Time-Dependent Metabolism of Luteolin by Human UDP

Sep 17, 2015 - Department of Urology, Nanfang Hospital, Southern Medical ... E-mail: [email protected]., *E-mail: [email protected]. ... This study inve...
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Time-Dependent Metabolism of Luteolin by Human UDPGlucuronosyltransferases and Its Intestinal First-Pass Glucuronidation in Mice Lili Wu,†,# Junjin Liu,†,# Weichao Han,† Xuefeng Zhou,‡ Xiaoming Yu,∥ Qiang Wei,∥ Shuwen Liu,*,†,§ and Lan Tang*,†,§ †

Guangdong Provincial Key Labortory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China ‡ Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China § State Key Laboratory of Organ Failure Research, Guangdong Provincial Institute of Nephrology, Southern Medical University, Guangzhou 510515, China ∥ Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China ABSTRACT: Luteolin is a well-known flavonoid with various pharmacological properties but has low bioavailability due to glucuronidation. This study investigated the time-course of luteolin glucuronidation by 12 human UDP-glucuronosyltransferases (UGTs) and its intestinal first-pass metabolism in mice. Six metabolites, including two novel abundant diglucuronides [3′,7-Odiglucuronide (diG) and 4′,7-diG] and four known ones, were identified. UGT1A6 and UGT1A9 generated almost only monoglucuronides (G’s). The production of 3′,7-diG followed a sequential time-dependent process along with decrease of 3′-G mainly by UGT1A1, indicating that 3′,7-diG was produced from 3′-G. Metabolism in mice intestine differed from that in humans. Probenecid, a nonspecific UGT inhibitor, did not affect absorption but significantly inhibited production of 7-, 4′-, and 3′-G, and enhanced the formation of another novel metabolite, 5-G, in mice. In conclusion, diglucuronide formation is timedependent and isoform-specific. UGT1A1 preferentially generates diG, whereas UGT1A6 and UGT1A9 share a preference for G production. KEYWORDS: luteolin, diglucuronidation, UGTs, time-dependent, absorption, metabolism

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degrading luteolin in UGT1A9 HeLa cells.10 The level of 3′,4′diG increased linearly with time, whereas that of 4′-G decreased with time after 30 min (p < 0.01). This trend was not the same across the three G’s; i.e., formation of diG’s in UGT1A9 was found to be a sequential process. In contrast to UGT1A9, UGTs 1A1, 1A3, 1A7, 1A8, and 1A10 are all isoforms responsible for luteolin glucuronidation. Therefore, we sought to optimize the experimental conditions to identify other novel luteolin metabolites and to uncover an alternative pathway for luteolin elimination. Furthermore, we aimed to further monitor the time course of luteolin metabolism in these UGTs. Previous studies explored the mechanism underlying luteolin absorption using in situ single-pass intestinal perfusion. Results showed that luteolin is passively absorbed in the intestine of rats, and the effective permeability and absorption rates of luteolin in duodenum and jejunum were markedly higher than those in the colon and ileum.11 One study suggested that the 5-, 7-, and 4′-hydroxyl groups of luteolin limited its apparent absorption in humans.12 Luteolin has more 3′-hydroxyl groups than apigenin, and the permeability of luteolin was found to be

INTRODUCTION Luteolin is a well-known flavone found in many natural plants, including some vegetables and food items such as Bryophyta, Pteridophyta, celery, carrots, peppers, olive oil, and peppermint.1 Luteolin has a multitude of beneficial pharmacological and biological properties, including anti-inflammation, antioxidation, anti-neuropathic pain, anti-allergy, and anti-cancer.2,3 Given its ability to penetrate into human skin, luteolin has also been considered as a candidate material to prevent and treat skin cancer.4 However, efforts at developing luteolin into a chemo-preventive agent have faced numerous challenges. Low bioavailability of luteolin because of extensive first-pass metabolism by phase II enzymes, including UGTs, remains a major challenge.5 Oral bioavailability of luteolin has been found to be only 26 ± 6% in rats.6 Theoretically, luteolin, which has four hydroxyl groups, can be metabolized to four monoglucuronides and six diglucuronides (diG’s). Monoglucuronides (G’s) are reportedly the main metabolites found in rat plasma and human serum.7−9 Three glucuronidated luteolin metabolites, namely, 7-, 3′-, and 4′-G, have been found in the phase II biotransformation of luteolin by human UGT isoenzymes.5 In our previous study, a novel 3′,4′-diG and three known G’s were found in 12 human UGT isoforms, and UGT1A9 was found to be the major isoform responsible for formation of glucuronides. Production of diG was speculated to be a compensatory pathway for © XXXX American Chemical Society

Received: June 9, 2015 Revised: September 16, 2015 Accepted: September 17, 2015

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Luteolin Glucuronidation by 12 Expressed Human UGTs and the Time-Dependent Experiment. Magnesium chloride (0.88 mM), D-saccharic-1,4-lactone monohydrate (4.4 mM), alamethicin (0.022 mg/mL), 12 UGT isoforms (UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17, with final concentrations between 0.0528 and 0.264 mg/mL), 1 mM luteolin (final concentration of 10 μM) in 50 mM KPi (pH 7.4), and uridine 5′diphospho-glucuronic acid (UDPGA, 3.5 mM, added last) were mixed together. The UGT reaction was incubated for different times at 37 °C in a shaking water bath (50 rpm). About 60 μL of iced methanol containing 0.5 μg/mL propiophenone was added to stop the reaction. All experiments were performed in triplicate. The samples were centrifuged at 13 000 rpm at 4 °C for 30 min, and the supernatant was then analyzed by ultra-performance liquid chromatography (UPLC). To elucidate the characteristics of luteolin glucuronidation by expressed human UGTs, the incubation time was set to 6 h. For the time-dependence metabolism experiment, the UGT reaction system was incubated at 37 °C for 30, 60, 90, and 120 min for each UGT isoform. The rest of the procedures were performed as described above. To identify the product of diglucuronides, the incubation time of luteolin in 12 UGTs was prolonged to 6 h, and the metabolite amount was calculated as nmol per mg. Animal Surgery. Male KM mice weighing between 22 and 25 g were obtained from the Laboratory Animal Centre of Southern Medical University. The mice were fasted overnight with free access to water before commencing the experiment. The experiments were done with permission from the Ethics Committee of Southern Medical University. Mice were then anesthetized with an i.p. injection of 0.026 mL/g mebubarbital (0.3% m/v). The intestinal surgical procedures were essentially the same as those described in previous publications with minor modifications.18 We perfused two segments of the intestine (small intestine and colon), with each segment 8−10 cm long. Blood circulation to the liver and intestine was not disrupted in this perfusion model. During the surgery, the body temperature was maintained at 37 °C using a heating lamp. The inlet cannulate was flushed with luteolin in HBSS, which was kept warm at 37 °C by a circulating water bath. Perfusion Experiments. First, mice gall bladders were obtained, and two segments of mice intestine (small intestine and colon) were simultaneously perfused with a perfusate containing 10 and 20 μM luteolin using an infusion pump at a flow rate of 10 mL/h. To terminate substrate hydrolysis of the enzymes contained in the perfused sample, 0.5 mL of acetonitrile (a stop solution) was added into the receiving container prior to sample collection. Addition of acetonitrile prevented hydrolysis of luteolin and stabilized it. After 15 min of washout period, the samples were collected every 30 min. At the end of the experiment, we measured the intestinal length as described in a previous study18 About 200 μL of acetonitrile containing 10 μg/mL propiophenone was added into 400 μL of perfusate. The samples were analyzed via UPLC after centrifugation at 13000 rpm at 4 °C for 30 min. Effect of UGT Inhibitor on Luteolin Glucuronidation in Situ. Probenecid is a potent nonspecific inhibitor of UGTs that significantly inhibits luteolin glucuronidation in vivo. Animal surgery was done as described above. Probenecid was added at 0.5 and 1 mM to perfusate containing 10 and 20 μM luteolin, respectively. About 200 μL of 10 μg/mL propiophenone was added to 400 μL of perfusate. The samples were centrifuged at 13 000 rpm at 4 °C for 30 min, and 10 μL of the supernatant was then analyzed via UPLC. The method for analyzing luteolin was the same as previously described.10 UPLC Analysis of Luteolin and Its Glucuronide. The validated method for analyzing luteolin was as follows: system, Waters Acquity UPLC; column, HSS T3, 1.8 μm, 2.1 mm × 100 mm; mobile phase A, 100% acetonitrile; mobile phase B, 0.1% (v/v) formic acid; flow rate, 0.25 mL/min; gradient 0−5 min, 95−45% B, 5−5.5 min, 45−30% B, 5.5−7 min, 30−95% B; wavelength, 340 nm for luteolin and the respective glucuronides, 254 nm for propiophenone; and injection volume, 10 μL. The tested linear response range was 0.3125−40 μM (for a total of eight concentrations) for luteolin. Analytical methods for each test compound were validated for inter-day and intra-day variation using six samples at three concentrations (40, 10, and 2 μM).

about 50% that of apigenin in an in situ rat intestinal single-pass perfusion model. This result is consistent with the observation that oral bioavailability of luteolin (30.4%) from a Flos chrysanthemi extract was significantly lower than that of apigenin (51.1%).13 Unfortunately, reports on diglucuronidation of luteolin in situ or in vivo are rare. This process might be a feasible method for eliminating luteolin and thereby controlling its bioavailability. Given that over 35% of all drugs are metabolized via phase II enzymes, especially UGTs,14 bioavailability of herbal medicines may be altered when their metabolism is modulated by coadministered drugs, namely, drug−drug interactions. The UGT inhibitor is also involved in many drug−drug interactions, and studies have reported that metabolic clearance of these drugs is inhibited by glucuronidation, such as zidovudine−fluconazole interactions. Fluconazole increases exposure to zidovudine, a typical probe for UGT2B7, to 74% owing to its inhibition of zidovudine glucuronidation.15 Thus, the effect of the UGT inhibitor on luteolin remains to be elucidated. In this study, we sought to systematically elucidate the time course of glucuronidation of luteolin by 12 human UDPglucuronosyltransferases and to further determine the absorption characteristics and metabolic processes of luteolin using an in situ mice intestinal single-pass perfusion model. Specifically, probenecid, a nonspecific UGT inhibitor,16 was used in this perfusion model. Our findings help us to understand first-pass metabolism and disposition of luteolin via enteric recycling.

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MATERIALS AND METHODS

Chemicals and Reagents. Luteolin, propiophenone, probenecid, formic acid (HPLC grade), and testosterone were purchased from Aladdin Reagent Company (Shanghai, China). Mebubarbital was purchased from Center of Reagent, Southern Medical University (Guangzhou, China). Uridine diphosphoglucuronic acid (UDPGA), Dsaccharic-1,4-lactone monohydrate, HEPES, and Hanks’ balanced salt (powder form) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alamethicin was purchased from Hui Cheng Biological Technology Co. Ltd. (Shanghai, China). Human-expressed UGT isoforms (UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) were also obtained from BD Gentest Corp. (Woburn, MA, USA). Acetonitrile and methanol were typically analytical grade or better (≥99%, HPLC grade), and were used as received. Animals. Use of all animals was approved by the Ethics Committee of Southern Medical University. Male KM mice weighing between 22 and 25 g were obtained from Laboratory Animal Centre of Southern Medical University. Mice were then fasted overnight with free access to water before the experiment. Determination of the Concentration Conversion Factor (K Values). To quantify each metabolite, the concentration conversion factor (K), which represents the ratio between the molar extinction coefficient of each glucuronide and luteolin, was determined following a previously described method.17 To calculate the K values, each metabolite was separated via HPLC, collected for blow drying in nitrogen, dissolved in 50 mM potassium phosphate (KPi, pH 7.4), and then divided into two parts, one of which was analyzed directly and the other was analyzed after being hydrolyzed with β-glucuronidase (100 units/mL) at 37 °C for 2 h. To determine the conjugation position of diG’s, the metabolites were separated, collected, and then hydrolyzed with β-glucuronidase for 1 h to ensure that the diG’s were not completely hydrolyzed and could produce two related G’s. Retention time of the metabolites is a determinant for the conjugation position of luteolin (e.g., 3′,7-diG and 4′,7-diG). Relationships between the peak areas of the metabolites before hydrolysis and the peak areas of aglycones after hydrolysis were used to establish the K values needed to quantify the amounts of luteolin conjugates. B

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Figure 1. continued

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Figure 1. UPLC and HRMS profiles of luteolin and its metabolites, and the structures of the metabolites. UPLC was used to separate and quantify luteolin and its metabolites in the experimental samples generated from incubation with human UGTs and perfusate. (A1) Chromatograms of luteolin and its metabolites in sample incubated with human UGTs. (A2) Chromatograms of luteolin and its metabolites in perfusate. (B1−B3) HRMS scan of diglucuronides (diG’s). (B4−B7) HRMS scans of monoglucuronides. Precision and accuracy for luteolin were in an acceptable range (80-fold higher). This surprising discovery demonstrated that UGT1A1 prefers 7-OH and 3′-OH more than 4′-OH. Furthermore, this result also revealed a sequential time-dependent process in which when the production of 7-G reaches a balance in time, UGT1A1 tends to catalyze glucuronic acid to the 3′-OH position of 7-G to produce 3′,7-diG (Figures 3A and 8). Unlike production of the three G’s, which peaked within 30 min of incubation and remained stable thereafter, production of 3′,4′diG slowly increased within 120 min of incubation. However, in UGTs 1A3, 1A4, 1A6, 1A7, 1A8, and 1A10, the amount of diG’s (3′,7-diG or 3′,4′-diG) increased steadily with the three G’s from the beginning to the end (0 to 120 min). In UGT2B’s, the amounts of all the glucuronides slowly increased after 120 min of incubation. Among these isoforms, UGT1A1 and UGT2B7 are well expressed in both the intestine and the liver, whereas UGT1A9 is well expressed in the liver and the kidneys24 but either poorly expressed14 or not expressed at all in the intestine.25 In contrast, UGT1A8 and UGT1A10 are well expressed in the intestine but not in the liver.24,25 Thus, we could speculate that the liver, kidney, and intestine are all major organs for luteolin glucuronidation. Moreover, since UGT1A1, UGT1A8, and

where the 7-OH group was found to be the most reactive for glucuronidation even in more complex flavones with two phenolic groups.17−23 In addition, the 3′-OH group was found to be highly reactive.21−23 A hypothesis that can explain the lack of a 3′ hydroxyl group in the flavonoids is that glucuronidation only occurred at position 7, and those containing this functional group also formed 3′-O-glucuronides and sometimes 4′-O-glucuronides. Thus, the presence or absence of the 3′-OH group is a major determinant of regioselectivity of glucuronidation. Moreover, the specific distribution of multiple glucuronide products (7-O, 3′-O, 4′O) is governed by the hydroxyl group of flavonoids.20 We also detected very small amounts of 4′,7-diG, produced mainly by UGT1A1. For 3′,4′-diG, another minor diG, UGTs 1A1, 1A7, 1A8, 1A9, and 1A10 were all identified to be the major isoforms responsible for its production. In UGT1A9, three G’s and 3′,4′diG (very small amounts) were identified. These results indicated that UGT1A9 was the most active isoform in catalyzing the formation of G’s (Figure 2A). UGT1A6 was one of the top six isoforms (UGTs 1A1, 1A6, 1A7, 1A8, 1A9, and 1A10) with high activity but could only catalyze 7-G formation (Figure 2B). All UGT isoforms catalyzed the biotransformation of luteolin to 7-G and 4′-G, except UGT1A4. These findings are consistent with those of our previous study, in which luteolin metabolism was found to be mediated chiefly by UGT1A1, UGT1A9, and UGT1A6.10 We systematically defined the time dependence of luteolin diglucuronidation in 12 human-expressed UGT isoforms. From the beginning of the incubation up until 30 min, the amounts of 3′-G, 4′-G, 7-G, and 3′,7-diG produced in UGT1A1 were all about 2 nmol/mg. However, the outcome changed after 30 min I

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Figure 7. Effect of the UGT-specific inhibitor probenecid on glucuronide excretion: amount of luteolin glucuronides excreted in the absence or presence of probenecid at 0.5 and 1 mM in the small intestine and colon. Each column represents the average of three determinations, with the error bar representing SD (n = 3). *p < 0.05; **p < 0.01.

Figure 8. Glucuronic pathways for conversion of luteolin to luteolin-3′,7-O-diglucuronide and the sequential time-dependent process in human UGT1A1.

UGT1A10 are not expressed in the kidney, we speculate that diG’s may not be produced in the kidney. Several studies have shown that some nuclear receptors, such as constitutive androstane receptor (CAR),26 pregnane X receptor (PXR),27 and aryl hydrocarbon receptor (AhR), are all involved in UGT modulation. These factors play important roles in the modulation of glucuronidation of natural products

through UGTs. It has been demonstrated that AhR, which can induce activities of human UGT1A1, is involved in modulation of UGT1A1 by luteolin.28 These results suggest that more attention should be paid to drug−drug interactions by UGTs, which have brought lots of studies upon the mechanism. In situ glucuronidation is quite different from the process that occurs in vitro because four G’s and 3′,4′-diG were found J

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metabolizing phenols might be responsible for metabolism of luteolin in mice.

in mice perfusate in the current study. The novel 5-G was not found in UGT reaction systems. Most research on flavonoid glucuronidation in vivo and in vitro indicates that metabolism of the 5-hydroxyl group is slow or lacking, which may be due to the fact that the 5-OH group favors the formation of an intramolecular hydrogen bond with the carbonyl group at the C-4 position.17,18,22,23 For 5-hydroxyflavone, in UGTs 1A1, 1A8, 1A9, 1A10, and 2B7, glucuronidation was apparent, but the other UGT isoforms did not metabolize it.17,18 Thus, 5-G could be detected in mice intestine, although it was not produced in the 12 UGT isoforms. Unexpectedly, the amount of 5-G was the highest for all glucuronides in situ. Two explanations are proposed: (1) difference in species might have caused this disparity; (2) other enzymes might be involved in the phase II reaction of luteolin. G’s are the major glycoside species of mammals, and members of UGT1 and UGT2 are involved in their formation. However, the UGTs involved in the formation of most glycosides have not been identified and may belong to the UGT3A family.29 Recently, a novel UGT2A1-mediated glucuronidation has been reported, and we speculate that 5-G may also be catalyzed by UGT2A1.30 Further refinement of our approach is needed by incorporating additional UGT isoforms, including the newly cloned UGT3A family (UGT3A1/2) and UGT2A1. The absorption/metabolism of luteolin was also investigated in mice. The absorption amount as well as the absorption rate of luteolin were significantly different (p < 0.05) at 10 and 20 μM, which were markedly higher in the small intestine than in the colon. These results are consistent with those reported previously in rat.11 Thus, luteolin was passively absorbed in the intestine of mice, and absorption was found to be more efficient in the small intestine than in the colon (Figure 4). The amount of excreted luteolin was significant in the small intestine and colon, thereby indicating that luteolin metabolism is concentration-dependent (Figure 6). Surprisingly, probenecid (0.5 and 1 mM) had no effect on luteolin absorption (Figure 5); the excreted amounts of 7-, 4′-, 3′-G, and 3′,4′-diG decreased, but that of 5-G increased when probenecid was used. Moreover, high concentrations of probenecid resulted in production of high amounts of 5-G. The amount of total metabolites remained almost unchanged (Figure 7). We speculate that probenecid is not an inhibitor of UGT3A, since it could not inhibit 5-G production. When UGT1A and UGT2B are inhibited, a compensatory pathway may turn on for activation of other UGTs. Many luteolin glucosides, such as luteolin 7-O-β-glucopyranoside and the C-glucoside homo-orientin (luteolin 6-Cglucoside),31 are found in vivo and in vitro. Therefore, cleavage of the glucoside by gut bacteria or at the intestinal wall associated with transporters would affect the overall uptake and metabolism/metabolic fate of luteolin. We note that it is important to conduct in vivo experiments with luteolin glucosides in mice to validate these findings. In conclusion, two novel luteolin diG’s, namely 3′,7- and 4′,7-diG, were detected in UGTs for the first time, and a novel G, namely 5-G, was found in situ. UGT1A1, UGT1A6, and UGT1A9 shared the same preference for production of monoglucuronides but displayed unique preferences in diG production such that UGT1A1 preferred to generate diG. Production of 3′,7-diG exhibited time dependence based on the consumption of 3′-G and 7-G by UGT1A1. A compensatory pathway for a newly discovered UGT isoform capable of

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AUTHOR INFORMATION

Corresponding Authors

*Tel./fax: +86 20-61648596. E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

L.W. and J.L. contributed equally to this paper.

Funding

This work was supported by the Foundation for Distinguished Young Teachers in Higher Education of Guangdong (Yq2013037), Foundation for graduate education innovation project of Guangdong (Sybzzxm201224), and the Foundation of science and technology of Guangdong (2014A010107012), as well as the Program for Pearl River New Stars of Science and Technology in Guangzhou (2012J2200048). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ai-Jun Sun and Yun Zhang at South China Sea Institute of Oceanology (SCSIO, CAS) for their assistance in LC-HRMS experiments.

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REFERENCES

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DOI: 10.1021/acs.jafc.5b02827 J. Agric. Food Chem. XXXX, XXX, XXX−XXX