, kaempferol is mainly exposed as K-3-G in vivo, which is driven by phase II metabolic enzymes and efflux transporters (i.e., BCRP and MRPs).
1. Introduction
Flavonoids are low-molecular-weight phenolic compounds widely distributed in natural plants and frequently consumed in daily diet (1). Their anti-aging and cancer-preventive benefits have been extensively investigated (2). Kaempferol (3,4′,5,7-trihydroxyflavone) is a bioactive flavonol found in many edible plants and beverages, such as tomato, grapefruit, broccoli, honey, and tea (3). The human dietary intake of this polyphenol may reach approximately 4 mg/day (4). Kaempferol provides many promising pharmacological activities, such as anti-oxidation (5, 6), anti-inflammation (7, 8), and antitumor (9–11). Epidemiological studies have revealed the preventive effects of dietary kaempferol intake on pancreatic cancer (4), lung cancer (12), ovarian cancer (13), and colorectal adenoma (14). Kaempferol exhibits anticarcinogenic activities through impairing cancer angiogenesis, disrupting cancer metastasis, and inducing cell apoptosis (15).
Flavonoids containing multiple phenolic hydroxyls easily undergo metabolism catalyzed by hepatic and intestinal metabolic enzymes; as a result, flavonoids yield low bioavailabilities (16). The conjugative reaction performed by phase II enzymes, such as UDP-glucuronosyltransferases (UGT) and sulfotransferases (SULT), is a major metabolic pathway (17). Many flavonoid phase II conjugates are substrates of membrane-bound efflux transporters, such as breast cancer resistance protein (BCRP) and multidrug resistance proteins (MRPs), which are abundantly expressed in the intestine and liver (18). These proteins transport drug molecules out of the cells and perform an important role in drug elimination.
The in vivo metabolism and exposure of kaempferol have been rarely investigated, although the in vitro metabolism of kaempferol has been extensively investigated. For example, rat cytochrome P450 1A1 catalyzes the oxidative metabolism of kaempferol (19); nevertheless, the tendency for uridine-diphosphate-glucuronic acid-dependent conjugation is evidently higher (20). Kaempferol-7-glucuronide (K-7-G) is the major product of conjugative metabolism determined by using rat liver microsomes (RLMs) (21). Kaempferol-3-glucuronide (K-3-G) is more produced in the small intestine (20). Different cell experiments have independently reported that kaempferol is probably a substrate and inhibitor of P-glycoprotein (P-gp) and BCRP (22–25). Kaempferol can be conjugated with glucuronic or sulfonic acid inside Caco-2 cells and subsequently transported across the monolayer or excreted back to the apical (AP) side (26); however, the transporters responsible for efflux of kaempferol conjugates remain unknown. The bioavailability of kaempferol is lower than 2% (20). Although liquid chromatography-tandem mass spectrometry (LC–MS/MS) is applied to determine plasma kaempferol, an indirect method should be proposed to quantify the total glucuronidated kaempferol through hydrolysis by using glucuronidases (27). Moreover, the lack of sensitivity leads to a large plasma usage, which requires excessive blood collection.
To understand the in vivo activities of kaempferol better, a comprehensive and thorough study of its in vivo disposition is important, which will also be suggestive for the investigation of its drug-like properties. In this study, a sensitive and reliable LC–MS/MS method was developed to determine kaempferol and phase II conjugates in various matrices directly and simultaneously. Kaempferol was initially subjected to pharmacokinetic analysis in rats to provide a general overview of its in vivo exposure. The pharmacokinetic analysis in knockout mice provided insights into the effect of efflux transporters on the exposure level of the conjugates. In situ intestinal perfusion in rats was conducted to investigate the metabolism of kaempferol in four intestinal segments. Our findings were then used to demonstrate the role of intestinal metabolism in the in vivo exposure of kaempferol. A Caco-2 cell model combined with selective inhibitors was used as an in vitro approach to confirm the role of efflux transporters.
2. Materials and methods
2.1. Chemicals and reagents
Kaempferol and genistein were purchased from Chengdu Must Biotech Co., Ltd. (Chengdu, China), and K-3-G was obtained from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China). Kaempferol-7-sulfate (K-7-S) was synthesized by KareBay™ Biochem, Inc. (Monmouth Junction, USA). The K-7-G standard was prepared in our laboratory. The purity of these standards was not less than 98% according to HPLC analysis. The water used for the LC–MS/MS analysis was obtained from a Milli-Q water purification system produced by Millipore (Billerica, MA, USA). HPLC-grade methanol, acetonitrile, and formic acid were acquired from Merck (Darmstadt, Germany). All other chemicals were of analytical grade. Transwell cell culture chamber insert (polycarbonate membrane and polystyrene plates, 24-mm diameter and 3-μm pore size) was obtained from Corning Inc. (Corning, NY, USA).
2.2. Animals
Adult male Sprague–Dawley (SD) rats weighing between 250 g and 300 g were purchased from The Experimental Animal Center of the Guangzhou University of Chinese Medicine (Guangzhou, China) and were used in the pharmacokinetic and intestinal perfusion experiments. Male wild-type FVB mice (Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) and knockout FVB mice (Biomodel Organism Science & Technology Development Co. Ltd., Shanghai, China) aged 8 weeks to 10 weeks were used in the experiment. These knockout mice include Bcrp1−/−, Mrp1−/−, Mdr1a−/−, and Mrp2−/−. The use of these animals was approved by the Institutional Animal Care and Use Committee of the Guangzhou University of Chinese Medicine. The animals were housed at an ambient temperature of 22 ± 2 °C with 12 h light/dark cycles and were fasted overnight with free access to water before the experiment.
2.3. Biosynthesis of K–7–G
The phase II metabolite K-7-G was biosynthesized using RLMs according to the approach previously employed in the laboratory (28). The K-7-G in the prepared solution was isolated by collecting post-column effluent using an Agilent 1200 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA), concentrated under nitrogen, and stored at −20 °C. We determined the concentration of K-7-G using the parent standard curve and conversion factor. The conversion factor was determined by comparing the peak area change in aglycone after hydrolyzing glucuronide with β-glucuronidases and the corresponding peak area change in glucuronide under the same UV detection wavelength. The conversion factor for K-7-G was 1.09.
2.4. Pharmacokinetic study
2.4.1. Pharmacokinetic study in rats and mice
Kaempferol was dissolved in 20% hydroxypropyl–beta–cyclodextrin aqueous solution. After oral administration in rats (high dose: 20 mg/kg; low dose: 10 mg/kg, n = 6), blood samples (0.1 mL) were collected from the postorbital venous plexus veins into heparinized tubes at 0, 3, 10, 20, 30, 60, 120, 180, 300, 480, 720, 900, 1440, and 2160 min. Before bleeding, isoflurane–oxygen-inhaled anesthesia was applied for pain relief. The plasma was separated by centrifuging at 8,000 rpm for 5 min and stored at −80 °C before use. Oral intake was resumed 12 h after dose. After oral kaempferol administration in mice (10 mg/kg, n = 6 for wild-type, n = 5 for knockout), blood samples (less than 40 μL) were collected from the caudal veins at 0, 3, 7, 15, 30, 60, 90, 120, 180, 240, 360, 540, and 720 min. The pharmacokinetic parameters were calculated via noncompartmental analysis using the WinNonlin® 3.3 software.
2.4.2. Plasma sample preparation
For calibration standards, blank plasma (10 μL) was added into standard working (10 μL) and internal standard (IS, 5 μL of 1 μM genistein) solutions. About 10, 10, and 5 μL of plasma, methanol, and IS were mixed for the samples, respectively. To extract the analytes and remove proteins, 180 μL of ethyl acetate and methanol mixture solution (5:1) was added and subsequently vortexed for 5 min. After centrifuging at 8,000 rpm for 5 min, the supernatant layer was removed, placed into new tubes, and evaporated to dryness in a thermostatic vacuum drier. The residues were redissolved using 100 μL of 50% methanol aqueous solution. After centrifuging at 13,500 rpm for 25 min, 5 μL of the solution was obtained for LC–MS/MS analysis.
2.5. In situ rat intestinal perfusion of kaempferol
We applied an established rat intestinal perfusion model that was approximately similar to that described in (29). Four segments (duodenum, jejunum, ileum, and colon) of the intestine were perfused with a bile duct cannulation. HBSS buffer with kaempferol was perfused from the inlet cannula of each segment using an infusion pump (model PHD2000; Harvard Apparatus, Cambridge, MA) at a 20 mL/h flow rate. The outlet perfusate in the cannula was driven into a receiving tube using an acetonitrile (2 mL) to denature the hydrolysis enzyme, except when blank perfusate (no flavonoid) was collected (no acetonitrile). Two kaempferol concentrations (5 μM and 20 μM) and four rats at each concentration were perfused. After a 20 min wash-out period, which was considered sufficient to achieve steady-state absorption, the perfusate and bile samples were collected from the outlet cannula every 15 min during the 60-min perfusion period. Blood samples were also collected at set intervals. The samples were diluted with IS solution and injected for LC–MS/MS analysis after centrifugation.
The major parameters, namely, permeability of kaempferol (P*eff), amounts of absorbed kaempferol, and amounts of excreted metabolites, were calculated as mentioned above (29).
2.6. In vitro incubation of kaempferol via hepatic and intestinal preparations
The hepatic and intestinal S9 fractions or microsomes from SD rats and knockout FVB mice were prepared according to the protocol described in (30). The glucurono-incubation system was applied as follows: S9 fractions or microsomes were mixed with D-saccharic-1,4-lactone monohydrate (4.4 mM), magnesium chloride (0.88 mM), alamethicin (0.022 mg/mL), and different kaempferol concentrations in a 50 mM Tris–HCl buffer (pH 7.4). The mixture with a final volume of 120 μL was incubated at 37 °C for 20 min. The sulfo-incubation system was applied as follows: S9 fractions were mixed with magnesium chloride (2.5 mM), 3′-phosphoadenosine-5′-phosphosulfate (0.1 mM), and different kaempferol concentrations in a 50 mM Tris–HCl buffer (pH 7.4). The mixture with a final volume of 120 μL was incubated at 37 °C for 30 min. The above reactions were terminated by adding 60 μL of IS-containing acetonitrile. The samples were analyzed by LC–MS/MS after centrifugation. All experiments were performed in triplicate.
2.7. Transport and inhibition experiments in the Caco-2 monolayer
The culture conditions for Caco-2 cells and the preparation of transwell-grown monolayers have been described in (31, 32). Kaempferol (10 μM) in HBSS buffer was loaded on the AP or BL side of the Caco-2 monolayer. Samples (500 μL) were collected from both sides of each Transwell at predetermined periods (0, 30, 60, 90, and 120 min), and 250 μL of acetonitrile was immediately added to each sample. The same volume of testing solution or blank HBSS was immediately added to supplement the removed samples. The cell membranes were carefully washed thrice with ice-cold blank HBSS (pH 7.4) at the end of the experiments. The cells attached to the polycarbonate membranes were cut off from the inserts, immersed in blank HBSS (1 mL), and sonicated for 30 min in an ice bath. After diluting with IS solution, the supernatant was injected into LC–MS/MS for analysis.
The inhibition experiments were performed as described in (31) with minor modifications. Verapamil was employed as a classical P-gp inhibitor, Ko143 was used as a specific inhibitor of BCRP, and LTC4 (33) and MK571 were used as selective inhibitors of MRP2 and BL-side MRPs, respectively. Verapamil (50 μM), Ko143 (10 μM), LTC4 (0.1 μM), and MK571 (50 μM) were employed to determine the effects of efflux transporters on the bidirectional transport. MK571 was added to the BL side, and other inhibitors were added to the AP side.
The major parameters, including the efflux rate of transport (Bt) and fraction of the metabolized dose (Fmet), were calculated according to (31).
2.8. Instrumentation and chromatographic conditions
LC–MS/MS was performed on an Agilent 1290 series UHPLC system and an Agilent 6490 Triple Quadrupole mass spectrometer equipped with an electrospray ionization source (Agilent Technologies). The data were recorded, and the system was controlled using the MassHunter software (version B.06.00, Agilent Technologies).
Chromatographic separation was achieved on a Zorbax C18 column (100 × 3.0 mm2, 1.8 μm; Agilent Technologies), with the column temperature maintained at 25 °C. The mobile phases comprised 0.05% formic acid water (A) and acetonitrile (B) by using a gradient elution of 23% (v/v) B at 0 min to 4.5 min, 52% B at 5 min, and 23% B at 7.5 min. The flow rate was 0.4 mL/min.
The mass spectrometer was operated in positive ion mode for the determination of glucuronides and in negative ion mode for the determination of kaempferol, sulfate, and IS. The MS/MS setting parameters in the positive mode (1 min to 5 min) were set as follows: 2.5 kV capillary voltage, 500 V nozzle voltage, 290 °C gas temperature, 340 °C sheath temperature, 11 L/min sheath gas flow, and 10 L/min gas flow. The fragmentation voltage was 380 V, and the Delta electron multiplier voltage (EMV) was 100 V. These parameters were slightly modified in the negative mode (5 min to 8 min) as follows: 3 kV capillary voltage, 1000 V nozzle voltage, and 200 V Delta EMV. Quantification was performed via the multiple reaction-monitoring of the transitions of m/z 463→287 for K-3-G and K-7-G, m/z 365→285 for K-7-S, m/z 285→93 for K, and m/z 269→132.9 for IS. Collision energy was set at 14 for K-3-G and K-7-G, 12 for K-7-S, 38 for K, and 27 for IS. The low limit of quantitative analysis (LLOQ) was 3.9 nM for K, 1.9 nM for K-3-G and K-7-G, and 0.39 nM for K-7-S.
2.9. Data analysis
Experimental data were expressed as mean ± SD. One-way ANOVA with or without Tukey–Kramer multiple comparison and Student’s t-test was used to evaluate statistical differences. Differences were considered significant at p < 0.05 or p < 0.01.
3. Results
3.1. Identification of kaempferol metabolites
For glucuronidation (Fig. 1A), the samples eluted at 3.58, 3.90, 4.38, and 4.87 min showed the expected UV spectra for a flavonoid with two distinct peaks of absorbance at 250 nm to 280 nm and 350 nm to 390 nm. The peak at 8.20 min corresponded to the aglycone kaempferol. The high-resolution mass spectrometry of these compounds indicated that the [M+H]+ = 463.0881 ion with [M+H]+ = 287.0555 accorded with the mono-glucuronide of kaempferol. For sulfation (Fig. 1C), only one peak was observed ahead of kaempferol and generated a [M+H]+ = 367.0123 ion, which suggested that this metabolite contained a kaempferol mono-sulfate moiety. Ion currents indicating kaempferol diglucuronide ([M+H]+ = 639) or kaempferol disulfate ([M+H]+ = 447) were not detected.
Fig. 1.
The UHPLC chromatograms of glucuronidation and sulfation of kaempferol and LC-MS/MS method for simultaneous determination of kaempferol and the metabolites. The chromatograms of glucuronidation via rat liver microsomes (a) and sulfation via rat liver S9 fraction (c), and high-resolution mass spectra of the four monoglucuronides (b) and mono-sulfate (d) were shown. Identification of these metabolites is shown in Table I. e LC-MS/MS method for determination of kaempferol and the three metabolites. Corresponding structures and MS/MS spectra of the tested compounds and internal standard (IS) are as follows: f kaempferol-3-glucuronide (K-3-G) and kaempferol-7-glucuronide (K-7-G); g kaempferol-7- sulfate (K-7-S); h IS (genistein); i kaempferol.
According to previous studies (34, 35), for flavones and flavonols, the extent of shift in the spectra of phase II metabolites in Band I and II regions as reflected by the changes in λmax can be analyzed to identify the position of glucuronidation and sulfation. Specifically, the substitution of 3- and 4′-hydroxyl resulted in Band I λmax hypsochromic shift of 13 nm to 30 nm and 5 nm to 10 nm, respectively. The substitution of 5-hydroxyl group caused a Band II λmax hypsochromic shift of 5 nm to 10 nm. In contrast, the substitution of 7-hydroxyl group did not cause any λmax change in Band I or II λmax. Several kaempferol conjugates with different hydroxyl substituted positions were identified using the above method as shown in Table. 1. We observed four mono-glucuronides, namely, K-5-G, K-3-G, K-7-G, and K-4′-G, and the mono-sulfate was identified as K-7-S. In addition, K-3-G and K-7-S were proven by comparing the retention time with the corresponding standard.
Table 1.
Summary of UV spectral data for kaempferol and the metabolites (shifts of less than 3 nm were not considered to be significant).
| Compound | Band I max (nm) | Band II max (nm) | Shifts in relation to aglycone (nm) | Identification |
|---|---|---|---|---|
| kaempferol | 365 | 266 | - | - |
| G1 | 362 | 258 | −8 (Band II) | kaempferol-5-glucuronide |
| G2 | 347 | 265 | −18 (Band I) | kaempferol-3-glucuronide |
| G3 | 365 | 265 | no change | kaempferol-7-glucuronide |
| G4 | 361 | 266 | −4 (Band I) | kaempferol-4′-glucuronide |
| S1 | 366 | 266 | no change | kaempferol-7-sulfate |
3.2. Pharmacokinetic study
3.2.1. Pharmacokinetic study in rats
A sensitive and reliable LC–MS/MS method was established and applied to determine the plasma concentrations of the parent and main metabolites after oral kaempferol administration (Fig. 1). Fig. 2 and Table 2 show the mean plasma concentration–time profiles and pharmacokinetic parameters of these compounds, respectively. After oral administration, kaempferol was quickly absorbed through the gastrointestinal tract and metabolized to phase II conjugates, including K-3-G, K-7-G, and K-7-S. The ion currents for diglucuronide, disulfate, or glucurono-sulfate were not detected in plasma. The proportion of free kaempferol was extremely small compared with the amount of metabolites. The maximum concentration (Cmax) of kaempferol was approximately 50 and 16 times lower than that of K-3-G and K-7-G, respectively. The concentration–time profile of K-7-S was different from those of kaempferol and glucuronides. Kaempferol and glucuronides reached Cmax within 30 min and were completely eliminated in 12 h, whereas sulfate had an evidently longer peak time (15 h to 19 h) and elimination, and was still detected in plasma 36 h after dose. K-3-G had higher plasma concentrations than K-7-G as reflected by its threefold to fivefold higher Cmax and AUC.
Fig. 2.
Mean plasma concentration–time curves of the tested compounds in rats after an oral administration of kaempferol (10 and 20 mg/kg). Data represent mean ± SD (n = 6). a kaempferol. b K-3-G. c K-7-G. d K-7-S.
Table 2.
Pharmacokinetic parameters of the four analytes in male SD rats after oral administration of 10 mg/kg and 20 mg/kg of kaempferol.
| Compounds | doses | Cmax (μmol/L) | Tmax (min) | AUC0-t (min•μmol/L) | AUC0−∞ (min•μmol/L) | MRT0-t (min) |
|---|---|---|---|---|---|---|
| Kaempferol | 10 mg/kg | 0.305 ± 0.106 | 26.7 ± 5.8 | 10.3 ± 3.8 | 11.4 ± 3.7 | 26.2 ± 1.8 |
| 20 mg/kg | 0.647 ± 0.244 | 18.0 ± 11.0 | 51.8 ± 17.2 | 57.9 ± 13.6 | 148 ± 68 | |
| K-3-G | 10 mg/kg | 24.6 ± 7.0 | 20.0 ± 8.9 | (1.74 ± 0.47) ×103 | (1.76 ± 0.46) ×103 | 88.8 ± 14.0 |
| 20 mg/kg | 30.9 ± 5.5 | 24.0 ± 5.5 | (2.79 ± 0.33) ×103 | (2.81 ± 0.33) ×103 | 114 ± 28 | |
| K-7-G | 10 mg/kg | 5.29 ± 2.97 | 17.5 ± 5.0 | 310 ± 95 | 314 ± 96 | 126 ± 37 |
| 20 mg/kg | 9.55 ± 3.37 | 16.0 ± 5.5 | 802 ± 231 | 811 ± 311 | 130 ± 43 | |
| K-7-S | 10 mg/kg | 0.332 ± 0.085 | (1.12 ± 0.57) ×103 | 454 ± 90 | 536 ± 164 | (1.05 ± 0.12) ×103 |
| 20 mg/kg | 0.703 ± 0.296 | 912 ± 768 | (1.15 ± 0.22) ×103 | (1.65 ± 0.56) ×103 | (1.09 ± 0.11) ×103 | |
Data represent mean ± SD, n = 6. Abbreviations: Cmax, maximum plasma concentration; Tmax, time to reach maximum drug concentration; AUC0-t, area under plasma concentration-time curve; AUC0−∞, predicted area under plasma concentration-time curve; MRT0-t, mean residence time.
3.2.2. Pharmacokinetic study in knockout mice
The pharmacokinetic studies in knockout and wild-type FVB mice were conducted after an oral administration of kaempferol (10 mg/kg). Fig. 3 and Table 3 show the data. Free kaempferol was below the LLOQ (3.9 nM) at most periods in all strains of mice. When Mrp1, which was expressed at BL membranes, was knocked out, the systemic parameters (Cmax, AUC0-t, and mean residence time or MRT) of K-3-G were dramatically reduced compared with those of the wild-type (p < 0.01). The lack of Mrp1 also significantly decreased the AUC0-t (p < 0.05) and MRT (p < 0.01) of K-7-G and K-7-S. The absence of Mrp2 resulted in a > 3.5-fold higher plasma Cmax and AUC0-t of K-3-G and K-7-G, respectively (p < 0.01), while the absence of Bcrp resulted in a ~2-fold higher plasma Cmax (p < 0.05) and AUC0-t of K-3-G (p < 0.01) as well as a > 4-fold higher Cmax (p < 0.01) and AUC0-t of K-7-G (p < 0.05). For K-7-S, Bcrp1−/− and Mrp2−/− significantly increased the Cmax and AUC0-t at p < 0.01 for Bcrp1−/− and at p < 0.05 for Mrp2−/−. When Mdr1a was knocked out, the AUC0-t of K-3-G significantly decreased compared with that of the wild-type (p < 0.05). The lack of Mdr1a did not cause any significant differences in the AUC0-t of K-7-G and K-7-S, but extended their MRT (p < 0.01 for K-7-G).
Fig. 3.
Mean plasma concentration-time curves of kaempferol metabolites in wild-type and knockout FVB mice after an oral administration of kaempferol (10 mg/kg). Data represent mean ± SD (n = 6, wild-type; n = 5, knockout). a K-3-G. b K-7-G. c K-7-S. Free kaempferol is below the low limit of quantitative analysis (3.9 nM) at most periods.
Table 3.
Pharmacokinetic parameters of the three analytes in wild-type and knockout FVB mice after an oral administration of kaempferol (10 mg/kg).
| Parameters | Compounds | FVB | Mrp1−/− | Mrp2−/− | Bcrp1−/− | Mdr1a−/− |
|---|---|---|---|---|---|---|
| Cmax | 3-G | 8.92 ± 1.69 | 1.75 ± 1.18** | 32.3 ± 9.7** | 18.0 ± 6.4** | 4.62 ± 1.14** |
| (μmol/L) | 7-G | 2.91 ± 1.94 | 0.839 ± 0.853 | 14.3 ± 4.8** | 14.9 ± 3.6** | 1.12 ± 0.68 |
| 7-S | 0.301 ± 0.185 | 0.153 ± 0.062 | 0.616 ± 0.059* | 1.09 ± 0.21** | 0.141 ± 0.101 | |
| AUC0-t | 3-G | 365 ± 195 | 38.0 ± 29.1** | (1.28 ± 0.27) ×103** | 848 ± 123** | 143 ± 83* |
| (min•μmol/L) | 7-G | 114 ± 79 | 13.9 ± 12.0* | 392 ± 78** | 464 ± 234* | 63.1 ± 15.3 |
| 7-S | 49.5 ± 38.6 | 6.72 ± 5.83* | 102 ± 10* | 156 ± 55** | 36.7 ± 37.0 | |
| MRT | 3-G | 103 ± 29 | 33.4 ± 19.9** | 90.5 ± 11.0 | 97.3 ± 21.7 | 113 ± 71 |
| (min) | 7-G | 81.3 ± 34.2 | 25.8 ± 16.6** | 90.9 ± 18.1 | 45.4 ± 22.3 | 244 ± 22** |
| 7-S | 165 ± 59 | 57.3 ± 23.9** | 197 ± 13 | 157 ± 8 | 237 ± 92 | |
Data represent mean ± SD, n = 5~6.
p<0.05;
p<0.01.
3.2.3. Glucuronidation and sulfation activities in knockout mice
A study was conducted to test whether the phase II metabolism was altered in knockout mice. The rates of kaempferol glucuronidation (combination of K-3-G and K-7-G) in the intestine of Bcrp1−/− mice were significantly decreased by ~55% compared with those in wild-type FVB mice, but the glucuronidation rates in their liver were increased by 32%. The lack of Mrp1, Mrp2, and Mdr1a decreased the hepatic glucurodination rates by ~26%, 11%, and 30%, respectively (Figs. 4A and B). For sulfation, the reaction rates from Mrp1−/−, Mrp2−/−, and Mdr1a−/− mice were significantly decreased by ~25%, 79%, and 47% in the intestine and by ~11%, 59%, and 39% in the liver (Fig. 4C).
Fig. 4.
Kaempferol glucuronidation and sulfation via hepatic and intestinal S9 fractions prepared from wild-type and knockout FVB mice. For glucuronidation (a, b), kaempferol (1.25 μM) was incubated with S9 fractions (0.0106 mg/mL). For sulfation (c), kaempferol (0.125 μM) was incubated with S9 fractions (0.02 mg/mL). Reaction rates were calculated and expressed as nanomole per minute per milligram of protein. Each column is the average of three determinations, and error bars represent the SD (n = 3). *p < 0.05; **p < 0.01.
3.3. Regional transport and metabolism of kaempferol in a perfused rat intestinal model
After perfusion with 5 μM or 20 μM kaempferol, kaempferol demonstrated faster absorption in colon than in jejunum and ileum at both concentrations. No significant differences were observed in the P*eff values between 5 μM and 20 μM concentrations (Fig. 5A). Kaempferol was converted into glucuronides and sulfate, thereby indicating that kaempferol could undergo at least two kinds of phase II metabolisms in the intestine. Large amounts of K-3-G and K-7-G were found in the perfusate, with the highest excretion in duodenum and jejunum and the lowest excretion in colon (Figs. 5C and D). In contrast, K-7-S had the highest excretion in colon and lowest excretion in ileum (Fig. 5E). The amount of K-7-G in the perfusate was slightly higher than that of K-3-G in duodenum and jejunum, but was significantly higher in ileum and colon (Fig. 5F). The glucuronides in the bile increased with time, and the amount of K-3-G was 3 to 6 times higher than that of K-7-G at each period (Figs. 5G and H). Only K-3-G was detected in plasma (Fig. 5I) because the other analytes were below the corresponding LLOQ.
Fig. 5.
Absorption and metabolism of kaempferol in a four-site rat intestinal perfusion model. Four segments of the intestine (i.e., duodenum (Duo), upper jejunum (Jej), terminal ileum (Ile), and colon (Col)) were perfused simultaneously at a flow rate of 20 mL/h using 5 or 20 μM kaempferol. Effective permeabilities of kaempferol (a), amounts of kaempferol absorbed (b), and kaempferol metabolites excreted (c–e) in theperfusate were determined and normalized over a 10-cm intestinal length. f Comparison of excreted K-3-G and K-7-G in four segments at 20 μM perfusion. The amounts of kaempferol-glucuronides excreted in bile at 5 μM perfusion (g) and at 20 μM perfusion (h), and the plasma concentration of K-3-G (i) were also determined. Each column (a–f) and each curve (g–i) represent the average of four determinations, and error bars represent the SD (n = 4). *, p < 0.05.
3.4. Glucuronidation and sulfation in rat hepatic and intestinal preparations
In vitro metabolism was conducted to investigate the metabolic differences of kaempferol in rat liver and intestinal segments. As shown in Fig. 6, high and low glucuronidation rates were observed in the small intestine and colon, respectively. Jejunum exhibited maximal formation rates for the two glucuronides under three kaempferol concentrations. Liver microsomes produced more K-7-G than K-3-G. Sulfation was relatively different from glucuronidation and achieved the highest reaction rates in liver. Among the four intestinal segments, colon showed the highest reaction rates and demonstrated statistically significant differences from duodenum and ileum (figure not shown).
Fig. 6.
Kaempferol glucuronidation and sulfation via rat hepatic and segmental intestinal preparations. For glucuronidation (a, b), three concentrations (1.25, 2.5, and 10 μM) of kaempferol were incubated with microsomes (0.00318 mg/mL). For sulfation (c), three concentrations (0.125, 0.5, and 1.25 μM) of kaempferol were incubated with S9 fraction (0.02 mg/mL). Reaction rates were calculated and expressed as nanomole per minute per milligram of protein. Each column is the average of three determinations, and error bars represent the SD (n = 3). *p < 0.05; **p < 0.01.
3.5. Metabolism and efflux of kaempferol in the Caco-2 cell monolayer
3.5.1. Efflux of K-3-G and K-7-G
The Caco-2 cell model was used as a classical model for simulating drug absorption and disposition in enterocytes. We monitored the amounts of kaempferol, K-3-G, K-7-G, and K-7-S in the two side buffers for 2 h after the AP loading (AP to BL) or BL loading (BL to AP) of kaempferol. Fig. 7 shows statistical differences between the K-3-G and K-7-G excretion in the two sides. The K-3-G efflux toward the BL side was approximately six times higher than that toward the AP side, while the K-7-G efflux toward the BL side was not more than twice higher than that toward the AP side, thereby indicating that K-3-G was more inclined to be transported into the BL side. No significant difference was observed in the intracellular amounts of two glucuronides.
Fig. 7.
Bidirectional efflux of kaempferol-glucuronides across the Caco-2 monolayer. After apical loading (AP to BL) or basolateral loading (BL to AP) of 10 μM kaempferol, the amounts of K-3-G and K-7-G in the apical (AP) and basolateral (BL) sides were determined. The efflux rates (a) and intracellular amounts (b) of K-3-G and K-7-G were calculated. Data are expressed as the mean of three determinations, and error bars represent the SD. *p < 0.05; **p < 0.01.
3.5.2. Effect of transporter inhibitors
Transport studies were performed in the presence of 50, 10, 0.1, and 10 μM verapamil, Ko143, LTC4, and MK571, respectively, to determine the role of P-gp, BCRP, MRP2, and BL-side MRPs in the transport of kaempferol conjugates. Fig. S1 (supplemental data) shows the time-related amounts of analytes in the two sides. Verapamil did not show any statistical difference in the efflux rates, intracellular concentrations, and Fmet of glucuronides (Figs. 8A to H). However, the bidirectional efflux rates and Fmet (BL to AP) of K-7-S were significantly reduced compared with the control (Figs. 8J and K). Ko143 significantly decreased the bidirectional efflux rates and Fmet of the two glucuronides (Figs. 8A to C and 8E to H). No statistical differences were observed in the efflux rates and Fmet of K-7-S. LTC4 reduced the amounts of the three metabolites in the AP side, except that of K-7-S (AP to BL). LTC4 also significantly increased the intracellular amounts of K-3-G and K-7-G (AP to BL), but decreased the Fmet (BL to AP) of the three metabolites (Figs. 8C, D, and G to I). In the presence of 10 μM MK571, the three metabolites transported into BL side were dramatically reduced. As expected, MK571 significantly lowered the efflux rates of the three metabolites toward the BL side, except that of K-7-G (AP to BL) but did not significantly influence their intracellular amounts (Fig. 8). Fmet (BL to AP) was also altered by MK571 (Figs. 8C, G, and K).
Fig. 8.
Effects of inhibitors on the efflux rates of K-3-G (a, b), K-7-G (e, f), and K-7-S (i, j); Fmet of K-3-G (c), K-7-G (g), and K-7-S (k); and intracellular amounts of K-3-G (d), K-7-G (h), and K-7-S (l) in the Caco-2 monolayer. Kaempferol (10 μM) without inhibitors and with verapamil (50 μM), Ko143 (10 μM), LTC4 (0.1 μM), and MK571 (10 μM), respectively, was added to the AP or BL side. Each column is the average of three determinations, and error bars represent the SD (n = 3). *p < 0.05; **p < 0.0.
4. Discussion
It is well proved by numerous studies that phase II metabolism contributes largely to the disposition of flavonoids including kaempferol (18); however, the role of phase II metabolic enzymes and efflux transporters in the in vivo exposure of kaempferol has not been systematically studied.
Our study is the first to investigate fully the pharmacokinetics and in vivo metabolism of kaempferol using LC–MS/MS. Glucuronides, followed by sulfate, are the main circulating forms of kaempferol in plasma. After oral kaempferol administration, the Cmax and AUC0-t of unchanged kaempferol are much smaller than those of the metabolites (Table 2). The main metabolites are K-3-G, K-7-G, and K-7-S, which suggests that 3- and 7-hydroxyls are active sites for the in vivo phase II conjugation of kaempferol.
K-3-G is the principal conjugate among the three metabolites. K-3-G possesses the highest AUC, which is five and four times higher than that of K-7-G and K-7-S, respectively, after 10 mg/kg administration in rats (Table 2). The intestinal perfusion in rats showed that K-3-G has three to six times higher concentrations than K-7-G in bile, and only K-3-G can be determined in plasma (K-3-G and K-7-G have the same LLOQ; Figs. 4G to I). Therefore, K-3-G has a higher tendency to be transported from the BL membrane into the portal vein. Coincidentally, in Caco-2 cells, the K-7-G efflux is significantly higher and lower than the K-3-G efflux in the AP and BL sides, respectively (Fig. 7). Therefore, efflux transporters exhibit regioselectivity to the positional isoforms of kaempferol–glucuronides from which we draw a conclusion that these transporters determine the in vivo exposure of kaempferol as large amounts of K-3-G.
Compared with glucuronidation, sulfation is a secondary metabolic pathway for kaempferol. The peak concentration of K-7-S is much lower than that of K-3-G and K-7-G no matter in rats or mice; however, K-7-S shows a continuous increase in plasma concentration over long periods when no parent or glucuronides are present in plasma (Fig. 2). This finding can be attributed to several reasons. First, metabolism via S9 fractions showed that the formation rates of K-7-S are highest in the colon among the four intestinal segments (Fig. 6C). A published study demonstrated that the expression of some sulfotransferases, such as Sult1a1/2, Sult1b1, and Sult1d1, was higher in the colon than in the small intestine (36). Second, K-7-S is mostly excreted in the colon in the rat perfusion model (Fig. 5E). This phenomenon is not driven by the efflux transporters in the AP side, such as MRP2 and BCRP, because these transporters are not abundantly expressed in the colon (36, 37). Therefore, the production of K-7-S is lagged compared with that of glucuronides. However, the time-concentration profile of K-7-S could not be fully explained by our findings. Kaempferol sulfation is being studied further in our laboratory.
Among the four surveyed efflux transporters, BCRP and MRPs (i.e., MRP1 and MRP2) have important roles in determining the in vivo exposure of kaempferol. In the Caco-2 monolayer, the glucuronides that efflux toward the AP side (BL to AP) are significantly reduced by Ko143 and LTC4, whereas Ko143 significantly decreases the glucuronides in the BL side and their Fmet, thereby suggesting that Ko143 could possibly suppress kaempferol glucuronidation. The K-7-S efflux toward the AP side (BL to AP) is significantly reduced by verapamil and LTC4, whereas verapamil significantly decreases K-7-S in the BL side and its Fmet, thereby suggesting that verapamil could possibly suppress kaempferol sulfation. MK571 hinders the efflux of the three metabolites toward the BL side, which indicates that inhibiting the BL-side MRPs can deter the absorption of kaempferol conjugates (Fig. 8). An inhibitor that can completely inhibit a specific transporter without affecting drug metabolism cannot be easily determined. Therefore, knockout mice provide a powerful means to investigate the assumed transporters directly. Bcrp1−/− and Mrp2−/− have significantly greater AUC0-t of the conjugates than wild-type, on the contrary, Mrp1−/− has much lower AUC0-t (Table 3), which may be attributed to several reasons. First, given that kaempferol conjugates are the likely substrates of BCRP and MRPs, the knockout of these transporters can influence the intestinal efflux and biliary clearance of the conjugates, thereby changing the blood concentrations. Second, given that a published assay identifies kaempferol as a BCRP substrate (24), blocking kaempferol flux via Bcrp knockout may increase the uptake of kaempferol aglycone. Changes in kaempferol flux may result in a higher formation of conjugates in the intestine or liver. We did not focus on kaempferol aglycone in this study because the amount of free kaempferol in vivo is extremely small. Future studies must be conducted to define the parent kaempferol by using other cell models, e.g., MDCKII cultured cells (33).
The plasma concentrations of the metabolites are lower in Mdr1a−/− mice. The lack of Mdr1a does not cause statistical differences in the AUC0-t of K-7-G and K-7-S, but significantly decreases the AUC0-t of K-3-G. Such phenomenon might be related to altered metabolism because Mdr1a knockout decreases the hepatic metabolic rates of K-3-G, K-7-G, and K-7-S (Fig. 4). Such metabolic changes are also observed in other knockout mice, which indicates that the knockout of efflux transporters may cause unexpected changes in UGT and SULT activities. Mrp1 knockout slightly decreases hepatic glucuronidation and sulfation rates, and intestinal sulfation rates, which indicates that the highly significant reduction in the plasma concentrations of the three metabolites in Mrp1−/− mice may be partly attributed to the decreased metabolism. Mrp2 knockout slightly lowers the hepatic glucuronidation rates and greatly reduces the intestinal and hepatic sulfation rates. However, the plasma concentrations of the metabolites are significantly increased in Mrp2−/− mice, which suggests the vital role of Mrp2 in increasing the exposure levels of kaempferol conjugates. For Bcrp1−/−, the intestinal glucuronidation rates decrease yet the hepatic glucuronidation rates increase. Another study reported that Bcrp1 knockout increased the genistein glucuronidation rates and decreased the sulfation rates (38). Therefore, pharmacokinetics in knockout mice only demonstrates that efflux transporters significantly affect the exposure levels of kaempferol conjugates in vivo.
In conclusion, a combined strategy was used to investigate the in vivo exposure of kaempferol. Our study revealed well-correlated in vitro, in situ, and in vivo data. In plasma, kaempferol is mostly exposed as phase II conjugates, including two glucuronides and one sulfate. K-3-G represents the major metabolite. Efflux transporters, namely, BCRP, MRP1, and MRP2, regulate the in vivo exposure levels of kaempferol conjugates. Therefore, the in vivo exposure of kaempferol is driven by phase II metabolic enzymes and efflux transporters, such as BCRP and MRPs.
Supplementary Material
Acknowledgments
This work was supported by the grants of National Natural Science Foundation of China (81120108025 and 81503466), Science and Technology Project of Guangzhou City (201509010004) and Guangdong Natural Science Foundation Province (2015AD030312012).
References:
- 1.Liu RH. Health-promoting components of fruits and vegetables in the diet. Adv Nutr. 2013;4(3):384S–392S. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Ravishankar D, Rajora AK, Greco F, Osborn HM. Flavonoids as prospective compounds for anti-cancer therapy. Int J Biochem Cell Biol. 2013;45(12):2821–31. [DOI] [PubMed] [Google Scholar]
- 3.Calderon-Montano JM, Burgos-Moron E, Perez-Guerrero C, Lopez-Lazaro M. A review on the dietary flavonoid kaempferol. Mini Rev Med Chem. 2011;11(4):298–344. [DOI] [PubMed] [Google Scholar]
- 4.Nothlings U, Murphy SP, Wilkens LR, Henderson BE, Kolonel LN. Flavonols and pancreatic cancer risk: The multiethnic cohort study. Am J Epidemiol. 2007;166(8):924–31. [DOI] [PubMed] [Google Scholar]
- 5.Wang L, Tu YC, Lian TW, Hung JT, Yen JH, Wu MJ. Distinctive antioxidant and antiinflammatory effects of flavonols. J Agric Food Chem. 2006;54(26):9798–804. [DOI] [PubMed] [Google Scholar]
- 6.Heijnen CG, Haenen GR, van Acker FA, van der Vijgh WJ, Bast A. Flavonoids as peroxynitrite scavengers: The role of the hydroxyl groups. Toxicol in Vitro. 2001;15(1):3–6. [DOI] [PubMed] [Google Scholar]
- 7.Kong L, Luo C, Li X, Zhou Y, He H. The anti-inflammatory effect of kaempferol on early atherosclerosis in high cholesterol fed rabbits. Lipids Health Dis. 2013;12:115. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Rajendran P, Rengarajan T, Nandakumar N, Palaniswami R, Nishigaki Y, Nishigaki I. Kaempferol, a potential cytostatic and cure for inflammatory disorders. Eur J Med Chem. 2014;86:103–12. [DOI] [PubMed] [Google Scholar]
- 9.Dang Q, Song W, Xu D, Ma Y, Li F, Zeng J, et al. Kaempferol suppresses bladder cancer tumor growth by inhibiting cell proliferation and inducing apoptosis. Mol Carcinog. 2015;54(9):831–40. [DOI] [PubMed] [Google Scholar]
- 10.Lee HS, Cho HJ, Yu R, Lee KW, Chun HS, Park JH. Mechanisms underlying apoptosis-inducing effects of Kaempferol in HT-29 human colon cancer cells. Int J Mol Sci. 2014;15(2):2722–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Yao K, Chen H, Liu K, Langfald A, Yang G, Zhang Y, et al. Kaempferol targets RSK2 and MSK1 to suppress UV radiation-induced skin cancer. Cancer Prev Res (Phila). 2014;7(9):958–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Cui Y, Morgenstern H, Greenland S, Tashkin DP, Mao JT, Cai L, et al. Dietary flavonoid intake and lung cancer-A population-based case-control study. Cancer-Am Cancer Soc. 2008;112(10):2241–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Gates MA, Tworoger SS, Hecht JL, De Vivo I, Rosner B, Hankinson SE. A prospective study of dietary flavonoid intake and incidence of epithelial ovarian cancer. Int J Cancer. 2007;121(10):2225–32. [DOI] [PubMed] [Google Scholar]
- 14.Bobe G, Sansbury LB, Albert PS, Cross AJ, Kahle L, Ashby J, et al. Dietary flavonoids and colorectal adenoma recurrence in the Polyp Prevention Trial. Cancer Epidemiol Biomarkers Prev. 2008;17(6):1344–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Chen AY, Chen YC. A review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention. Food Chem. 2013;138(4):2099–107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Chen Z, Zheng S, Li L, Jiang H. Metabolism of flavonoids in human: A comprehensive review. Curr Drug Metab. 2014;15(1):48–61. [DOI] [PubMed] [Google Scholar]
- 17.Chen J, Lin H, Hu M. Metabolism of flavonoids via enteric recycling: Role of intestinal disposition. J Pharmacol Exp Ther. 2003;304(3):1228–35. [DOI] [PubMed] [Google Scholar]
- 18.Ma Y, Zeng M, Sun R, Hu M. Disposition of flavonoids impacts their efficacy and safety. Curr Drug Metab. 2014;15(9):841–64. [DOI] [PubMed] [Google Scholar]
- 19.Silva ID, Rodrigues AS, Gaspar J, Maia R, Laires A, Rueff J. Involvement of rat cytochrome 1A1 in the biotransformation of kaempferol to quercetin: Relevance to the genotoxicity of kaempferol. Mutagenesis. 1997;12(5):383–90. [DOI] [PubMed] [Google Scholar]
- 20.Barve A, Chen C, Hebbar V, Desiderio J, Saw CL, Kong AN. Metabolism, oral bioavailability and pharmacokinetics of chemopreventive kaempferol in rats. Biopharm Drug Dispos. 2009;30(7):356–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Yodogawa S, Arakawa T, Sugihara N, Furuno K. Glucurono- and sulfo-conjugation of kaempferol in rat liver subcellular preparations and cultured hepatocytes. Biol Pharm Bull. 2003;26(8):1120–4. [DOI] [PubMed] [Google Scholar]
- 22.Romiti N, Tramonti G, Donati A, Chieli E. Effects of grapefruit juice on the multidrug transporter P-glycoprotein in the human proximal tubular cell line HK-2. Life Sci. 2004;76(3):293–302. [DOI] [PubMed] [Google Scholar]
- 23.Wang Y, Cao J, Zeng S. Involvement of P-glycoprotein in regulating cellular levels of Ginkgo flavonols: Quercetin, kaempferol, and isorhamnetin. J Pharm Pharmacol. 2005;57(6):751–8. [DOI] [PubMed] [Google Scholar]
- 24.An G, Gallegos J, Morris ME. The bioflavonoid kaempferol is an abcg2 substrate and inhibits Abcg2-Mediated quercetin efflux. Drug Metab Dispos. 2011;39(3):426–32. [DOI] [PubMed] [Google Scholar]
- 25.Limtrakul P, Khantamat O, Pintha K. Inhibition of P-glycoprotein function and expression by kaempferol and quercetin. J Chemother. 2005;17(1):86–95. [DOI] [PubMed] [Google Scholar]
- 26.Barrington R, Williamson G, Bennett RN, Davis BD, Brodbelt JS, Kroon PA. Absorption, conjugation and efflux of the flavonoids, kaempferol and galangin, using the intestinal CaCo-2/TC7 cell model. J Funct Foods. 2009;1(1):74–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Zhang W, Wang X, Zhou S, Gu Y, Wang R, Zhang T, et al. Determination of free and glucuronidated kaempferol in rat plasma by LC-MS/MS: Application to pharmacokinetic study. J Chromatogr B. 2010;878(23):2137–40. [DOI] [PubMed] [Google Scholar]
- 28.Shi J, Zheng L, Lin Z, Hou C, Liu W, Yan T, et al. Study of pharmacokinetic profiles and characteristics of active components and their metabolites in rat plasma following oral administration of the water extract of Astragali radix using UPLC-MS/MS. J Ethnopharmacol. 2015;169:183–94. [DOI] [PubMed] [Google Scholar]
- 29.Dai P, Zhu L, Luo F, Lu L, Li Q, Wang L, et al. Triple recycling processes impact systemic and local bioavailability of orally administered flavonoids. AAPS J. 2015;17(3):723–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Zhu W, Xu H, Wang SWJ, Hu M. Breast cancer resistance protein (BCRP) and sulfotransferases contribute significantly to the disposition of genistein in mouse intestine. AAPS J. 2010;12(4):525–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Ye L, Lu L, Li Y, Zeng S, Yang X, Chen W, et al. Potential role of ATP-binding cassette transporters in the intestinal transport of rhein. Food Chem Toxicol. 2013;58:301–5. [DOI] [PubMed] [Google Scholar]
- 32.Liu W, Feng Q, Li Y, Ye L, Hu M, Liu Z. Coupling of UDP-glucuronosyltransferases and multidrug resistance-associated proteins is responsible for the intestinal disposition and poor bioavailability of emodin. Toxicol Appl Pharm. 2012;265(3):316–24. [DOI] [PubMed] [Google Scholar]
- 33.Dai P, Zhu L, Yang X, Zhao M, Shi J, Wang Y, et al. Multidrug resistance-associated protein 2 is involved in the efflux of Aconitum alkaloids determined by MRP2-MDCKII cells. Life Sci. 2015;127:66–72. [DOI] [PubMed] [Google Scholar]
- 34.Singh R, Wu B, Tang L, Liu Z, Hu M. Identification of the position of mono-O-glucuronide of flavones and flavonols by analyzing shift in online UV spectrum (λmax) generated from an online diode array detector. J Agr Food Chem. 2010;58(17):9384–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Tang L, Zhou J, Yang C, Xia B, Hu M, Liu Z. Systematic studies of sulfation and glucuronidation of 12 flavonoids in the mouse liver S9 fraction reveal both unique and shared positional preferences. J Agr Food Chem. 2012;60(12):3223–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Enokizono J, Kusuhara H, Sugiyama Y. Regional expression and activity of breast cancer resistance protein (Bcrp/Abcg2) in mouse intestine: Overlapping distribution with sulfotransferases. Drug Metab Dispos. 2007;35(6):922–8. [DOI] [PubMed] [Google Scholar]
- 37.Cherrington NJ, Hartley DP, Li N, Johnson DR, Klaassen CD. Organ distribution of multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and hepatic induction of Mrp3 by constitutive androstane receptor activators in rats. J Pharmacol Exp Ther. 2002;300(1):97–104. [DOI] [PubMed] [Google Scholar]
- 38.Yang Z, Zhu W, Gao S, Yin T, Jiang W, Hu M. Breast cancer resistance protein (ABCG2) determines distribution of genistein phase II metabolites: Reevaluation of the roles of ABCG2 in the disposition of genistein. Drug Metab Dispos. 2012;40(10):1883–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
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