Mutual interactions between flavonoids and enzymatic and transporter elements responsible for flavonoid disposition via phase II metabolic pathways

Abstract

Flavonoids, existing mainly as glycosides in nature, have multiple “claimed” beneficial effects in humans. Flavonoids are extensively metabolized in enterocytes and hepatocytes by phase II enzymes such as UGTs and SULTs to form glucuronides and sulfates, respectively. These glucuronides and sulfates are subsequently excreted via ABC transporters (e.g., MRP2 or BCRP). Therefore, it is the interplay between phase II enzymes and efflux transporters that affects the disposition of flavonoids and leads to the low bioavailability of flavonoid aglycones. Flavonoids can also serve as chemical regulators that affect the activity or expression levels of phase II enzymes including UGTs, SULTs and GSTs, and transporters including P-gp, MRP2, BCRP, OATP and OAT. In general, flavonoids may exert the inhibitory or inductive effects on the phase II enzymes and transporters via multiple mechanisms that may involve different nuclear receptors. Since flavonoids may affect the metabolic pathways shared by many important clinical drugs, drug–flavonoid interaction is becoming an increasingly important concern. This review article focuses on the disposition of flavonoids and effects of flavonoids on relevant enzymes (e.g. UGTs and SULTs) and transporters (e.g. MRP2 and BCRP) involved in the interplay between phase II enzymes and efflux transporters. The effects of flavonoids on other metabolic enzymes (e.g. GSTs) or transporters (e.g. P-gp, OATP and OAT) are also addressed but that is not the emphasis of this review.

---

Wen Jiang

Wen Jiang graduated from Nanjing University of Technology with a Masters degree in Biochemical Engineering in 2003 (P.R.China). She received her PhD degree in Pharmaceutics from the Department of Pharmacological and Pharmaceutical Sciences at the University of Houston in 2011 (USA), under primary direction of Dr Ming Hu. Her main research work is focused on the mechanisms of interplay between UGTs and efflux transporters in flavonoid disposition.

Ming Hu

Ming Hu received his PhD in Pharmaceutics from College of Pharmacy at the University of Michigan. Currently, he is a full professor in the Department of Pharmacological and Pharmaceutical Sciences of University of Houston (USA). His primary research interests include bioavailability of drugs, nutrients and micronutrients with emphasis on mechanisms of absorption and metabolism of flavonoids and phenolic drugs. He published and contributed numerous pioneer papers and book chapters. He has served in editorial boards from multiple journals. Most of his current research are funded by the National Institutes of Health of USA.

---

1. Introduction

Flavonoids are a class of phenolic and polyphenolic compounds produced as secondary plant metabolites. Flavonoids are widely distributed in nature. The typical sources of flavonoids include vegetables, fruits, beans, grains and herbs. Therefore, an average person could have a significant intake of flavonoids from their daily diet. Published studies indicate that the typical daily intake of flavonoids is in the range of 20–190 mg day⁻¹ in typical Western populations.^1,2,3

In nature, flavonoids are usually presented as glycosides and to a less extent as aglycones as well as their methylated derivatives. For aglycones, they share the backbone consisting of three carbon rings: a 1,4-benzopyran ring (A), in which the 2 position is linked to a phenyl ring (B) as a substituent.⁴ For glycosides, the sugars are normally attached to the hydroxyl groups at position 3 or 7 of the A ring. The common sugars or carbohydrates linked to aglycones are D-glucose, glucorhamnose, galactose, L-rhamnose, or arabinose.⁴ For methylated derivatives, the methylation usually occurs at position 5′ and 3′ of the B ring or at position 7 of the A ring.⁵ According to their chemical structures, flavonoids (aglycones) are classified into several subfamilies: flavones, flavonols, flavanone, flavanonol, flavanol, isoflavones, anthocyanidin and chalcones (Table 1).^6,7,8,9

Table 1 Classification of flavonoids and typical compounds of each subfamily

Subclass	Flavonoid	Substitution pattern
a Silybin: (2R,3R)-3,5,7-trihydroxy-2-[(2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl]chroman-4-one. b Ellagic acid: 2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione. c Cyanidin: 2-(3,4-dihydroxyphenyl) chromenylium-3,5,7-triol. d Phloretin: 3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one.
Flavones	Chrysin	5,7-OH
	Apigein	5,7,4′-OH
	Luteolin	5,7,3′,4′-OH
	Eupatilin	5,7-OH, 6,4′,5′-OMe
	Baicalein	5,6,7-OH
	Nobiletin	5,6,7,8,4′5′-OMe
	Tangeritin	5,6,7,8,4′-OMe
	Tricin	5,7,4′-OH,3′,5′-OMe
	Wogonin	5,7-OH,8-OMe
Flavonol	Isorhamnetin	3,5,7,4′-OH,3′-OMe
	Kaempferol	3,5,7,4′-OH
	Myricetin	3,5,7,3′,4′,5′-OH
	Quercetin	3,5,7,3′,4′-OH
	Morin	3,5,7,2′,4′-OH
	Galangin	3,5,7-OH
	Fisetin	3,7,4′,5′-OH
Flavanone	Naringenin	5,7,4′-OH
	Hesperetin	5,7,3′-OH,4′-OMe
	Eriodictyol	5,7,3′,4′-OMe
Flavanonol	Silybin^a	3,5,7,-OH
Flavanol	(−)-Epicatechin (EC)	3,5,7,3′,4′-OH (2-S, 3-R)
	(+)-Catechin	3,5,7,3′,4′-OH (2-S, 3-S)
	Epigallocatechin gallate (EGCG)	3,5,7,3′,4′,5′,-OH, 3-gallate
	Ellagic acid^b	2,3,7,8-OH
Isoflavones	Daidzein	7,4′-OH
	Genistein	5,7,4′-OH
	Glycitein	6,4′-OH,7-OMe
	Formononetin	7-OH,4′-OMe
	Biochanin A	5,7-OH,4′-OMe
	Prunetin	5,4′-OH,7-OMe
Anthocyanidin	Cyanidin^c	3,5,7,3′,4′-OH
Chalcones	Phloretin^d	2,4,6, 4′-OH

---

Table 2 Interactions between UGTs and flavonoids

Isoform	Expression^a^45,47,48	Flavonoids as substrates	Flavonoids as modulators		Ref.
			Inducer or activator	Inhibitor
a Expression was mostly referred to expression in those organs, which leads to metabolism and excretion.
UGT1A1	Liver, small intestine, colon, stomach	Quercetin, fisetin, naringenin, luteolin, genistien, daidzein, eupatilin, glycitein, formononetin, biochanin A, prunetin	Chrysin, apigenin, quercetin	Chrysin	44,54,78–80,85,87
UGT1A3	Liver, small intestine, colon	Isorhamnetin, sylibin, kaempferol, daidzein, morin, avicularin, eupatilin quercetin xylopyranoside quecetin-3′,4′–OCHO–	—	—	51,54
UGT1A6	Liver, small intestine, colon, stomach	Luteolin and chrysin	Quercetin	Silymarin	52,54,32,87
UGT1A7	Colon Stomach Esophagus	Eupatilin	—	—	54
UGT1A8	Small intestine Colon Esophagus	Naringenin, genistein, daidzein, eupatilin, glycitein, formononetin, biochanin A, prunetin, apigenin, chrysin, 7-hydroxyflavone	—	—	44,50,54
UGT1A9	Liver, colon, esophagus, (kidney)	Luteolin, genistein, glycitein, formononetin, biochanin A, prunetin, daidzein,morin, avicularin, quercetin, eupatilin, quercetin xylopyranoside, quecetin-3′,4′–OCHO–	—	Silymarin	44,51,54,84
UGT1A10	Small intestine, colon, stomach, esophagus	Naringenin, genistein, apigenin, chrysin, 7-hydroxyflavone, eupatilin	—	—	44,49,54
UGT2B7	Liver, small intestine, colon, esophagus	Luteoin and quercetin	—	—	52
UGT2B15	Liver, small intestine, colon, stomach, esophagus	Luteoin and quercetin	—	—	52
UGT2B17	Liver, small intestine, colon, stomach	Galangin, chrysin, naringin, 7-hydroxyflavone	—	—	55
Unspecified isoforms	—		Biochanin A, daidzein, formononetin, genistein, prunetin, apigenin, galangin, kaempferol, naringenin, quercetin, chrysin, nobiletin, silymarin, tangeritin	Chrysin, nobiletin, silymarin, baicalein, wogonin	77,83,80,88,89–91

---

Table 3 Interactions between SULTs and flavonoids

Isoform	Expression^a^58–61	Flavonoids as substrates	Flavonoids as modulators		Ref
			Inducer or activator	Inhibitor
a Expression was mostly referred to expression in those organs, which leads to metabolism and excretion.
SULT1A1	Liver, small intestine, colon, stomach	EC, galangin, hesperetin, eriodictyol, (+)catechin	Genistein, biochanin A	Apigenin, chrysin, quercetin, myricetin kaempferol, genistein, daidzein, hesperetin, eriodictyol, catechin EC, luteolin 3,4′-dihydroxyflavone, 3′,4′,7-trihydroxyisoflavone	62,63,40,67,92–96,99,100
SULT1A3	Liver, small intestine, colon	Galangin, hesperetin, eriodictyol, (+)catechin, EC Similar to SULT1A1	—	Baicalein, hesperetin, eriodictyol	62,63,40,66,95,97
SULT1E1	Liver, small intestine, colon, stomach	EC, galangin Similar to SULT1A1	Biochanin A	Tricin, galangin, genistein, equol, daidzein, quercetin	40,95,97,98,100
Unspecified isoform	—	Genistein, apigenin, baicalein	—	—	23,64,65
SULT2A1	Liver, small intestine, colon	—	Genistein, biochanin A	Apigenin, myricetin, baicalein, galangin, 7-hydroxyflavone	96,99,100

---

Table 4 Interactions between transporters and flavonoids

Transporters	Flavonoids as substrates	Flavonoids as modulators			Ref.
		Inducer or activator	Inhibitor	No effect
P-gp	Flavonoid aglycone	Catechin, epicatechin, grapefruit juice, quercetin and kaempherol (low concentration)	EGCG, quercetin kaempherol (high concentraion), chrysin, flavones, hesperetin, naringenin, genistein	Grapefruit juice, narigin, hesperidin, rutin	109–121
MRP2	Flavonoid glucuronides and sulfates	Chrysin	Genistein, kaempferol, flavopiridol, chrysin, quercetin, biochanin A, catechin, EGCG, quercetin-7-O-glucuronide	Genistin	125–131
BCRP	Flavonoid glucuronides and sulfates	Quercetin, chrysin and flavone	Chrysin, biochanin A, apigenin, genistein, fisetin, kaempferol, hesperetin, naringenin, quercetin, luteolin-4-glucoside, daidzein-7-glucuronide, daidzein-4-sulfate, daidzin, ononin, genistin, sissotrin, glycitin, coumestrin	Naringin and phloridzin	139–147
OATP	—	Rutin	Naringin, naringenin, quercetin, hesperidin, biochanin A, genistein, EGCG	Genistin and quercetin	149,151,153
OAT	Ellagic acid quercetin-3-O-glucuronide, quercetin-3′-O-glucuronide, quercetin-3′-O-sulfate	—	Ellagic acid, naringenin, morin, silybin, quercetin, quercetin-3-O-glucuronide, quercetin-3′-O-glucuronide, quercetin-3′-O-sulfate	—	156,157,148

---

There is growing scientific and public interest in flavonoids because of their potential uses for improving human health. Multiple investigations showed that higher consumption of a flavonoid-rich diet was associated with a lower incidence and mortality rates of various degenerative diseases such as cancer, cardiovascular disease and immune dysfunction.^1,10,11,12 Extensive research has uncovered that flavonoids have a myriad of biological activities (e.g. anti-cancer, anti-inflammation, antioxidant, anti-microbial, cardiotonic and lipid lowering activities), which could affect a plethora of enzyme systems and signaling cascades involved in the diseases.⁴ Therefore, the beneficial impacts of flavonoids are likely to be derived from their pleiotropic biological activities.

To exert these biological activities, flavonoids must be absorbed and remain as aglycones in the body, since phase II metabolites of flavonoids are rarely reported as the active species.¹³ It is believed that flavonoid aglycones are able to passively diffuse across the gut wall, though evidence suggests that the absorption of intact glycosides may also occur, albeit at a significantly slower rate.^14,15 For most flavonoid glycosides, they must be first hydrolyzed to aglycones either by microorganisms residing in the intestine or by brush-border enzymes (e.g. lactase phloridzin hydrolase, LPH) before their absorption.^16,17,18,19 Because the vast majority of flavonoids are present as glycosides in nature, absorption of flavonoids present in most fruits, vegetables, and herbs are slow and often incomplete. The exception to this rule is certain flavonoid mono-glucosides, which are rapidly hydrolyzed by intestinal LPH, releasing aglycones that are rapidly absorbed.^20,21,22 On the other hand, for flavonoid aglycones, which are normally given at a dose that is intended to be pharmacologically active, the dose could be quite high (e.g., 1000 mg), which could result in higher concentration in the intestinal lumen. In general, the absorption of flavonoids (aglycones) is a rapid process. After entering the enterocytes, flavonoids are subjected to extensive metabolism (especially phase II) by intestine conjugating enzymes.²³ For flavonoids that have passed through the intestinal epithelium intact, they remain to be subject to the equally rapid metabolism by liver conjugating enzymes.²⁴

After absorption, phase I metabolism (e.g. cytochrome P450) other than hydrolysis of glycosides makes only a minor contribution to flavonoid clearance. In contrast, phase II metabolism plays a significant role in flavonoid clearance. Most of the flavonoids undergo glucuronidation by uridine-5′-diphosphate glucuronosyltransferases (UGTs) and/or sulfation (or sulfonation) by sulfotransferases (SULTs) in either enterocytes or hepatocytes, which convert them to glucuronides and sulfates, respectively.^25,26 These glucuronides and sulfates of flavonoids could be subsequently excreted into the lumen or bile by efflux transporters such as multi-drug resistance protein 2 (MRP2) and breast cancer resistance protein (BCRP).^26,27 Interestingly, the excreted glucuronides and sulfates in the intestine can be hydrolyzed by microflora β-glucuronidases and sulfatases back to aglycones, which can again undergo absorption and metabolism. Therefore, it is not surprising to speculate that the low bioavailability of flavonoids (usually, the oral bioavailability of flavonoid aglycones with a range of 2–20%) is due to the presence of a potent disposition network that mainly consists of phase II conjugating enzymes and efflux transporters of phase II enzyme conjugates.^28,29,30,31 In this paper, the bioavailability of flavonoids refers to intact aglycones in the systematic circulation, according to definition of the US Food and Drug Administration. Conjugates of flavonoids in the blood are not considered as bioavailable according to this definition.

Flavonoids are not only substrates of drug metabolizing enzymes (DMEs) and active efflux transporters; they are also regulators for some of those DMEs and transporters. Substantial amounts of evidence demonstrated that flavonoids could inhibit the activities of phase II enzymes (e.g. UGTs, SULTs and glutathione-S-transferases (GSTs)) and active transporters such as P-gp (P-glycoprotein), BCRP, MRP2 or OATP (organic anion-transporting peptide) by a variety of mechanisms (e.g., competitive inhibition).^32,33,34,35 In addition, flavonoids can also modify the expression of phase II enzymes and transporters at both the mRNA and the protein level. Thus, the enzymes and transporters involved in the flavonoid disposition could be affected by flavonoids. Due to the fact that many clinical drugs share the same metabolic pathways with flavonoids, potential drug–flavonoid interactions are expected. Specifically, when large amounts of flavonoids, taken up from dietary or supplementary sources, are administered with prescription or OTC (over the counter drugs) for a prolonged period of time, the pharmacokinetics of therapeutic drugs can be altered. As a result, it is meaningful to investigate the effects of flavonoids on functions and expression of the DMEs and transporters.

Based on the above considerations, we will present a current overview of flavonoid disposition and the effects of flavonoids on phase II enzymes and transporters, most of which are involved in their disposition as well. The paper reviews the literature mainly from the period 1990 to 2011. The effects of DMEs and transporters on flavonoid disposition will be addressed in detail, the effects of flavonoids on the activity or expression of DMEs and transporters will be discussed, and lastly the potential impact of the interactions between flavonoids and DMEs as well as transporters on the approved drugs will be discussed. The focus of this review is on the in vitro or in vivo observations from human enzymes and transporters, though animal studies were also summarized.

Unless otherwise specified, flavonoids refer to aglycones. Since phase I enzymes lead to minor metabolism of flavonoids and many other reviews have described their modulation by flavonoids in detail,^32,35,36,37 the effects of flavonoids on phase I enzymes were not included in this review. Though phase II enzymes like GSTs and transporters like P-gp, OATP and OAT, are not mainly involved in flavonoid disposition on the basis of current knowledge, their modulation by flavonoids are still covered in this review, considering that many clinical drugs and endogenous compounds are their substrates. We believe that the information discussed in this review can be used as an effective and updated research reference on flavonoid study, especially for flavonoid disposition and drug–flavonoid interactions. Hopefully, the efforts made on flavonoid disposition will eventually lead to an effective way to improve flavonoid bioavailability, and allow flavonoids to exert more therapeutic or disease-prevention effects on humans.

2. Disposition of flavonoids

2.1. Phase I metabolism of flavonoids

Flavonoids, like other drugs or xenobiotics, can be metabolized by cytochrome P450 (CYP450) enzymes. It was reported that CYP2C9 was the most efficient isoform for the oxidation of galangin followed by CYP1A3 and CYP1A1.³⁸ For kaempferide, CYP1A2 was predominantly responsible for its oxidation followed by CYP2C9 and CYP1A1.³⁹

Compared with phase II metabolic pathways, however, CYP450-mediated metabolism usually contributes little to the overall metabolism of flavonoids. In addition, CYP-mediated metabolism of flavonoids has never been shown to be important in vivo or in intact cells. For example, apigenin was metabolized by rat liver microsomes to form three monohydroxylated derivatives, one of which was luteolin. Further investigation indicated the involvement of CYP1A1, CYP3B and CYP2E1 in apigenin hydroxylation in vitro.²⁴ However, when apigenin was perfused through an isolated rat liver, none of the phase I metabolites could be recovered in the effluent perfusate, suggesting that CYP-mediated metabolism has little impact on in vivo flavonoid clearance, when the phase II metabolic pathway was functioning.²⁴ Therefore, metabolism via CYP is not a major clearance mechanism for flavonoids and is not the main reason for their poor bioavailability in vivo.

2.2. Phase II metabolism of flavonoids

In contrast to minor contributions of phase I metabolism, phase II metabolism is the predominant pathway for the flavonoid disposition both in vitro and in vivo. There are two main types of phase II metabolites of flavonoids present in vivo: glucuronides and sulfates, which are produced by UGTs and SULTs, respectively. In a study of galangin metabolism, glucuronides are found to be the primary metabolites followed by sulfates, and then oxidative metabolites.⁴⁰ These results supported the hypothesis that the efficiency of glucuronidation of flavonoids was very high, followed by sulfation, with only a very minor contribution by CYP-mediated oxidation. This metabolism rank (glucuronidation > sulfation > oxidation) could be extended to most flavonoids known in the literature.

Both enterohepatic and enteric recycling plays a critical role in flavonoid disposition. Therefore, UGT and SULT isoforms expressed in the liver or intestine are functionally more important than other isoforms in flavonoid metabolism. It is expected that different UGT and SULT isoforms can convert the same flavonoid at different rates, and conjugations at different positions will produce regiospecific glucuronides and sulfates at different ratios.^41–43 On the other hand, flavonoids with different structural features are targets of different UGT or SULT isoforms.^41–44 Lastly, the conditions of glucuronidation or sulfation reactions can also affect the structural preference of UGT or SULT isoforms.

2.2.1 UGT-mediated metabolism of flavonoids. Enzymes from the UGT superfamily are located in the endoplasmic reticulum with molecular weights of 50–60 KD. These UGTs are able to transfer the glucuronyl group from uridine-5′-diphosphoglucuronic acid to the substrate molecules with a functional group of oxygen, nitrogen, sulphur or carbon.⁴⁵ As for flavonoids, which belong to polyphenolic compounds, glucuronidation occurs mostly at OH groups and rarely at the C position.⁴⁶

The human UGT superfamily consists of UGT1, UGT2, UGT3 and UGT8 families. The UGT1 family currently contains only the UGT1A subfamily, while the UGT2 family can be classified into UGT2A (including UGT2A1, 2A2 and 2A3) and UGT2B subfamilies. The UGT3 family is currently composed of the UGT3A subfamily (including UGT3A1 and 3A2). The only known member of the UGT8 family is UGT8A1.⁴⁷

Among all the UGT subfamilies, the UGT1A and UGT2B subfamilies, which are classified according to their primary amino acid sequences, are predominantly responsible for metabolism of flavonoids and have more clinical relevance than other subfamilies with respect to drug metabolism. As a result of different exon splicing the same gene, transcription of the UGT1A gene cluster can produce the following isoforms: UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9 and UGT1A10.⁴⁷ Unlike isoforms from the UGT1A subfamily, each isoform (UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15 and UGT2B17) from the UGT2B subfamily is encoded by an individual gene.⁴⁷ UGTs are widely distributed in the human body. The liver, intestines, kidney and stomach are enriched with various and sometimes unique UGT isoforms.⁴⁸

Flavonoids are extensively metabolized by variations of UGT isoforms (Table 2). It was reported that UGT1A1, UGT1A8, UGT1A9 and UGT1A10 mainly contribute to the glucuronidation of isoflavones including daidzein, genistein, glycitein, formononetin, biochanin A and prunetin in a concentration-dependent manner.⁴⁴ Under different concentrations, the glucuronidation pattern of each isoflavone by UGT isoforms was sometimes changed. For example, genistein was mostly metabolized by UGT1A9 at 2.5 μM, whereas it was catalyzed mainly by UGT1A8 at 10 and 35 μM.⁴⁴ In fact, isoflavones were the best substrates among all the 42 flavonoids tested on the basis of the structure–function relationship of UGT1A10, suggesting that the hydroxyl group at C6 or C7 other than C5 of the A-ring or on C4′ of the B ring was preferred.⁴⁹ UGT1A8, which had a 94% similarity to the primary amino acid sequence of UGT1A10, not only had maximal overlapping substrates with UGT1A10, but also displayed a higher glucuronidation rate toward flavones (7-hydroxyflavone, chrysin and apigenin), a flavanone (naringenin) and an isoflavone (genistein) than UGT1A10.⁵⁰ In another study, UGT1A9 and UGT1A3 were able to catalyze both flavonoid aglycones and glycoside, though their substrate preference was clearly aglycones.⁵¹ The newly reported substrates of UGT1A3 included isorhamnetin, silybin, kaempferol, daidzein, morin, quercetin-3′, 4′-OCHO-, quercetin xylopyranoside, and avicularin, most of which were also the substrates of UGT1A9 except isorhamnetin, silybin and kaempferol.⁵¹ With respect to shared substrates, UGT1A9 always had a higher catalytic efficiency or intrinsic clearance (V_max/K_m) than UGT1A3.⁵¹ Other than the above UGT isoforms, UGT1A6 and UGT1A7 are also involved in glucuronidation of flavonoids. UGT1A6 was the predominant isoform responsible for glucuronidation of luteolin at the 7 position.⁵² It was also found that chrysin was metabolized by UGT1A6, an isoform highly expressed in Caco-2 cells.⁵³ In another study, UGT1A7 was found to be involved in glucuronidation of eupatilin, which was also the substrate of UGT1A1, UGT1A3, UGT1A8, UGT1A9 and UGT1A10.⁵⁴

In addition to UGT1A isoforms, UGT2B isoforms can also metabolize flavonoids, though they may not be as efficient as UGT1A isoforms. Evidence showed that galangin, chrysin, 7-hydroxyflavone and naringin were substrates for UGT2B17, among which galangin had the highest conversion value, whereas apigenin, baicalein, fisetin, quercetin, genistein and biochanin A were not conjugated by UGT2B17.⁵⁵ Another example of flavonoids as UGT2B substrates was from the study of the regioselectivity of the phase II metabolism of luteolin and quercetin. It was demonstrated that luteoin and quercetin were substrates of UGT2B15 and UGT2B7 with glucuronidation primarily occurring at position 7 and 3′, respectively.⁵² Between these two isoforms, UGT2B7 had a relatively higher conversion than UGT2B15. Generally, as substrates of UGT2B7, flavonol has a higher glucuronidation activity than flavones and isoflavones.⁵⁶

After glucuronidation mediated by various UGT isoforms, flavonoids are converted to corresponding glucuronides. These glucuronides are more hydrophilic and can be easily excreted via urine or feces. Usually, the biological activity of glucuronides is less than corresponding flavonoids with a few exceptions (e.g. quercetin-7-O-glucuronide).⁵⁷

2.2.2. SULT-mediated metabolism of flavonoids. SULTs, another important superfamily of phase II enzymes for flavonoid metabolism, can catalyze the formation of a flavonoid sulfate by transferring a sulfonate group from the coenzyme 3-phosphoadenosine-5′-phosphosulfate (PAPS) to the substrate. SULTs expressed in the cytosol are responsible for the metabolism of flavonoids (Table 3). In humans, the SULT superfamily can be divided into three subfamilies consisting of thirteen members: SULT1 family (SULT1A1, 1A2, 1A3, 1A4, 1B1, 1C2, 1C4 and 1E1), SULT 2 family (SULT2A1, SULT2B1_v1 and _v2), and SULT4A1 (SULT4A1_v1 and _v2).⁵⁸ Among all the SULTs, SULT1A1, SULT1A2, SULT1A3 and SULT1E1, which are distributed in the liver and gastrointestinal tract, are well studied.^58–61

Compared with the glucuronidation of flavonoids, research on the sulfation of flavonoids, which is regarded as a metabolic pathway that can compete with glucuronidation, is relatively scant. Previously, sulfation primarily catalyzed by SULT1A1 and SULT1A3, was the major pathway in the metabolism of (−)-epicatechin (EC) in human liver and intestine without detectable glucuronidation.⁶² This sulfation result was consistent with another report showing that SULT1A1 and SULT1A3 metabolized EC in addition to hesperetin, eriodictyol, (+)-catechin.⁶³ On the other hand, rapid sulfation and glucuronidation of EC in rats implied a large species difference in metabolism compared to humans.⁶² As mentioned previously, sulfation of galangin was one of metabolic pathways contributing to metabolism of galangin. Three SULT isoforms (SULT1A1, SULT1A3 and SULT1E1) were involved in sulfation of galangin, among which SULT1A1 displayed the most sulfation efficiency. On the contrary to the above three SULT isoforms, SULT2A1 had no activity against galangin.⁴⁰ In Caco-2 cells, it is common to observe that flavonoids (e.g. genistein, apigenin and baicalein) undergo both glucuronidation and sulfation.^23,64,65 Sulfation might be attributed to SULT isoforms being expressed in Caco-2 cells such as SULT1A1, SULT1A2 and SULT1A3.⁶⁶ Recently, Meng et al. showed the SULT1A3 exclusively metabolized flavonoids at the 7-OH position.⁴¹

2.3. Efflux of flavonoids and their phase II metabolites

In addition to extensive metabolism of flavonoids, efflux of flavonoids and their metabolites is another factor that leads to low bioavailability of flavonoids. ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that outwardly transport various ligands actively across the membrane with hydrolysis of ATP.⁶⁷ Accumulating evidence showed that members of ABC such as P-gp, MRP2 and BCRP were involved in transportation of flavonoids or their phase II metabolites out of cells.^68–71 Experiments on Caco-2 cells demonstrated that both quercetin and naringenin were substrates of MRP2 while only naringenin was substrate of P-gp.⁶⁸ Moreover, glucuronides of quercetin (quercetin-7-O-β-D-glucuronide, -3-O-β-D-glucuronide, -3′-O-β-D-glucuronide and -4′-O-β-D-glucuronide) were also substrates of MRP2.⁶⁹ Kaempferol, a major constituent of Ginkgo biloba extract, was found to be a substrate of BCRP by An and co-workers.⁷¹ In fact, glucuronides and sulfates of flavonoids are more likely to be substrates of MRP2, MRP3 and BCRP.^{26,27,69–70,72}

2.4. Interplay between phase II enzymes and efflux transporters in disposition of flavonoids

As an extension to the idea of interplay between CYP3A and P-gp, interplay between phase II enzymes and BCRP/MRP describes a sequential process in which substrates are metabolized by phase II enzymes (e.g., UGTs) and the produced hydrophilic phase II metabolites are transported out of cells by the efflux transporters (e.g., BCRP or MRP). This kind of interplay was commonly identified in the study of flavonoid disposition in both animal and cell models. This is because flavonoids as substrates can passively diffuse into cells and subsequently be metabolized to glucuronides and sulfates via glucuronidation and sulfation, respectively. The glucuronides and sulfates, which are too hydrophilic to diffuse across the cell membrane, can only exit cells via the action of the efflux transporters [Fig. 1]. The engaged efflux transporters include BCRP, MRP2 and MRP3.^{26,27,69,70,72} Previously, the efflux step of flavonoid conjugates (phase II metabolites) was regarded most likely as the rate-limiting step for the interplay due to the discrepancy of results between in vitro glucuronidation assay and the Caco-2 cell model or rat perfusion model. For example, glucuronidation of biochanin A was found to be faster than formononetin in rat intestinal and liver microsomes; however, excretion of formononetin conjugates was more efficient than biochanin A in the rat intestine perfusion model and Caco-2 cell model.⁷³ Recently, the idea that the rate-limiting step of efflux is challenged when it was applied to a simple model of one phase II enzyme (UGT1A9) and one dominant efflux transporter (BCRP). In this simple model, we showed clearly that the efflux transporter was simply a gate-keeper and kinetic interplay between UGT1A9 and BCRP ultimately determined how much metabolite was formed and then excreted.⁷⁴ Efforts are underway to understand more detail about the interplay between phase II enzymes and efflux transporters.

Fig. 1 Cellular interplay between phase II enzymes and efflux transporters in flavonoid disposition.

3. Effects of flavonoids on phase II enzymes

Flavonoids are likely to be metabolized by phase II enzymes as described previously. Reciprocally, flavonoids can also exert some effect on phase II enzymes [Fig. 2]. As an activator or inducer, a flavonoid may induce or activate the target enzymes via multiple mechanisms, which possibly require the involvement of various nuclear receptors. For example, some flavonoids could induce phase II enzymes by disrupting the interaction between Nrf2 and Keap1 and by making Nrf2 relocate to the nucleus and bind to the antioxidant/electrophile response element (ARE).^35,75 In addition, flavonoids may interact with constitutive androstane receptor (CAR) and pregnane X receptor (PXR), which were reported to regulate phase II enzymes and transporters.⁷⁶ On the other hand, flavonoids can act as inhibitors of phase II enzymes, more likely through simple mechanisms such as competitive inhibition. In the following parts, we will discuss the research developments on how flavonoids affect the phase II enzymes, including UGTs, SULTs and GSTs.

Fig. 2 Effects of flavonoids on phase I and II enzymes and transporters in cells. The solid line represents the inhibition, whereas the dotted line represents induction via various nuclear receptors.

3.1. Effects of flavonoids on UGTs (Table 2)

Early study on the prostate cancer cell line LNCaP showed that certain glucuronidation activities (i.e., glucuronidation of testosterone via UGT2Bs) were increased after the cells were exposed to the flavonoids including biochanin A, daidzein, formononetin, genistein, prunetin, apigenin, galangin, kaempferol, naringenin and quercetin.⁷⁷ Among the tested flavonoids, biochanin A was the most potent inducer of UGTs, increasing the glucuronidation activity by 10 fold.⁷⁷ At the same time, due to less availability of testosterone resulting from glucuronidation, the production and release of prostate specific antigen (PSA) as a prostate tumor marker were significantly decreased in the presence of biochanin A.⁷⁷ It suggested that flavonoids could play an important role in modulation of sex hormones in humans and provide us with a possible means to prevent and manage prostate cancers. In addition, chrysin was reported to be able to induce UGT1A1 specifically, both in Caco-2 and HepG2 cells.^78–80 However, UGT1A6, UGT1A9 and UGT2B7 were not affected by the chrysin treatment.⁸⁰ Further study from the same group showed that induction of UGT1A1 by chrysin was not dependent on an aryl hydrocarbon receptor (AhR) since galangin and isorhamnetin, both of which are inducers of CYP1A1 via AhR, did not display any induction of UGT1A1.⁸¹ More recently, UGT1A1 was believed to be under the regulation of Nrf2.⁸² In addition to be an inducer of UGT1A1, chrysin was also an inhibitor for the glucuronidation of estradiol at substrate concentrations above 25 μM.⁸³ Similar inhibitory effects on glucuronidation of estradiol were also observed when nobiletin and silymarin were treated at substrate concentrations over 25 μM.⁸³ On the contrary, the same three flavonoids had little effect or possibly even slight stimulation effects on estradiol glucuronidation at the lower concentrations (5 and 10 μM). As for tangeritin and quercetin, both exerted inhibitory effects on glucuronidation of estrodiol. Tangeritin was the most potent inhibitor, while quercetin was a relatively weak inhibitor.⁸³ In addition, naringenin could inhibit glucuronidation of estradiol to a similar extent at all tested naringenin concentrations (5–100 μM), suggesting that it did not act as a competitive inhibitor.⁸³ Furthermore, the milk thistle extract silymarin and its major ingredient silybin could inhibit UGT1A6 and UGT1A9 in human hepatocytes at a concentration of 100 μM.^32,84 As for apigenin, it was found to be able to induce UGT1A1 transcription 4 fold in undifferentiated Caco-2 cells.⁸⁵ The combination of apigenin and sulforaphane could further lead to significant induction of UGT1A1 mRNA (up to 12 fold).⁸⁵ Considering that SN50 (a NF-kB translocation inhibitor) could enhance the induction of UGT1A1 by apigenin, it was believed that UGT1A1 induction by apigenin might be associated with NF-kB translocation.⁸⁵ In addition to apigenin, quercetin was also shown to markedly increase the glucuronidation activity in Caco-2 cells.⁸⁰ The significant increase in glucuronidation activity might result from increasing expression levels of UGT1A6 (at 50 μM of quercetin) and UGT1A1.^86,87

Regulation studies done on the cell culture could be replicated in vivo, although the results were not always consistent. For example, feeding rats with a diet containing 1% quercetin significantly increased the activity of UGTs in liver and to a lesser extent in intestine.⁸⁸ Similar results of increasing hepatic UGT activity were obtained after flavone, flavanone and tangeritin were fed to rats at a dietary concentration of 0.3% (w/w) or soy isoflavones (0.81 mg kg⁻¹ diet).^89,90 In contrast, hepatic UGT activity was inhibited after exposing mice to a liquid diet containing 5 mM baicalein or wogonin for 1 week.⁹¹ In one study, where a human jejunum was perfused with an onion and broccoli extract containing 60 μM quercetin for 2 h, mRNA of UGT1A1 in shed enterocytes was increased.⁸⁷ The results from humans were consistent with the observation in Caco-2 cells treated with pure quercetin.

3.2. Effects of flavonoids on SULTs (Table 3)

It is not surprising to observe the inhibition of SULT activities at higher flavonoid concentrations (> 10 μM). Indeed, auto-inhibition or substrate-inhibition is a very common phenomenon in SULT-mediated metabolism of flavonoids. For example, metabolism of genistein and daidzein by SULT1A1 followed the substrate-inhibition kinetic profile. When substrate concentrations were over 1 μM, the sulfation activities were substantially decreased.⁹² In addition to substrate-inhibition, a large body of studies showed that flavones (e.g. apigenin and chrysin), flavonols (e.g. quercetin, myricetin and kaempferol), isoflavones (e.g. genistein and daidzein), flavanone (e.g. hesperetin and eriodictyol) and flavanols (e.g. catechin and epicatechin) were potent inhibitors of SULT1A1.^92,93,94,63

In an investigation conducted by Harris and co-workers, 37 flavonoids were tested to determine their inhibition potency against SULT1A1.⁹⁵ The results indicated that flavonoids could inhibit SULT1A1 significantly at submicromolar concentrations. Among all the flavonoids selected, 3,4′-dihydroxyflavone was the most potent inhibitor with an IC₅₀ less than 1 nM, whereas luteolin was regarded as the most potent naturally occurring inhibitor with IC₅₀ of 3 nM.⁹⁵ In contrast to the above flavones and flavonols, isoflavones such as genistein and daidzein had relatively high IC₅₀, which was in the range of 500–600 nM except for 3′,4′,7-trihydroxyisoflavone, which had an IC₅₀ of 20 nM.⁹⁵ Therefore, the 3′,4′-dihydroxy motif might be a critical feature for flavonoids to have potent inhibitory effects of SULT1A1 based on the fact that flavonoids with the above feature motif had an IC₅₀ of 100 nM or less against SULT1A1-mediated sulfonation of the standard substrate 4-nitrophenol (3 μM).^95,96 Though various flavonoids were shown to have potent inhibition of SULT1A1, few of them displayed powerful inhibitory effects on SULT1A3. Baicalein was found to be the only one having substantial inhibition (IC₅₀ 500 nM) at less than 1 μM against 10 μM dopamine as the standard substrate.^95,96 Recently, hesperetin and eriodictyol were also reported to be potent inhibitors of SULT1A3. Hesperetin and eriodictyol had IC₅₀ values of 20 μM and 15 μM, respectively, against SULT1A3-mediated sulfonation of its typical substrate dopamine (50 μM).⁶³ In the case of SULT1E1, which was inhibited relatively easily by many simple phenols, tricin (a derivative of flavonoid, 3′,5′-dimethoxy-4′,5,7-trihydroxyflavone) was found to be the most potent inhibitor.⁹⁷ It could competitively inhibit SULT1E1 with an inhibition constant of approximately 1 nM.⁹⁷ Another potent inhibitor of SULT1E1 was galangin with IC₅₀ of <1 μM against 10 nM estradiol as standard substrate.⁹⁵ Similarly, genistein and equol are as potent as galangin, they exhibited inhibitory constants at the active site of 400 and 500 nM and at an allosteric site of 2 and 5 μM, respectively (substrate 10 nM estradiol).⁹⁵ On the other hand, daidzein was less potent than above flavonoids, and had an IC₅₀ of 5 μM against 10 nM estradiol even though its chemical structure was most close to equol and distantly related to genistein.⁹⁵ In the same system, formononetin was found to be the least potent inhibitor among all the tested compounds.⁹⁵ In another published study, quercetin had surprisingly 10 times more inhibitory potency for SULT activity in the intact cultured human mammary epithelial cells (HME) (an IC₅₀ of about 0.1 μM) than in recombinant SULT isoform.⁹⁸ Last but not least, SULT2A1, another SULT isoform expressed in the intestine, was reported to be inhibited by apigenin with a K_i value of around 2.4 μM.⁹⁶ Similar to apigenin, myricetin, baicalein, galangin and 7-hydroxyflavone also had K_i values between 2–3 μM.⁹⁶ Possibly, a 7-hydroxyflavone substituent seemed to be a requirement for some inhibition.⁹⁶ On the contrary, fisetin, kaempferol, luteolin, hesperetin, morin, quecetin, rutin, catechin, and daidzein did not show any inhibitory effect on SULT2A1 even at a concentration of 25 μM.⁹⁶

Flavonoids not only inhibit the SULT activity, but also induce the expression of SULTs in various model systems. Recently in both HepG2 and Caco-2 cells, genistein was shown to induce SULT1A1 and SULT2A1 at both mRNA and protein levels in a dose- and time-dependent manner.⁹⁹ In Sprague–Dawley rats, biochanin A significantly induced expression of rat SULT1A1, SULT2A1 and SULT1E1 in liver and intestine after a 7 day treatment period (doses: 0, 2, 10, and 50 mg kg⁻¹ day⁻¹).¹⁰⁰

3.3. Effects of flavonoids on GSTs

Glutathione-S-transferases (GSTs) are an another group of phase II enzymes. However, GSTs are not primarily responsible for flavonoid metabolism. They are able to catalyze the binding of a wide range of electrophiles to the sulfydryl group of glutathione in order to neutralize the reactive oxygen species. As a result, GSTs play an important role in the detoxification of various chemical carcinogens. GSTs are widely distributed in many tissues and present in relatively large amounts in the epithelial tissues of the human gastrointestinal tract.^35,101 Volumes of evidence showed that considerable numbers of flavonoids contribute to the regulation of GSTs, either in gene expression or protein activities or both. An example from a cell study in vitro indicated that effects of genistein on expression of GSTs through the ER/ARE signaling pathway were cell-type specific.¹⁰² In COS1 I cells expressing ERα and ERβ, genistein (1 μM) repressed the GST Ya ARE-dependent gene expression; however, in C4-12-5 cells, which are ER-negative breast cancer cells derived from the MCF-7 cells, genistein (1 μM) showed modest induction of the GST gene following transfection with ERα and ERβ.¹⁰² In addition, evidence from an in vivo study showed that GSTs in the stomach were significantly induced when the mice were fed with citrus limonoids and citrus flavonoids (hesperidin, naringin and crude flavonoids mixture) for one week at dose of 20 mg.¹⁰³ Moreover, in another study, Swiss Webster mice were exposed to flavone, morin, naringenin, (+)-catechin and quercetin for 20 days at 2.5 g kg⁻¹ diet.¹⁰⁴ It was revealed that only groups fed with a flavone showed the gender and isozyme-specific induction of GSTs. In female mice, GST activity was increased 7 fold, while in male mice, GST activity was increased 4 fold. With regard to enzyme specific assay, it was found that the activities of GSTM and GSTP increased to a greater extent in females (8 fold and 4 fold respectively) than in males (6 fold and 2 fold).¹⁰⁴ In humans, the modification of GSTs by flavonoids is more complicated than that in mice. In a study involving healthy volunteers, who drank juice containing several flavonoids (e.g., quercetin, quercetrin, myricetrin, kaempferol, hesperidin, eriodictyol and naringenin), GSTP1 protein in leucocytes was first suppressed at weeks 4 and 6. However, at week 8, expression of GSTP1, which was reversibly linked with DNA damage, significantly increased.¹⁰⁵ The results were contrary to in vitro experiments. In vitro, when leucocytes were treated with polyphenol mixtures containing flavonoids, no change of GST gene expression or activity was observed, though up-regulation of certain CYP450 and SULTs isoforms were observed.¹⁰⁵

4. Effects of flavonoids on transporters

Currently, the interaction between flavonoids and ABC transporters (Table 4) is drawing more and more attention of scientific researchers [Fig. 2]. In addition to ABC transporters, another class of transporters called solute carrier family (SLC), is also involved in interaction with flavonoids [Fig. 2]. In contrast to ABC transporters, which are mainly responsible for efflux, members of the SLC primarily manage cellular uptake. Flavonoids and their metabolites might act as substrates or inhibitors of both ABC and SLC transporters. As an inducer of transporters either at the mRNA or at the protein level, long exposure to flavonoids is necessary. Various mechanisms underpinning the interaction between ABC transporters and flavonoids have been postulated. Some flavonoids can affect the ATPase activity by inhibition or stimulation; other flavonoids, which are substrates of transporters, can compete for the binding site of transporters and result in competitive inhibition; and still a more selected group of flavonoids can modulate the transporters through post translational modifications (e.g., phosphorylation).

4.1. Effects of flavonoids on P-gp

P-glycoprotein (P-gp), encoded by the ABCB1 gene with a molecular weight of 170 KD, is the first human ABC transporter that was cloned and characterized through its ability to confer a multi-drug resistant phenotype to cancer cells.¹⁰⁶ As an efflux transporter expressed in many tissues and apical membrane of epithelial cells, P-gp is mainly responsible for outward transportation (i.e., efflux) of hydrophobic, cationic or neutral compounds with a planar structure with some notable exception (e.g., methotrexate and phenytoin, both with negative charges).^33,107P-gp consists of two homologous segments, each of which contains six transmembrane domains (TMDs) and a nucleotide binding domain (NBDs) with ATPase activity.¹⁰⁸

There were several controversial opinions with regard to effects of flavonoids on P-gp. The first controversy was about the effects of green tea. One study supported the notion that P-gp activity was inhibited by green tea polyphenols,¹⁰⁹ whereas the other suggested that P-gp function was elevated by catechin in green tea.¹¹⁰ Experiments from the first group indicated that green tea polyphenols at a concentration of 30 μg ml⁻¹ could inhibit the photolabeling of P-gp by 75% and increase the accumulation of rhodamine-123 3 fold in the multidrug-resistant cell line CH^RC5.¹⁰⁹ Moreover, EGCG as a representative of green tea polyphenols exhibited an inhibitory effect on P-gp activity not only in CH^RC5 cells but also in Caco-2 cells.¹⁰⁹ In the second study, although some catechins like EGCG displayed inhibitory effects on P-gp, others like (−)-epicatechin were shown to facilitate the P-gp-mediated transport of the fluorescent markers LDS-751 via a heterotropic allosteric mechanism.¹¹⁰ Currently, there is no explanation for these controversial findings.

Another controversy deals with the grapefruit juice effect.¹¹¹ Some studies show that there were some effects whereas other showed none. Earlier studies from Dr Lown’s group indicated that twice daily consumption of grapefruit juice (8 oz) for 6 days resulted in 62% reduction of CYP3A4 levels without any change of P-gp level in 10 healthy volunteers.¹¹² This result was consistent with the conclusion that naringin and 6′,7′-dihydroxybergamottin, both of which were components of grapefruit juice, improved saquinavir transport in Caco-2 cells by inhibition and down-regulation of the CYP3A4 rather than by modulation of P-gp.¹¹³ On the contrary, some papers showed that grapefruit juice had either inhibitory or activating effect on P-gp. Dr Benet’s group found that grapefruit juice stimulated the efflux of a couple of P-gp substrates across the MDCK-MDR1 cells.¹¹⁴ In the study of vincristine transport across MBEC4 cells (cultured mouse brain capillary endothelial cells), quercetin and kaempherol were found to decrease the uptake of vincristine at low concentration (10 μM), but increased the uptake of vincristine at high concentration (50 μM).¹¹⁵ This biphasic in vitro results were further confirmed by the study on ddY mouse in vivo with co-administration of quercetin (0.1 mg kg⁻¹) and (1.0 mg kg⁻¹).¹¹⁵ The reasons were due to changes in P-gp functionality. At a low concentration, P-gp activity was stimulated as a result of enhancing its phosphorylation; while at a high concentration, P-gp function was hindered.¹¹⁵ In addition to quercetin and kaempherol, other flavonoid components, including chrysin, flavone, hesperetin, naringenin, were found to increase the vincristine uptake in MBEC4 cells by inhibiting P-gp at the 10–50 μM,. However, their corresponding glycosides such as hesperidin, naringin and rutin did not affect P-gp.¹¹⁵ Furthermore, P-gp was reported to be inhibited by kaempferol and naringenin in the human immortalized tubular cell line HK-2 cells by down-regulating the protein level and mRNA expressions of P-gp.¹¹⁶

Apart from above controversies, the majority of findings supported the inhibitory role of flavonoids against P-gp. It was reported that genistein (200 μM) could elicit an elevation in intracellular accumulation of rhodamine 123 and daunorubicin in P-gp-expressing cell lines. In addition, genistein could also decrease photoaffinity labeling of P-gp by [³H] azidopine.¹¹⁷ Other related studies also pointed out that flavonoids had an inhibitory effect on P-gp in MDCKII-MDR1 cells, Caco-2 cells and K562 cells, based on their ability to the increase cellular uptake of various testing substrates.^118–120

On the basis of accumulating interaction data, the structure–function relationship between flavonoids and P-gp was established. More and more flavonoid derivatives were synthesized to modulate P-gp functions.¹²¹ Recently, a 3D linear solvation energy model was developed to quantify the affinity of flavonoid derivatives toward P-gp. According to this 3D model, two major physicochemical parameters (shape parameters and hydrophobicity), were identified to be primarily responsible for the affinity of flavonoid derivatives towards P-gp. At the same time, the hydrogen-bonding capacity was identified as a minor modulator.¹²²

4.2. Effect of flavonoids on MRP2

MRP2 is an efflux transporter with molecular weight of 190 KD, which belongs to the ABCC (also called MRP) subfamily of ABC transporters. MRP2 has two nucleotide binding domains (NBDs) and seventeen transmembrane domains (TMDs).¹²³ As a mechanism leading to the multidrug resistance, MRP2 is highly expressed in carcinoma cells or tumor tissues as well as normal tissues such as liver, kidney and intestine.¹²⁴ The main function of MRP2, which is located to apical membrane of polarized cells, is to actively transport endogenous (e.g. bilirubin and its polar conjugates) and xenobiotic (e.g. drugs) substances out of the cells.¹²⁴

Early investigation showed that genistein inhibited the efflux of daunorubicin from small-cell lung cancer GL4/ADR cells, in which MRP was overexpressed, in a competitive manner.¹²⁵ The same result of inhibition of daunorubicin by genistein competitively was also observed in plasma membrane vesicles from marine MRP-transfected NIH3T3 cells.¹²⁶ Follow-up study confirmed that genistein but not genistin, together with kaempferol and flavopiridol (synthetic flavonoid derivative) could affect MRP-mediated transport of anticancer drugs by a direct interaction with MRP.¹²⁷ Moreover, chrysin was reported to inhibit the accumulation of the MRP2 substrate CMFDA in Caco-2 cells in a dose-dependent manner.¹²⁸ The maximal accumulation, which could also be achieved by specific MRP inhibitor-MK571, was observed in the presence of 250 μM chrysin. Interestingly, chrysin was also found to increase the expression of MRP2 5 fold in Caco-2 cells after long-term treatment.¹²⁸ Over a time period of 48 h, the inhibition of transporter function overtook the enhanced expression of MRP2 by chrysin, resulting in an increased accumulation of topotecan.¹²⁸ Therefore, chrysin seemed to play a dual role in regulating MRP2 in Caco-2 cells. In fact, the dual role of chrysin was also extended to other efflux transporters—P-gp and BCRP. Evidence from other investigators confirmed the inhibition of MRP2 by chrysin in Caco-2 cells.¹²⁹ It was found that chrysin, genistein, quercetin and biochanin A (all at 50 μM) could increase transportation of ochratoxin A from the apical side to basolateral side. Enhanced ochratoxin A uptake by the above flavonoids was also observed in Caco-2 cells. Moreover, quercetin displayed concentration-dependent inhibition on ochratoxin A absorption.¹²⁹ A later study further identified that major phase II metabolites of quercetin inhibited MRP2 function with a similar potency to quercetin itself.¹³⁰ The results by using Sf9 inside-out vesicles revealed that glucuronides, especially 7-O-glucuronosyl-quercetin significantly increased the potential of quercetin to inhibit MRP2-mediated calcein transport. On the contrary, methylation at the 4′ position of quercetin resulted in a reduction of the potential to inhibit MRP2.¹³⁰

Quantitative structure–function relationship has been built according to flavonoid-mediated inhibition of MRP2 by using MDCKII-MRP2 cells.¹³¹ A total 29 flavonoids with various structures were used in the study. It was revealed that robinetin and myricetin, both of which had an IC₅₀ much lower than 50 μM (15 and 22 μM), respectively, were the two best inhibitors of MRP2 among all the tested flavonoids.¹³¹ Furthermore, the kinetic mechanism for robinetin to inhibit MRP2-mediated transportation of calcein was competitive. As a result, a flavonol B-ring pyrogallol group appeared to be an essential structure for inhibition of MRP2-mediated calcein efflux.¹³¹

4.3. Effect of flavonoids on BCRP

Breast cancer resistance protein (BCRP), which is encoded by the ABCG2 gene, consists of 655 amino acids with molecular weight of 72 kD.¹³² Unlike many other members from the ABC transporter family, BCRP is a “half ABC transporter” because it only contains one NBD and six TMDs.¹³³ To function properly, BCRP must form homodimers or homooligomers.¹³⁴ In addition to its physiological function, BCRP has a role in limiting oral bioavailability of drugs and drug transport across the blood–brain barrier, blood–testis barrier and the maternal–fetal barrier of selected substrates.^135–137 Though BCRP was identified originally from cancer cells, it is broadly distributed in humans including kidney, liver and intestine.¹³⁸ The substrates of BCRP include various compounds from endogenous to exogenous, which are generally organic anion or neutral substances, often time similar to the substrates of MRP2.

Currently, the inhibition of BCRP by flavonoids was most extensively investigated among all the ABC transporters. A substantial amount of work has been done to study the interactions between flavonoids and BCRP. It was Zhang and coworkers who first identified chrysin and biochanin A as the most potent inhibitors of BCRP among all the 20 naturally occurring flavonoids.¹³⁹ In fact, it was found that most tested flavonoids (e.g. apigenin, genistein, fisetin, kaempferol, hesteretin, naringenin and quercetin) could increase mitoxantrone accumulation in BCRP-overexpressing cells (MCF-7-MX100 and NCI-H460-MX20) at concentration of 50 μM, but none of the flavonoid glycosides (e.g.naringin and phloridzin) were effective.¹³⁹ Specifically, the concentration-dependent enhancement of accumulation of mitoxantrone were observed for apigenin, biochanin A, chrysin, genistein and kaempferol.¹³⁹ Furthermore, several combinations of multiple flavonoids showed an additive (inhibitory) effect on BCRP.¹⁴⁰ For example, apigenin (A), biochanin A (B) and chrysin (C) actively inhibited BCRP-mediated transport in combinations of AB, BC and ABC with equal molar concentration of each individual constituent.¹⁴⁰ The combinations were not limited to 3 flavonoids. The additive inhibition of BCRP could be obtained when 5 or 8 flavonoids were combined together.¹⁴⁰ Recently, the pharmacokinetic and tissue distributions of mitoxantrone with or without co-administration of 5,7-dimethoxyflavone (5,7-DMF) or multiple flavonoids with low EC50 (7,8-benzoflavone, 5,6,7-trimethoxyflavone and 8-methylflavone) were studied in mice to evaluate the potentially additive or synergistic effect of multiple flavonoids on BCRP inhibition.¹⁴¹ It revealed that there was no significant change of pharmacokinetic parameters with or without flavonoids. However, significant changes in mitoxantrone distribution were observed in several tissues with co-administration of flavonoids. In the presence of 5,7-DMF, the AUC values of mitoxantrone were significantly increased in liver (94.5%), kidney (61.9%) and lung (18.4%); on the other hand, co-administration of multiple flavonoids resulted in increasing AUC values in heart (30.5%), liver (95.9%), kidney (63.3%), lung (30.7%) and muscle (33.8%).¹⁴¹ Therefore, inhibition of BCRP by either individual flavonoid or multiple flavonoids can occur both in vitro and in vivo. However, conclusions from in vitro studies were not always consistent with in vivo studies. Discrepancy was observed when biochanin A was used as inhibitor to treat MDCKII-BCRP or MDCKII-Bcrp cells or given to mice.¹⁴²In vitro, accumulation of cellular mitoxantrone was enhanced with biochanin A at concentration of 2.5 or 25 μM in MDCK cells overexpressing human BCRP or murine Bcrp. In contrast to in vitro data, biochanin A administrated intravenously at 10 mg kg⁻¹ had no impact on the plasma concentration of mitoxantrone and AUC values in most tissues (brain, heart, liver and lung).¹⁴² Thus, when extrapolating results of in vitro inhibition on BCRP by flavonoids to in vivo, we should pay particular attention to the fact the low bioavailability may severely limit their in vivo levels necessary to achieve observed inhibition effects (in vitro).

Unlike flavonoid aglycones, most flavonoid glycosides were reported to have little or no effect on BCRP-mediated drug resistance. Only a few flavonoid glycosides were shown to affect BCRP-mediated drug resistance. Naringenin-7-glucoside and luteolin-4-glucoside displayed moderate effects to reverse the resistance to SN-38 and mitoxantrone in BCRP-transduced K562 cells.¹⁴³ However, the inhibition of BCRP by naringin might be controversial. Recently, experiments indicated that 6 isoflavonoid glucosides (daidzin, ononin, genistin, sissotrin, glycitin and coumestrin) had 10 to 100 fold lower inhibitory potency (against BCRP) than their corresponding aglycones.¹⁴⁴ Interestingly, it was also found that the inhibitory potency of daidzein-7-glucuronide was 100 fold lower than daidzein while daidzein-4-sulfate had a inhibitory potency comparable to daidzein.¹⁴⁴ It is very clear that there is a strong structure–inhibition relationship between BCRP and flavonoids. It appeared that flavonoids with following structural features might inhibit BCRP effectively: a hydroxyl group at position 5, double bond between position 2 and 3, and a methoxyl moiety at position 3 or 6.¹⁴⁵ With regard to the inhibition mechanisms, interference with substrate-binding sites of BCRP might account for flavonoid inhibition of BCRP since quercetin and daidzein were able to stimulate vanadate-inhibitable ATPase activity in membranes prepared from bacteria expressing BCRP.¹⁴⁶ In contrast to inhibition, expression of BCRP in Caco-2 cells can also be induced by flavonoids.¹⁴⁷ Evidence indicated that quercetin (25 μM) could strongly induce the protein expression of BCRP in Caco-2 cells. At mRNA level, quercetin, chrysin and flavone had a pronounced induction of BCRP, while genistein showed no effect. The induction of BCRP was also believed to be via regulation of AhR (aryl hydrocarbon receptor).¹⁴⁷

4.4. Effects on OATP and OAT

Compared to abundant studies related to interactions of flavonoids with ABC transporters, little is known about the interactions of flavonoids with transporters from the solute carrier family (SLC). Organic anion-transporting peptide (OATP, also called SLC21A) and organic anion transporter (OAT, also called SLC22A) are two subfamilies, which are reported to have an interaction with flavonoids or flavonoid conjugates.^148–151

Members of the OATP family mediate sodium-independent uptake of a broad spectrum of amphipathic organic anions, organic cations and conjugates. OATP2B1 and OATP1A2 are expressed in the intestine while OATP1B1 and OATP1B3 are expressed in liver.^152,153 According to published papers, OATP1B1, OATP1B3 and OATP2B1 are localized on the basolateral membrane, while OATP1A2 is localized on the apical membrane.¹⁵³ A large volume of evidence showed that various juices, which contained plenty of flavonoids, could inhibit the function of OATP. It was demonstrated that several citrus juice constituents (10 μM), including naringin, naringenin, quercetin, bergamottin and 6,7-dihydroxybergamottin, tangeretin and nobiletin, significantly inhibited the OATP2B1-mediated uptake of estrone-3-sulfate by about 20-60% in OATP2B1-expressing HEK293 cells.¹⁵⁴ Another study also found that naringin (grapefruit) and hesperidin (orange) inhibit the OATP1A2-mediated transport of fexofenadine in vitro and in vivo.¹⁵¹ Further analysis of human studies showed that consumption of grapefruit juice concomitantly or 2 h before fexofenadine administration was linked with reduced oral fexofenadine plasma concentration without affecting protein expressions of OATP1A2 and P-gp in intestine.¹⁵² In OATP1B1-expressing HeLa cells, 20 naturally occurring flavonoids were tested for their impacts on uptake of [³H]dehydroepiandrosterone sulfate (DHEAS).¹⁴⁹ Most flavonoids (e.g. biochanin A, genistein, and epigallocatechin-3-gallate) significantly inhibited DHEAS uptake in a concentration-dependent manner.¹⁴⁹ Not surprisingly, flavonoid glycosides showed comparable, opposite or no effect on uptake of DHEAS in contrast to their corresponding flavonoid aglycones. Genistin could not inhibit the uptake but genistein could; rutin, on the other hand, showed activation of uptake, whereas quercetin had no effect on uptake.¹⁴⁹ As the most potent inhibitor among 20 flavonoids, biochanin A was selected for in vitro kinetic study. The results suggested that biochanin A inhibited uptake of DHEAS in a noncompetitive way as it was not the substrate for OATP1B1.¹⁴⁹

Members of OAT play an important role in the sodium-dependent renal uptake of organic anions. OAT1 and OAT3, which are highly expressed on the basolateral membrane of proximal tubules, are two relatively well-studied transporters within the OAT family.¹⁵⁵ Their substrates are highly variable from endogenous metabolites to drugs and toxicants. At present, only a few studies have focused on the interaction of OAT with flavonoids. In one study, ellagic acid was identified to be a substrate and potent inhibitor of OAT1.¹⁵⁶ In another study, naringenin, morin, silybin and quercetin were reported to inhibit both OAT1 and OAT3.¹⁵⁷ Most recently, quercetin phase II conjugates (quercetin-3-O-glucuronide, 3′-O-glucuronide and 3′-O-sulfate) were found to potently inhibit OAT3-mediated transport of 5-carboxyfluorescein. In addition, quercetin-3′-O-sulfate could also strongly inhibited transport of p-aminohippuric acid, which is a substrate of OAT1.¹⁴⁸

5. Clinical significance of flavonoid interaction with disposition machinery

Flavonoids can exert various biological and pharmacological effects that benefit humans. This can be achieved since humans can easily ingest large amounts of flavonoids from their daily dietary intake because of their ubiquitous distribution in vegetables, fruits, herbal supplements and beverages deriving from plants. A couple of clinical studies indicated that oral or intravenous administration of flavonoids, either as individual components (e.g. quercetin alone) or mixtures, could inhibit lymphocyte tyrosine kinase activity (resulting in antitumor effects) or prevent bone loss in late postmenopausal women (without a detectable hyperplasia effect on the endometrium as the side effects of many drugs do).^158,159 These and other clinical study results suggest that some flavonoids can reach the effective concentrations in vivo at which their pharmacological activity is exhibited. Although there is extensive phase II metabolism mediated mainly by UGTs and SULTs, when multiple flavonoids are taken together, the bioavailability of one flavonoid might be improved. In an animal study, co-administration of quercetin and EGCG together with biochanin A significantly enhanced the bioavailability of biochanin A due to decrease of clearance by extending enterohepatic cycling.¹⁶⁰ Therefore, we should pay more attention to flavonoid–flavonoid interactions in pharmacokinetic and efficacy studies in vivo.

Drug–flavonoid interaction is another important issue, especially from a clinical point of view. In one instance, patients with long-term exposure to foods containing considerable amounts of flavonoids may experience changes in their physiological status including expression of drug metabolizing enzymes and transporters. On the other hand, the immediate inhibitory effects on drug metabolizing enzymes and transporters can also be observed when flavonoid-enriched edibles are consumed with medication. As a result, the pharmacokinetics of the medication is changed. For example, an aqueous solution of naringin that contained the same amount of the flavanone as 300 ml grapefruit juice decreased the bioavailability of co-administered fexofenadine.¹⁵¹ In another study, an intake of daidzein (200 mg twice daily) for ten consecutive days increased the bioavailability, maximal plasma concentration and elimination half-life of the bronchodilator theophylline in humans.¹⁶¹ Since flavonoids can interact with multiple drug metabolizing enzymes and transporters, which are involved in absorption, distribution, metabolism and excretion of drugs, drug–flavonoid interaction has become and will remain an important area for clinical research and studies.

6. Conclusion

In conclusion, the mutual interactions between flavonoids and drug-metabolizing enzymes and transporters are very complex and multifaceted. Flavonoids can be substrates of and/or chemical modulators of certain enzymes and transporters that will impact the disposition processes of various compounds including flavonoids themselves. The extensive metabolism of flavonoids by phase II enzymes and subsequent excretion of conjugates by the associated efflux transporters, especially those from the ABC transporter superfamily, explain why flavonoid aglycones are present at very low levels in the systemic circulation. Recent research further suggested that it was the interplay between phase II enzymes (e.g. UGTs and SULTs isoforms) and efflux transporters (e.g. MRP2 and BCRP) that drives the metabolic disposition and clearance processes.

On the other hand, flavonoids can also modify expression or activity of those enzymes and transporters relevant for the interplay. For most flavonoids, these mean that they could inhibit the activity of phase II enzymes or transporters acutely; but induce the expression or activate the function of phase II enzymes or efflux transporters on a chronic basis. These interactions are often mediated via the action of various chemosensing mechanisms involving partners such as nucleic receptors and their co-regulators.

Therefore, mutual interactions between flavonoids and enzymes as well as efflux transporters are critical factors that should be taken into considerations when flavonoids are co-administered with medication. This is because pharmacokinetics of the medication can be altered. A better understanding of mutual interactions between disposition of flavonoids and elements responsible for their disposition will greatly help us to identify potential drug–flavonoid interactions and flavonoid–flavonoid interactions. In addition, the mutual interaction study can accelerate the process of building structure–metabolism or structure–activity relationships, allowing us to identify critical structural features that are not subjected to metabolism but may retain essential biological activity.

Abbreviation list

ABC	ATP-binding cassette

UGT	Uridine-5′-diphosphate glucuronosyltransferases

SULT	Sulfotransferases

CYP450

Cytochrome P450

P-gp	P-glycoprotein

MRP	Multi-drug resistance protein

BCRP	Breast cancer resistance protein

GST	Glutathione-S-transferases

OATP	Organic anion-transporting peptide

OAT	Organic anion transporter

SLC	Solute carrier
DME	Drug metabolizing enzymes

References

1. O. K. Chun, S. J. Chung and W. O. Song, J Nutr, 2007, 137, 1244.
2. J. H. de Vries, P. L. Janssen, P. C. Hollman, W. A. van Staveren and M. B. Katan, Cancer Lett., 1997, 114, 141.
3. M. G. Hertog, P. C. Hollman, M. B. Katan and D. Kromhout, Nutr. Cancer, 1993, 20, 21.
4. K. R. Narayana, M. S. Reddy, M. R. Chaluvadi and D. R. Krishna, Indian Journal of Pharmacology, 2001, 33, 2.
5. G. Schroder, E. Wehinger, R. Lukacin, F. Wellmann, W. Seefelder, W. Schwab and J. Schroder, Phytochemistry, 2004, 65, 1085.
6. Y. C. Wong, L. Zhang, G. Lin and Z. Zuo, Expert Opin. Drug Metab. Toxicol., 2009, 5, 1399.
7. J. B. a. W. C. A. Harborne, Nat. Prod. Rep., 1998, 15, 631.
8. N. C. a. G. R. J. Veitch, Nat. Prod. Rep., 2011, 28, 1626.
9. J. B. Harborne, The flavonoids: advances in research since 1986, Chapman & Hall/CRC, 1994.
10. C. Bosetti, L. Spertini, M. Parpinel, P. Gnagnarella, P. Lagiou, E. Negri, S. Franceschi, M. Montella, J. Peterson, J. Dwyer, A. Giacosa and C. La Vecchia, Cancer Epidemiol., Biomarkers Prev., 2005, 14, 805.
11. D. Grassi, G. Desideri, G. Croce, S. Tiberti, A. Aggio and C. Ferri, Curr. Pharm. Des., 2009, 15, 1072.
12. B. N. Ames, M. K. Shigenaga and T. M. Hagen, Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 7915.
13. T. Koga and M. Meydani, Am J Clin Nutr, 2001, 73, 941.
14. T. Walle, Free Radical Biol. Med., 2004, 36, 829.
15. R. A. Walgren, J. T. Lin, R. K. Kinne and T. Walle, J Pharmacol Exp Ther, 2000, 294, 837.
16. T. Walle, Y. Otake, U. K. Walle and F. A. Wilson, J Nutr, 2000, 130, 2658.
17. A. J. Day, M. S. DuPont, S. Ridley, M. Rhodes, M. J. Rhodes, M. R. Morgan and G. Williamson, FEBS Lett., 1998, 436, 71.
18. A. J. Day, F. J. Canada, J. C. Diaz, P. A. Kroon, R. McLauchlan, C. B. Faulds, G. W. Plumb, M. R. Morgan and G. Williamson, FEBS Lett., 2000, 468, 166.
19. Y. Liu, Y. Liu, Y. Dai, L. Xun and M. Hu, J. Altern. Complementary Med., 2003, 9, 631.
20. Y. Liu and M. Hu, Drug Metab. Dispos., 2002, 30, 370.
21. A. J. Day, J. M. Gee, M. S. DuPont, I. T. Johnson and G. Williamson, Biochem. Pharmacol., 2003, 65, 1199.
22. K. Nemeth, G. W. Plumb, J. G. Berrin, N. Juge, R. Jacob, H. Y. Naim, G. Williamson, D. M. Swallow and P. A. Kroon, Eur. J. Nutr., 2003, 42, 29.
23. J. Chen, H. Lin and M. Hu, J. Pharmacol. Exp. Ther., 2003, 304, 1228.
24. A. Gradolatto, M. C. Canivenc-Lavier, J. P. Basly, M. H. Siess and C. Teyssier, Drug Metab. Dispos., 2004, 32, 58.
25. C. Manach and J. L. Donovan, Free Radical Res., 2004, 38, 771.
26. W. Zhu, H. Xu, S. W. Wang and M. Hu, AAPS J., 2010, 12, 525.
27. S. R. Miranda, J. K. Lee, K. L. Brouwer, Z. Wen, P. C. Smith and R. L. Hawke, Drug Metab. Dispos., 2008, 36, 2219.
28. K. D. Setchell, N. M. Brown, P. Desai, L. Zimmer-Nechemias, B. E. Wolfe, W. T. Brashear, A. S. Kirschner, A. Cassidy and J. E. Heubi, J Nutr, 2001, 131, 1362S.
29. A. Crozier, D. Del Rio and M. N. Clifford, Mol. Aspects Med., 2010, 31, 446.
30. M. Hu, Mol. Pharmaceutics, 2007, 4, 803.
31. A. Crozier, I. B. Jaganath and M. N. Clifford, Nat. Prod. Rep., 2009, 26, 1001.
32. R. Cermak, Expert Opin. Drug Metab. Toxicol., 2008, 4, 17.
33. M. E. Morris and S. Zhang, Life Sci., 2006, 78, 2116.
34. A. I. Alvarez, R. Real, M. Perez, G. Mendoza, J. G. Prieto and G. Merino, J Pharm Sci, 2010, 99, 598.
35. Y. J. Moon, X. Wang and M. E. Morris, Toxicol. in Vitro, 2006, 20, 187.
36. P. Hodek, P. Trefil and M. Stiborova, Chem.-Biol. Interact., 2002, 139, 1.
37. R. Z. Harris, G. R. Jang and S. Tsunoda, Clin. Pharmacokinet., 2003, 42, 1071.
38. Y. Otake and T. Walle, Drug Metab. Dispos., 2002, 30, 103.
39. U. K. Walle and T. Walle, Drug Metab. Dispos., 2007, 35, 1985.
40. Y. Otake, F. Hsieh and T. Walle, Drug Metab. Dispos., 2002, 30, 576.
41. S. Meng, B. Wu, R. Singh, T. Yin, J. K. Morrow, S. Zhang and M. Hu, Mol. Pharmaceutics, 2012, 9, 862.
42. L. Tang, L. Ye, R. Singh, B. Wu, C. Lv, J. Zhao, Z. Liu and M. Hu, Mol. Pharmaceutics, 2010, 7, 664.
43. B. Wu, S. Basu, S. Meng, X. Wang and M. Hu, Curr. Drug Metab., 2011, 12, 900.
44. L. Tang, R. Singh, Z. Liu and M. Hu, Mol. Pharmaceutics, 2009, 6, 1466.
45. R. H. Tukey and C. P. Strassburg, Annu. Rev. Pharmacol., 2000, 40, 581.
46. T. B. Joseph, S. W. Wang, X. Liu, K. H. Kulkarni, J. Wang, H. Xu and M. Hu, Mol. Pharmaceutics, 2007, 4, 883.
47. P. I. Mackenzie, K. W. Bock, B. Burchell, C. Guillemette, S. Ikushiro, T. Iyanagi, J. O. Miners, I. S. Owens and D. W. Nebert, Pharmacogenet. Genomics, 2005, 15, 677.
48. A. Nakamura, M. Nakajima, H. Yamanaka, R. Fujiwara and T. Yokoi, Drug Metab. Dispos., 2008, 36, 1461.
49. R. H. Lewinsky, P. A. Smith and P. I. Mackenzie, Xenobiotica, 2005, 35, 117.
50. Z. Cheng, A. Radominska-Pandya and T. R. Tephly, Drug Metab Dispos, 1999, 27, 1165.
51. Y. Chen, S. Xie, S. Chen and S. Zeng, Biochem. Pharmacol., 2008, 76, 416.
52. M. G. Boersma, H. van der Woude, J. Bogaards, S. Boeren, J. Vervoort, N. H. Cnubben, M. L. van Iersel, P. J. van Bladeren and I. M. Rietjens, Chem. Res. Toxicol., 2002, 15, 662.
53. A. Galijatovic, Y. Otake, U. K. Walle and T. Walle, Xenobiotica, 1999, 29, 1241.
54. H. S. Lee, H. Y. Ji, E. J. Park and S. Y. Kim, Xenobiotica, 2007, 37, 803.
55. D. Turgeon, J. S. Carrier, S. Chouinard and A. Belanger, Drug Metab. Dispos., 2003, 31, 670.
56. S. Xie, L. You and S. Zeng, Pharmazie, 2007, 62, 625.
57. K. M. Janisch, G. Williamson, P. Needs and G. W. Plumb, Free Radical Res., 2004, 38, 877.
58. N. Gamage, A. Barnett, N. Hempel, R. G. Duggleby, K. F. Windmill, J. L. Martin and M. E. McManus, Toxicol. Sci., 2006, 90, 5.
59. K. Nagata and Y. Yamazoe, Annu. Rev. Pharmacol., 2000, 40, 159.
60. D. Ung and S. Nagar, Drug Metab. Dispos., 2007, 35, 740.
61. S. Nowell and C. N. Falany, Oncogene, 2006, 25, 1673.
62. J. B. Vaidyanathan and T. Walle, Drug Metab. Dispos., 2002, 30, 897.
63. C. Huang, Y. Chen, T. Zhou and G. Chen, Xenobiotica, 2009, 39, 312.
64. M. Hu, J. Chen and H. Lin, J. Pharmacol. Exp. Ther., 2003, 307, 314.
65. H. Sun, L. Zhang, E. C. Chow, G. Lin, Z. Zuo and K. S. Pang, J. Pharmacol. Exp. Ther., 2008, 326, 117.
66. W. Meinl, B Ebert, H. Glatt and A. Lampen, Drug Metab. Dispos., 2008, 36, 276.
67. K. J. Linton, Physiology, 2007, 22, 122.
68. M. Nait Chabane, A. Al Ahmad, J. Peluso, C. D. Muller and G. Ubeaud, J. Pharm. Pharmacol., 2009, 61, 1473.
69. G. Williamson, I. Aeberli, L. Miguet, Z. Zhang, M. B. Sanchez, V. Crespy, D. Barron, P. Needs, P. A. Kroon, H. Glavinas, P. Krajcsi and M. Grigorov, Drug Metab. Dispos., 2007, 35, 1262.
70. W. Brand, P. A. van der Wel, M. J. Rein, D. Barron, G. Williamson, P. J. van Bladeren and I. M. Rietjens, Drug Metab. Dispos., 2008, 36, 1794.
71. G. An, J. Gallegos and M. E. Morris, Drug Metab. Dispos., 2011, 39, 426.
72. K. van de Wetering, W. Feddema, J. B. Helms, J. F. Brouwers and P. Borst, Gastroenterology, 2009, 137, 1725.
73. X. Jia, J. Chen, H. Lin and M. Hu, J. Pharmacol. Exp. Ther., 2004, 310, 1103.
74. W. Jiang, B. Xu, B. Wu, R. Yu and M. Hu, Drug Metab Dispos, 2011.
75. Y. Zhang and G. B. Gordon, Mol Cancer Ther, 2004, 3, 885.
76. B. L. Urquhart, R. G. Tirona and R. B. Kim, J. Clin. Pharmacol., 2007, 47, 566.
77. X. Y. Sun, C. A. Plouzek, J. P. Henry, T. T. Wang and J. M. Phang, Cancer Res, 1998, 58, 2379.
78. T. Walle, Y. Otake, A. Galijatovic, J. K. Ritter and U. K. Walle, Drug Metab Dispos, 2000, 28, 1077.
79. A. Galijatovic, U. K. Walle and T. Walle, Pharm. Res., 2000, 17, 21.
80. A. Galijatovic, Y. Otake, U. K. Walle and T. Walle, Pharm. Res., 2001, 18, 374.
81. U. K. Walle and T. Walle, Drug Metab. Dispos., 2002, 30, 564.
82. M. F. Yueh and R. H. Tukey, J. Biol. Chem., 2007, 282, 8749.
83. J. A. Williams, B. J. Ring, V. E. Cantrell, K. Campanale, D. R. Jones, S. D. Hall and S. A. Wrighton, Drug Metab. Dispos., 2002, 30, 1266.
84. R. Venkataramanan, V. Ramachandran, B. J. Komoroski, S. Zhang, P. L. Schiff and S. C. Strom, Drug Metab Dispos, 2000, 28, 1270.
85. V. Svehlikova, S. Wang, J. Jakubikova, G. Williamson, R. Mithen and Y. Bao, Carcinogenesis, 2004, 25, 1629.
86. K. W. Bock, T. Eckle, M. Ouzzine and S. Fournel-Gigleux, Biochem. Pharmacol., 2000, 59, 467.
87. N. Petri, C. Tannergren, B. Holst, F. A. Mellon, Y. Bao, G. W. Plumb, J. Bacon, K. A. O'Leary, P. A. Kroon, L. Knutson, P. Forsell, T. Eriksson, H. Lennernas and G. Williamson, Drug Metab. Dispos., 2003, 31, 805.
88. E. M. van der Logt, H. M. Roelofs, F. M. Nagengast and W. H. Peters, Carcinogenesis, 2003, 24, 1651.
89. M. C. Canivenc-Lavier, M. F. Vernevaut, M. Totis, M. H. Siess, J. Magdalou and M. Suschetet, Toxicology, 1996, 114, 19.
90. L. C. Appelt and M. M. Reicks, J Nutr, 1999, 129, 1820.
91. Y. F. Ueng, C. C. Shyu, Y. L. Lin, S. S. Park, J. F. Liao and C. F. Chen, Life Sci., 2000, 67, 2189.
92. H. Nakano, K. Ogura, E. Takahashi, T. Harada, T. Nishiyama, K. Muro, A. Hiratsuka, S. Kadota and T. Watabe, Drug Metab. Pharmacokinet., 2004, 19, 216.
93. E. A. Eaton, U. K. Walle, A. J. Lewis, T. Hudson, A. A. Wilson and T. Walle, Drug Metab Dispos, 1996, 24, 232.
94. T. Walle, E. A. Eaton and U. K. Walle, Biochem. Pharmacol., 1995, 50, 731.
95. R. M. Harris, D. M. Wood, L. Bottomley, S. Blagg, K. Owen, P. J. Hughes, R. H. Waring and C. J. Kirk, J. Clin. Endocrinol. Metab., 2004, 89, 1779.
96. R. M. Harris and R. H. Waring, Curr. Drug Metab., 2008, 9, 269.
97. R. H. Waring, S. Ayers, A. J. Gescher, H. R. Glatt, W. Meinl, P. Jarratt, C. J. Kirk, T. Pettitt, D. Rea and R. M. Harris, J. Steroid Biochem. Mol. Biol., 2008, 108, 213.
98. Y. Otake, A. L. Nolan, U. K. Walle and T. Walle, J. Steroid Biochem. Mol. Biol., 2000, 73, 265.
99. Y. Chen, C. Huang, T. Zhou and G. Chen, Basic Clin. Pharmacol. Toxicol., 2008, 103, 553.
100. Y. Chen, C. Huang, T. Zhou, S. Zhang and G. Chen, J. Biochem. Mol. Toxicol., 2010, 24, 102.
101. W. H. Peters, L. Kock, F. M. Nagengast and P. G. Kremers, Gut, 1991, 32, 408.
102. P. J. Ansell, C. Espinosa-Nicholas, E. M. Curran, B. M. Judy, B. J. Philips, M. Hannink and D. B. Lubahn, Endocrinology, 2004, 145, 311.
103. B. S. Patill, J. S. Brodbelt, E. G. Miller, N. D. Turner, in Potential Health Benefits of Citrus, ed. B. S. Patil, N. D. Turner , E. G. Miller and J. S. Brodbelt, 2006, vol. 936.
104. A. E. Mitchell, S. A. Burns and J. L. Rudolf, Arch. Toxicol., 2007, 81, 777.
105. T. Hofmann, U. Liegibel, P. Winterhalter, A. Bub, G. Rechkemmer and B. L. Pool-Zobel, Mol. Nutr. Food Res., 2006, 50, 1191.
106. M. Dean, The human ATP-binding cassette (ABC) transporter superfamily, National center for biotechnology information, 2002.
107. M. M. Gottesman and I. Pastan, Annu. Rev. Biochem., 1993, 62, 385.
108. U. A. Germann, I. Pastan and M. M. Gottesman, Seminars in Cell Biology, 1993, 4, 63.
109. J. Jodoin, M. Demeule and R. Beliveau, Biochim. Biophys. Acta, Mol. Cell Res., 2002, 1542, 149.
110. E. J. Wang, M. Barecki-Roach and W. W. Johnson, Biochem. Biophys. Res. Commun., 2002, 297, 412.
111. S. Zhou, L. Y. Lim and B. Chowbay, Drug Metab. Rev., 2004, 36, 57.
112. K. S. Lown, D. G. Bailey, R. J. Fontana, S. K. Janardan, C. H. Adair, L. A. Fortlage, M. B. Brown, W. Guo and P. B. Watkins, J. Clin. Invest., 1997, 99, 2545.
113. V. A. Eagling, L. Profit and D. J. Back, Br. J. Clin. Pharmacol., 1999, 48, 543.
114. A. Soldner, U. Christians, M. Susanto, V. J. Wacher, J. A. Silverman and L. Z. Benet, Pharm. Res., 1999, 16, 478.
115. Y. Mitsunaga, H. Takanaga, H. Matsuo, M. Naito, T. Tsuruo, H. Ohtani and Y. Sawada, Eur. J. Pharmacol., 2000, 395, 193.
116. N. Romiti, G. Tramonti, A. Donati and E. Chieli, Life Sci., 2004, 76, 293.
117. A. F. Castro and G. A. Altenberg, Biochem. Pharmacol., 1997, 53, 89.
118. X. L. Liu, H. W. Tee and M. L. Go, Bioorg. Med. Chem., 2008, 16, 171.
119. R. Hayeshi, C. Masimirembwa, S. Mukanganyama and A. L. Ungell, Eur. J. Pharm. Sci., 2006, 29, 70.
120. T. Ikegawa, H. Ohtani, N. Koyabu, M. Juichi, Y. Iwase, C. Ito, H. Furukawa, M. Naito, T. Tsuruo and Y. Sawada, Cancer Lett., 2002, 177, 89.
121. M. Hadjeri, M. Barbier, X. Ronot, A. M. Mariotte, A. Boumendjel and J. Boutonnat, J. Med. Chem., 2003, 46, 2125.
122. J. Boccard, F. Bajot, A. Di Pietro, S. Rudaz, A. Boumendjel, E. Nicolle and P. A. Carrupt, Eur. J. Pharm. Sci., 2009, 36, 254.
123. E. M. Leslie, R. G. Deeley and S. P. Cole, Toxicology, 2001, 167, 3.
124. E. M. Leslie, R. G. Deeley and S. P. Cole, Toxicol. Appl. Pharmacol., 2005, 204, 216.
125. C. H. Versantvoort, H. J. Broxterman, J. Lankelma, N. Feller and H. M. Pinedo, Biochem. Pharmacol., 1994, 48, 1129.
126. S. Paul, M. G. Belinsky, H. Shen and G. D. Kruh, Biochemistry, 1996, 35, 13647.
127. J. H. Hooijberg, H. J. Broxterman, M. Heijn, D. L. Fles, J. Lankelma and H. M. Pinedo, FEBS Lett., 1997, 413, 344.
128. M. Schumacher, A. Hautzinger, A. Rossmann, S. Holzhauser, D. Popovic, A. Hertrampf, S. Kuntz, M. Boll and U. Wenzel, Biochem. Pharmacol., 2010, 80, 471.
129. T. Sergent, S. Garsou, A. Schaut, S. De Saeger, L. Pussemier, C. Van Peteghem, Y. Larondelle and Y. J. Schneider, Toxicol. Lett., 2005, 159, 60.
130. J. J. van Zanden, H. van der Woude, J. Vaessen, M. Usta, H. M. Wortelboer, N. H. Cnubben and I. M. Rietjens, Biochem. Pharmacol., 2007, 74, 345.
131. J. J. van Zanden, H. M. Wortelboer, S. Bijlsma, A. Punt, M. Usta, P. J. Bladeren, I. M. Rietjens and N. H. Cnubben, Biochem. Pharmacol., 2005, 69, 699.
132. L. A. Doyle, W. Yang, L. V. Abruzzo, T. Krogmann, Y. Gao, A. K. Rishi and D. D. Ross, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 15665.
133. R. Allikmets, L. M. Schriml, A. Hutchinson, V. Romano-Spica and M. Dean, Cancer Res, 1998, 58, 5337.
134. C. Ozvegy, T. Litman, G. Szakacs, Z. Nagy, S. Bates, A. Varadi and B. Sarkadi, Biochem. Biophys. Res. Commun., 2001, 285, 111.
135. K. M. Giacomini, S. M. Huang, D. J. Tweedie, L. Z. Benet, K. L. Brouwer, X. Chu, A. Dahlin, R. Evers, V. Fischer, K. M. Hillgren, K. A. Hoffmaster, T. Ishikawa, D. Keppler, R. B. Kim, C. A. Lee, M. Niemi, J. W. Polli, Y. Sugiyama, P. W. Swaan, J. A. Ware, S. H. Wright, S. W. Yee, M. J. Zamek-Gliszczynski and L. Zhang, Nat. Rev. Drug Discovery, 2010, 9, 215.
136. A. E. van Herwaarden and A. H. Schinkel, Trends Pharmacol. Sci., 2006, 27, 10.
137. M. L. Vlaming, J. S. Lagas and A. H. Schinkel, Adv. Drug Delivery Rev., 2009, 61, 14.
138. R. W. Robey, K. K. To, O. Polgar, M. Dohse, P. Fetsch, M. Dean and S. E. Bates, Adv. Drug Delivery Rev., 2009, 61, 3.
139. S. Zhang, X. Yang and M. E. Morris, Mol. Pharmacol., 2004, 65, 1208.
140. S. Zhang, X. Yang and M. E. Morris, Pharm. Res., 2004, 21, 1263.
141. G. An, F. Wu and M. E. Morris, Pharm. Res., 2011, 28, 1090.
142. G. An and M. E. Morris, Biopharm Drug Dispos, 2010, 31, 340.
143. Y. Imai, S. Tsukahara, S. Asada and Y. Sugimoto, Cancer Res., 2004, 64, 4346.
144. H. Tamaki, H. Satoh, S. Hori, H. Ohtani and Y. Sawada, Drug Metab. Pharmacokinet., 2010, 25, 170.
145. A. Pick, H. Muller, R. Mayer, B. Haenisch, I. K. Pajeva, M. Weigt, H. Bonisch, C. E. Muller and M. Wiese, Bioorg. Med. Chem., 2011, 19, 2090.
146. H. C. Cooray, T. Janvilisri, H. W. van Veen, S. B. Hladky and M. A. Barrand, Biochem. Biophys. Res. Commun., 2004, 317, 269.
147. B. Ebert, A. Seidel and A. Lampen, Toxicol. Sci., 2007, 96, 227.
148. C. C. Wong, N. P. Botting, C. Orfila, N. Al-Maharik and G. Williamson, Biochem. Pharmacol., 2011, 81, 942.
149. X. Wang, A. W. Wolkoff and M. E. Morris, Drug Metab. Dispos., 2005, 33, 1666.
150. G. K. Dresser, D. G. Bailey, B. F. Leake, U. I. Schwarz, P. A. Dawson, D. J. Freeman and R. B. Kim, Clin. Pharmacol. Ther., 2002, 71, 11.
151. D. G. Bailey, G. K. Dresser, B. F. Leake and R. B. Kim, Clin. Pharmacol. Ther., 2007, 81, 495.
152. H. Glaeser, D. G. Bailey, G. K. Dresser, J. C. Gregor, U. I. Schwarz, J. S. McGrath, E. Jolicoeur, W. Lee, B. F. Leake, R. G. Tirona and R. B. Kim, Clin. Pharmacol. Ther., 2007, 81, 362.
153. B. Hagenbuch and P. J. Meier, Pfluegers Arch., 2004, 447, 653.
154. H. Satoh, F. Yamashita, M. Tsujimoto, H. Murakami, N. Koyabu, H. Ohtani and Y. Sawada, Drug Metab. Dispos., 2005, 33, 518.
155. R. Kojima, T. Sekine, M. Kawachi, S. H. Cha, Y. Suzuki and H. Endou, J Am Soc Nephrol, 2002, 13, 848.
156. A. C. Whitley, D. H. Sweet and T. Walle, Drug Metab. Dispos., 2005, 33, 1097.
157. S. S. Hong, K. Seo, S. C. Lim and H. K. Han, Pharmacol. Res., 2007, 56, 468.
158. D. R. Ferry, A. Smith, J. Malkhandi, D. W. Fyfe, P. G. deTakats, D. Anderson, J. Baker and D. J. Kerr, Clin Cancer Res, 1996, 2, 659.
159. G. Zhang, L. Qin and Y. Shi, J. Bone Miner. Res., 2007, 22, 1072.
160. Y. J. Moon and M. E. Morris, Mol. Pharmaceutics, 2007, 4, 865.
161. W. X. Peng, H. D. Li and H. H. Zhou, Eur. J. Clin. Pharmacol., 2003, 59, 237.

---