Inhibitory Effect of Hesperetin and Naringenin on Human UDP-Glucuronosyltransferase Enzymes: Implications for Herb–Drug Interactions

Abstract

Hesperetin (HET) and naringenin (NGR) are flavanones found in citrus (oranges and grapefruit) and Aurantii Fructus Immaturus. The present study aims to investigate the inhibition potential of HET and NGR derivatives towards one of the most important phase II drug-metabolizing enzymes-uridine diphosphate (UDP)-glucuronosyltransferases (UGTs). We used trifluoperazine as a probe substrate to test UGT1A4 activity, and recombinant UGT-catalyzed 4-methylumbelliferone glucuronidation was used as a probe reaction for other UGT isoforms. Data show that HET and NGR displayed broad-spectrum inhibition against human UGTs. Besides, HET exhibited strong inhibitory effects on UGT1A1, 1A3 and 1A9 (both IC₅₀ and K_i values lower than 10 µM), and the inhibitory effects of NGR against three major UGTs, including UGT1A1, 1A3 and 2B7. In a combination of inhibition parameters (K_i) and in vivo concentration of HET and NGR, the potential in vivo inhibition magnitude was predicted. Based on the reported maximum plasma concentration of HET and NGR in vivo, these findings indicate the potential herb–drug interactions (HDI) between HET or NGR and the drugs mainly undergoing UGT1A3 or UGT2B7 catalyzed metabolic elimination. Considering the variety of citrus that contains HET and NGR, so caution should be applied when taking drugs that utilize UGTs for metabolism and clearance with citrus fruits.

Hesperetin (HET; 4′-methoxy-3′,5,7-trihydroxyflavanone) and naringenin (NGR; 4,5,7-trihydroxyflavanone), aglycones of the flavanone glycoside hesperidin (chiefly in oranges) and naringin (grapefruits) are also used as dietary flavonoids.¹⁾ Moreover, flavanones are bioactive components of Aurantii Fructus Immaturus, which is reported to be used in traditional Chinese medicine.²⁾ Several animal studies have been performed to document pharmacokinetics of HET and NGR.^3,4) The role of HET and NGR in the treatment of disease has received considerable attention, and it is reported to have anti-oxidant, anti-inflammatory, anti-hypertensive, anti-atherogenic, anti-diabetic, antiviral, and neuroprotective activities.^5–8) Thus, HET and NGR have multiple uses and pharmacokinetic studies of both compounds may elucidate information about efficacy and safety. Chemical structures of HET and NGR appear in Fig. 1.

Fig. 1. HET and NGR Structure

Herb–drug interaction (HDI) strongly limits the clinical application of herbs and drugs, and the inhibition of herbal components towards important drug-metabolizing enzymes (DMEs) has been regarded as one of the most important reasons.⁹⁾ CYP450 and uridine diphosphate (UDP)-glucuronosyltransferase (UGT) are two enzymes responsible for oxidative phase I and conjugative phase II reactions, respectively.¹⁰⁾ In the past decades, the CYP450 inhibition by HET and NGR has been widely studied, the effects of HET and NGR on UGTs activity have not been fully characterized. Moreover, a large number of different UGTs that exhibit a wide range of substrates are distributed throughout the human body, which makes them a particularly important group of phase II metabolizing enzymes.¹¹⁾ Thus, addressing this deficit in the literature would better inform investigators about UGT metabolism of various xenobiotics (e.g., flavonoids, drugs, chemical carcinogens) and endogenous substances (e.g., bilirubin, estradiol, bile acids, and fat-soluble vitamins), which accounts for >35% of all phase II drug metabolism events.¹²⁾ Studies indicate that some herbal components can potently or modestly inhibit UGT isoforms. For example, quercetin potent to inhibits UGT1A1 and UGT1A9¹³⁾ and deoxyschizandrin and schisantherin A inhibit UGT1A3.¹⁴⁾ Inhibition of UGT activity not only induce HDI, but also cause troublesome side effects. For example, sorafenib, approved for treating renal cell and unresectable hepatocellular carcinomas, has been reported to induce hyperbilirubinemia via inhibition of UGT1A1 activity.¹⁵⁾ Inhibition of UGT1A1 can decrease bilirubin conjugation to induce hyperbilirubinemia, so it was worth to study how herbs interact with UGTs.

Pharmacokinetic investigations of interactions between herbs and DMEs indicate that HET and NGR inhibited CYP450s.^16–18) However, the effects of HET and NGR on UGT activity have not been fully characterized. Understanding the effects of HET and NGR on UGT activities is important to ensure their safe administration and development new co-administration therapies with both drugs. The aim of this study was to investigate and compare the effects of HET and NGR on the activities of human UGTs. The potential for HDI in vivo was also quantitatively predicted and compared by using the area under the curve (AUC) ratios.

MATERIALS AND METHODS

Chemicals and Reagents

HET (purity: 98%) and NGR (purity: 98%) were purchased from Yi Fang Technology Co., Ltd. (Tianjin, China). Trifluoperazine hydrochloride (purity: 99.4%) and trifluoperazine-N-glucuronide were purchased from Toronto Research Chemicals (North York, ON, Canada). 4-Methylumbelliferone (4-MU), 4-methylumbelliferone-β-D-glucuronide (4-MUG), and 5′-diphospho-glucuronic acid trisodium salt (UDPGA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Isoimperatorin (purity: 99.6%), clarithromycin (purity: 99.4%) and magnesium chloride were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Tris–HCl buffer (pH 7.4) was prepared by using Millipore filtered water (Millipore, Bedford, U.S.A.) and stored at 4°C until use. All other reagents were of the highest purity commercially available or HPLC grade.

Recombinant Human UGTs

Recombinant human UGT supersomes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in baculovirus-infected insect cells were obtained from BD Gentest Corp. (Woburn, MA, U.S.A.) and stored at −80°C until use.

Incubation of 4-MU Glucuronidation Assay

4-MU, a nonselective substrate of UGTs, was used as a probe substrate for all tested UGT isoforms (except trifluoperazine was used for UGT1A4). 4-MU was incubated with recombinant UGTs in the presence/absence of HET or NGR (1, 10, and 100 µM) in a reaction mixture (200 µL) containing 50 mM Tris–HCl buffer (pH 7.4), 5 mM MgCl₂, and 3 mM UDPGA. All components were dissolved in methanol and the final concentration of methanol in incubation system was no more than 0.5% (v/v). After a 5 min pre-incubation at 37°C, UDPGA was added in the mixture to initiate the reaction. Incubation time and protein measurements were measured according to published methods with slight modifications^19,20) details appear in Table 1.

Table 1. Incubation Conditions of 4-MU and Trifluoperazine Glucuronidation by Recombinant UGTs

UGTs	Protein (mg/mL)	Incubation (min)	4-MU concentration (µM)
UGT1A1	0.10	30	100
UGT1A3	0.05	60	1000
UGT1A4	0.10	20	TFP (40 µM)
UGT1A6	0.05	30	100
UGT1A7	0.05	30	15
UGT1A8	0.05	15	750
UGT1A9	0.025	30	10
UGT1A10	0.10	60	30
UGT2B4	0.20	120	1000
UGT2B7	0.025	60	300
UGT2B15	0.10	30	250
UGT2B17	0.20	120	2000

4-MU and trifluoperaezine concentrations corresponded to known K_m or S₅₀ values for each enzyme.^20,21) Reactions were quenched with adding 100 µL ice-cold acetonitrile with isoimperatorin (4 µM) as an internal standard, then the mixture was placed on ice for 20 min. Mixtures were vortexed for 5 min and then centrifuged at 20000×g for 10 min and then an aliquot of supernatant was transferred to an auto-injector vial for HPLC analysis. All experiments were performed in duplicate.

4-MUG concentrations were quantified using an HPLC system (Agilent Technologies, Santa Clara, CA, U.S.A.) equipped with four G1310A pumps, a G1329A auto injector, a G1314B VWD UV detector. Chromatographic separation was performed on a Luna C₁₈ column (150×4.6 mm, 5 µm, Phenomenex) with the mobile phase consisted of acetonitrile (A) and 0.5% formic acid (B) at a flow rate of 1 mL/min. The following gradient was used: 0–7 min, 15–90% A; 7–9 min, 90% A; 9–15 min, 15% A. The column temperature was maintained at 25°C. The UV detector was set at 316 nm. The total run time was 15.0 min per sample for each metabolite, with a retention time for the peaks of 4-MUG (4.06 min), 4-MU (5.58 min), and isoimperatorin (9.27 min, internal standard). Peak shapes and separation efficiency for three compounds were achieved.

Incubation of Trifluoperazine Glucuronidation Assay

Trifluoperazine was used as a probe substrate for UGT1A4 because it does not conjugate 4-MU. Trifluoperazine hydrochloride was dissolved in H₂O and then incubated with recombinant UGT1A4 in presence/absence of HET or NGR (1, 10, and 100 µM) in a reaction mixture (200 µL total volume). Then samples were treated and assayed as described above with clarithromycin (0.4 µM) as an internal standard.

Trifluoperazine-N-glucuronide was analyzed on an LC/MS system (Shimadzu, Kyoto, Japan) equipped with UPLC and LCMS-2010EV instruments. Chromatographic separations were carried out using Luna C₁₈ column (50×2.0 mm, 5 µm) with a single quadrupole mass spectrometer. The mobile phase consisted of acetonitrile (A) and H₂O containing 5 mmol/L ammonium (pH 3.7, B) (50 : 50, v/v) at a flow rate of 0.2 mL/min. The column temperature was maintained at 25°C. MS parameters were: negative mode, curved desolvation line (CDL) temperature 250°C, heat block temperature 200°C, interface voltage 40 V, detector voltage 1.3 kV, and nebulizing gas (N₂) 1.5 L/min. Quantification was performed with the selective ion monitoring mode of ions at m/z 584.52 for trifluoperazine-N-glucuronide and m/z 748.70 for clarithromycin (internal standard), and retention times were 1.26 and 1.21 min, respectively.

Kinetic Analyses for HET and NGR-Associated Inhibition on Recombinant Human UGTs

IC₅₀ values were obtained with various concentrations of HET and NGR. IC₅₀ values were calculated by non-linear regression analysis using Graphpad Prism 5.0. Glucuronidation velocity was measured using various concentrations of HET, NGR and 4-MU or trifluoperazine (TFP). K_i values were obtained using nonlinear regression and equations for competitive inhibition (Eq. 1), non-competitive inhibition (Eq. 2), or mixed inhibition (Eq. 3) as previously described.²²⁾ Actual experimental examples of uncompetitive inhibition are hard to find, so there is no equation to show here. Dixon and Lineweaver plots (and plot slope) were used to confirm the inhibition type and calculate K_i values.   

(1)

  

(2)

  

(3)

Where V is the velocity of the reaction; S and I are the substrate and inhibitor concentrations, respectively; K_i is the inhibition constant describing the affinity of the inhibitor to the enzyme; K_m is the substrate concentration at half of the maximum velocity (V_max) of the reaction. K_i is the constant describing the affinity of enzyme-substrate complex for inhibitor.

In Vitro–in Vivo Extrapolation (IV–IVE) to Predict in Vivo HET/NGR–Drug Interaction

The magnitudes of inhibitory interactions of HET and NGR were estimated as the equation of the area under the plasma concentration-time curve in the presence and absence of the inhibitor (AUC_i/AUC).

Ratios were calculated with the following Eq. 4:   

(4)

Where I is the concentration of inhibitor at the enzyme active site, and f_m is the fraction of drug metabolized by the inhibited enzyme. K_i is the inhibition constant describing the affinity of the inhibitor to the enzyme. When a drug’ clearance is solely mediated by a single enzyme (f_m=1), the AUC_i/AUC equation can be simplified to Eq. 5 as previously utilized.¹⁰⁾   

(5)

Terms are defined as follows: AUC_i/AUC is the predicted ratio of in vivo exposure of xenobiotics or endogenous substances with/without co-administration of HET or NGR. K_i values are in vitro inhibition kinetic constants. [I]_{in vivo} is the in vivo unbound concentration of HET or NGR. Ratios of [I]_{in vivo}/K_i values were calculated to evaluate risks of UGT inhibition by HET and NGR. Based on the evaluation standard for HDI ([I]_{in vivo}/K_i<0.1, not possible; 0.1< [I]_{in vivo}/K_i<1, possible; [I]_{in vivo}/K_i>1, highly possible).²³⁾

RESULTS

Inhibition Screening of UGT Activities by HET and NGR in Recombinant Human UGTs

As seen in Fig. 2, the addition of HET (100 µM, final concentration) exhibited a strong or moderate inhibition on UGT1A1, 1A3, 1A4, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and the corresponding residual activities were 1.17, 3.56, 45.88, 23.71, 34.27, 6.74, 55.94, 51.78, 59.77, 59.73%, respectively. Likewise, as shown in Fig. 3, 100 µM of NGR exhibited a strong or moderate inhibition on UGT1A1, 1A3, 1A4, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and the corresponding residual activities were 3.25, 5.68, 38.72, 23.13, 29.71, 12.03, 38.55, 52.20, 3.71, 21.89%, respectively. Both HET and NGR had a negligible effect on the activities of UGT1A6 and 2B17. For UGT isoforms whose activity is inhibited by >50% by individual HET or NGR, IC₅₀ values of HET and NGR were further estimated.

Fig. 2. HET Inhibition of 12 Tested rhUGT Isoforms: HET (0, 1, 10, and 100 µM) as Percent of Control

Fig. 3. NGR Inhibition of 12 Tested rhUGT Isoforms: NGR (0, 1, 10, and 100 µM) as Percent of Control

To characterize inhibitory effects of HET and NGR towards UGT activities, dose-dependent inhibition curves were plotted with different HET and NGR concentrations and IC₅₀ data appear in Figs. 4A, 5A, 6A, 7A, 8A, 9A. Nilotinib (IC₅₀ for UGT1A1: 0.10 µM) and androsterone (IC₅₀ for UGT2B7: 12.8 µM) were used as positive controls for inhibition assays.^22,24) These results showed that HET strongly inhibited UGT1A1, UGT1A3, and UGT1A9 (IC₅₀<10 µM) and moderately inhibited UGT1A4, UGT1A7, UGT1A8 (IC₅₀ values 29.68–63.87 µM). NGR exhibited a strong inhibition on UGT1A1, UGT1A3, UGT2B7 (IC₅₀<10 µM) and a moderate inhibition on UGT1A4, UGT1A7, UGT1A8, UGT1A9, UGT1A10, and UGT2B15 (IC₅₀: 15–55 µM).

Fig. 4. K_i of UGT1A1 by Hesperetin

Dose-dependent inhibition of HET against rhUGT1A1-catalyzed 4-MU glucuronidation and IC₅₀ value (A), Dixon plot for HET inhibition of rhUGT1A1-catalyzed 4-MU glucuronidation (B), Lineweaver–Burk plot for HET inhibition of rhUGT1A1-catalyzed 4-MU glucuronidation (C), slopes from Lineweaver–Burk plot of HET inhibition of rhUGT1A1-catalyzed 4-MU glucuronidation (D).

Fig. 5. K_i of UGT1A3 by Hesperetin

Dose-dependent inhibition of HET against rhUGT1A1-catalyzed 4-MU glucuronidation and IC₅₀ value (A), Dixon plot for HET inhibition of rhUGT1A3-catalyzed 4-MU glucuronidation (B), Lineweaver–Burk plot for HET inhibition of rhUGT1A3-catalyzed 4-MU glucuronidation (C), slopes from Lineweaver–Burk plot of HET inhibition of rhUGT1A3-catalyzed 4-MU glucuronidation (D).

Fig. 6. K_i of UGT1A9 by Hesperetin

Dose-dependent inhibition of HET against rhUGT1A9-catalyzed 4-MU glucuronidation and IC₅₀ value (A), Dixon plot for HET inhibition of rhUGT1A9-catalyzed 4-MU glucuronidation (B), Lineweaver–Burk plot for HET inhibition of rhUGT1A9-catalyzed 4-MU glucuronidation (C), slopes from Lineweaver–Burk plot of HET inhibition of rhUGT1A9-catalyzed 4-MU glucuronidation (D).

Fig. 7. K_i of UGT1A1 by Naringenin

Dose-dependent inhibition of NGR against rhUGT1A1-catalyzed 4-MU glucuronidation and IC₅₀ value (A), Dixon plot for NGR inhibition of rhUGT1A1-catalyzed 4-MU glucuronidation (B), Lineweaver–Burk plot for NGR inhibition of rhUGT1A1-catalyzed 4-MU glucuronidation (C), slopes from Lineweaver–Burk plot of NGR inhibition of rhUGT1A1-catalyzed 4-MU glucuronidation (D).

Fig. 8. K_i of UGT1A3 by Naringenin

Dose-dependent inhibition of NGR against rhUGT1A3-catalyzed 4-MU glucuronidation and IC₅₀ value (A), Dixon plot for NGR inhibition of rhUGT1A3-catalyzed 4-MU glucuronidation (B), Lineweaver–Burk plot for NGR inhibition of rhUGT1A3-catalyzed 4-MU glucuronidation (C), slopes from Lineweaver–Burk plot of NGR inhibition of rhUGT1A3-catalyzed 4-MU glucuronidation (D).

Fig. 9. K_i of UGT2B7 by Naringenin

Dose-dependent inhibition of NGR against rhUGT2B7-catalyzed 4-MU glucuronidation and IC₅₀ value (A), Dixon plot for NGR inhibition of rhUGT2B7-catalyzed 4-MU glucuronidation (B), Lineweaver–Burk plot for NGR inhibition of rhUGT2B7-catalyzed 4-MU glucuronidation (C), slopes from Lineweaver–Burk plot of NGR inhibition of rhUGT2B7-catalyzed 4-MU glucuronidation (D).

Inhibition Kinetics of HET and NGR on Recombinant UGTs

Based on above IC₅₀ values (<10 µM), the inhibition types and inhibition parameters of HET on UGT1A1, 1A3, 1A9 and NGR on UGT1A1, 1A3, 2B7 were investigated. The results (Figs. 4B & C, 5B & C, 6B & C) indicated that HET competitively inhibited UGT1A1-catalyzed 4-MU glucuronidation, and exerted mixed inhibition towards UGT1A3 and 1A9-catalyzed 4-MU glucuronidation, the K_i values were calculated to be 9.63, 0.99 and 3.41 µM, respectively. Likewise, NGR was found to be a strong competitive inhibitor of UGT2B7 with a K_i of 1.39 µM (Figs. 9B & C). It also exerted intermediate non-competitive inhibition against UGT1A1 with K_i of 7.61 µM (Figs. 7B & C), as well as an intermediate uncompetitive inhibition against UGT1A3 with K_i of 85.76 µM (Figs. 8B & C).

Data Extrapolation

In vivo extrapolation was used to generate pharmacokinetic data after ingestion of orange or grapefruit juice, and these data appear in Table 2. The results indicated that HET has demonstrated the potential risk of strong inhibition against UGT1A3, and NGR has shown the potential risk of inhibition towards UGT2B7.

Table 2. Calculation of IV–IVE of HET and NGR

	UGT	IC50 (µM)	[I]in,u/Ki	Ki (µM)	[I]in,u (µM)	Cmax (ng/mL)	Dosing	Reference
HET	UGT1A1	4.75±0.178	0.014	9.62±0.397	0.132	655±479	8 mL/kg orange juice	Iris Erlund, 200133)
	UGT1A3	9.23±0.156	0.134	0.995±0.634	0.132
	UGT1A9	3.55±0.377	0.039	3.41±1.01	0.132
NGR	UGT1A1	8.58±0.286	0.024	7.61±0.293	0.179	1628±1459	8 mL/kg grapefruit juice	Iris Erlund, 200133)
	UGT1A3	9.87±0.156	0.002	87.35±0.274	0.179
	UGT2B7	3.47±0.347	0.129	1.392±0.137	0.179

DISCUSSION

The present study investigated whether dietary flavonoids from citrus may cause herb–drug interactions. Our results demonstrated that both HET and NGR displayed broad-spectrum and strong or medium inhibition against human UGT isoforms except UGT1A6 and 2B17. Notably, 100 µM of HET exhibited more than 90% inhibition towards UGT1A1, 1A3, and 1A9. In contrast with HET, NGR had a strong inhibitory effect against UGT1A1, 1A3, and 2B7, and the residual activities were 4.395, 6.13, 6.04% at 100 µM, respectively. Both of HET and NGR are showing moderate inhibitory effects against UGT1A7, UGT1A8. To understand the potential of HDI mediated by UGT enzymes, it is important to know the tissue distribution of these enzymes and the drug/metabolite concentrations in specific tissues. Moreover, UGT1A1, UGT1A3, UGT1A9, UGT2B7 are expressed in human liver. In contrast, UGT1A8 are expressed in the gastrointestinal tract, whereas UGT1A7 is present only in the esophagus, stomach, and lung. The HDI via inhibition of these UGTs may not only take place in human liver but also in the small intestine. UGTs in the gastrointestinal tract may contribute significantly to the first-pass metabolism of orally administered drugs that undergo glucuronidation. Our results showed that HET and NGR might affect the glucuronidation and first-pass metabolism of orally administered drugs.

Through the IV–IVE of HET and NGR, highly possible HDI between HET/NGR and drugs mainly undergoing UGT1A3/UGT2B7-catalysed metabolism might occur. It is well known that UGT1A3 and UGT2B7 are two major metabolizing enzymes responsible for the metabolic elimination of mitiglinide, a new potassium channel antagonist for the treatment of type 2 diabetes mellitus.²⁵⁾ The likelihood of the interactions between HET or NGR containing herbals and mitiglinide is very high when they are administered concomitantly. UGT1A3 efficiently catalyzes the glucuronidation of endogenous substances, such as estrogen and bile acids, and inhibition of UGT1A3 activity may disrupt the metabolism of these compounds.²⁶⁾ UGT2B7 can also conjugate endogenous substances, including bile acids, androgens, and estrogens.²⁷⁾ Many compounds have been reported to inhibit UGT2B7, such as arbidol and herbal andrographolide derivatives.^28,29)

In vitro data tend to underestimate inhibition of drug glucuronidation in vivo.²¹⁾ Thus, HET and NGR might be more potent than data here suggest and individuals with greater systemic concentrations of HET and NGR may have increased the risk for herb-drug interactions, so IV–IVE should be done with care.³⁰⁾ Also the inhibition behavior may be probe substrate-dependent and selection of different substrates changes the data regarding inhibition type and amount.³¹⁾ Moreover, complex herb constituents, processing methods, and environmental factors (soil, altitude, seasonal variation in temperature, length of daylight, rainfall pattern, shade etc.) may influence in vivo predictions.³²⁾ Finally, individual differences in pharmacokinetics may change in vivo doses of HET or NGR and this may alter susceptibility to adverse effects with HET or NGR co-administration.

In summary, our finding shows that HET and NGR is a potent and broad-spectrum inhibitor against human UGT activity. Our prediction results for in vivo HDI demonstrate that the interaction is much likely to occur between high the dose of HET/NGR and the drugs which are substrates for the UGT1A3/UGT2B7. These data should be useful for guiding applications of orange juice or grapefruit juice, HET or NGR and related pharmaceutical preparations.

Acknowledgments

This work was supported by National Nature Science Foundation of China (No. 81160411, 81560606), Jiangxi Provincial Nature Science Fund (No. 20151BAB205084) and Jiangxi Province Young Scientist Supporting Project (No. 20133BCB23006).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Krogholm KS, Bredsdorff L, Knuthsen P, Haraldsdóttir J, Rasmussen SE. Relative bioavailability of the flavonoids quercetin, hesperetin and naringenin given simultaneously through diet. Eur. J. Clin. Nutr., 64, 432–435 (2010).
• 2) Yan Chen. Wang Jin yan, Jia Xiao bin, Xin Ran, Tan Xiao bin. Advances in studies on absorption, metabolism, and drug interaction of dihydrof lavones in Aurantii Fructus Immaturus. Chin. Tradit. Herbal Drugs, 41, 1564–1566 (2010).
• 3) Bredsdorff L, Nielsen ILF, Rasmussen SE, Cornett C, Barron D, Bouisset F, Offord E, Williamson G. Absorption, conjugation and excretion of the flavanones, naringenin and hesperetin from α-rhamnosidase-treated orange juice in human subjects. Br. J. Nutr., 103, 1602–1609 (2010).
• 4) Pingili R, Vemulapalli S, Mullapudi SS, Nuthakki S, Pendyala S, Kilaru N. Pharmacokinetic interaction study between flavanones (hesperetin, naringenin) and rasagiline mesylate in wistar rats. Drug Dev. Ind. Pharm., 2015, 1–33 (2015).
• 5) Choi EJ, Ahn WS. Neuroprotective effects of chronic hesperetin administration in mice. Arch. Pharm. Res., 31, 1457–1462 (2008).
• 6) Olsen HT, Stafford GI, van Staden J, Christensen SB, Jäger AK. Isolation of the MAO inhibitor naringenin from Mentha aquatica L. J. Ethnopharmacol., 117, 500–502 (2008).
• 7) Ribeiro IA, Rocha J, Sepodes B, Mota-Filipe H, Ribeiro MH. Effect of naringin enzymatic hydrolysis towards naringenin on the anti-inflammatory activity of both compounds. J. Mol. Catal., B Enzym., 52-53, 13–18 (2008).
• 8) Bacanli M, Basaran AA, Basaran N. The antioxidant and antigenotoxic properties of citrus phenolics limonene and naringin. Food Chem. Toxicol., 81, 160–170 (2015).
• 9) Ma HY, Sun DX, Cao YF, Ai CZ, Qu YQ, Hu CM, Jiang C, Dong PP, Sun XY, Hong M, Tanaka N, Gonzalez FJ, Ma XC, Fang ZZ. Herb–drug interaction prediction based on the high specific inhibition of andrographolide derivatives towards UDP-glucuronosyltransferase (UGT) 2B7. Toxicol. Appl. Pharmacol., 277, 86–94 (2014).
• 10) Williams JA, Hyland R, Jones BC, Smith DA, Hurst S, Goosen TC, Peterkin V, Koup JR, Ball SE. Drug–drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUC_i/AUC) ratios. Drug Metab. Dispos., 32, 1201–1208 (2004).
• 11) Kiang TKL, Ensom MHH, Chang TKH. UDP-glucuronosyltransferases and clinical drug–drug interactions. Pharmacol. Ther., 106, 97–132 (2005).
• 12) Evans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science, 286, 487–491 (1999).
• 13) Williams JA, Ring BJ, Cantrell VE, Campanale K, Jones DR, Hall SD, Wrighton SA. Differential modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to human liver microsomes. Drug Metab. Dispos., 30, 1266–1273 (2002).
• 14) Liu X, Huang T, Chen JX, Zeng J, Fan XR, Xu-Zhu, Yu ZW, Sun XY, Hong M, Sun HZ. Arbidol exhibits strong inhibition towards UDP-glucuronosyltransferase (UGT) 1A9 and 2B7. Pharmazie, 68, 945–950 (2013).
• 15) Peer CJ, Sissung TM, Kim A, Jain L, Woo S, Gardner ER, Kirkland CT, Troutman SM, English BC, Richardson ED, Federspiel J, Venzon D, Dahut W, Kohn E, Kummar S, Yarchoan R, Giaccone G, Widemann B, Figg WD. Sorafenib is an inhibitor of UGT1A1 but is metabolized by UGT1A9: implications of genetic variants on pharmacokinetics and hyperbilirubinemia. Clin. Cancer Res., 18, 2099–2107 (2012).
• 16) Fang ZZ, Cao YF, Hu CM, Hong M, Sun XY, Ge GB, Liu Y, Zhang YY, Yang L, Sun HZ. Structure–inhibition relationship of ginsenosides towards UDP-glucuronosyltransferases (UGTs). Toxicol. Appl. Pharmacol., 267, 149–154 (2013).
• 17) Quintieri L, Bortolozzo S, Stragliotto S, Moro S, Pavanetto M, Nassi A, Palatini P, Floreani M. Flavonoids diosmetin and hesperetin are potent inhibitors of cytochrome P450 2C9-mediated drug metabolism in vitro. Drug Metab. Pharmacokinet., 25, 466–476 (2010).
• 18) Lu WJ, Ferlito V, Xu C, Flockhart DA, Caccamese S. Enantiomers of naringenin as pleiotropic, stereoselective inhibitors of cytochrome P450 isoforms. Chirality, 23, 891–896 (2011).
• 19) Zhu L, Ge G, Liu Y, He G, Liang S, Fang Z, Dong P, Cao Y, Yang L. Potent and selective inhibition of magnolol on catalytic activities of UGT1A7 and 1A9. Xenobiotica, 42, 1001–1008 (2012).
• 20) Uchaipichat V, Mackenzie PI, Guo XH, Gardner-Stephen D, Galetin A, Houston JB, Miners JO. Human UDP-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid. Drug Metab. Dispos., 32, 413–423 (2004).
• 21) Uchaipichat V, Mackenzie PI, Elliot DJ, Miners JO. Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) “probes” for human UDPglucuronosyltransferases. Drug Metab. Dispos., 34, 449–456 (2006).
• 22) Zhou J, Tracy TS, Remmel RP. Glucuronidation of dihydrotestosterone and trans-androsterone by recombinant UDP-glucuronosyltransferase (UGT) 1A4: evidence for multiple UGT1A4 aglycone binding sites. Drug Metab. Dispos., 38, 431–440 (2010).
• 23) Fang ZZ, Wang H, Cao YF, Sun DX, Wang LX, Hong M, Huang T, Chen JX, Zeng J. Enantioselective Inhibition of Carprofen towards UDP-glucuronosyltransferase (UGT) 2B7. Chirality, 27, 189–193 (2014).
• 24) Ai LM, Zhu LL, Yang L, Ge GB, Cao Y, Liu Y, Fang Z, Zhang Y. Selectivity for inhibition of nilotinib on the catalytic activity of human UDP-glucuronosyltransferases. Xenobiotica, 44, 320–325 (2014).
• 25) Yu L, Lu S, Lin Y, Zeng S. Carboxyl-glucuronidation of mitiglinide by human UDP-glucuronosyltransferases. Biochem. Pharmacol., 73, 1842–1851 (2007).
• 26) Lankisch TO, Gillman TC, Erichsen TJ, Ehmer U, Kalthoff S, Freiberg N, Munzel PA, Manns MP, Strassburg CP. Aryl hydrocarbon receptor-mediated regulation of the human estrogen and bile acid UDP-glucuronosyltransferase 1A3 gene. Arch. Toxicol., 82, 573–582 (2008).
• 27) Gall WE, Zawada G, Mojarrabi B, Tephly TR, Green MD, Coffman BL, Mackenzie PI, Radominska-Pandya A. Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7. J. Steroid Biochem. Mol. Biol., 70, 101–108 (1999).
• 28) Liu X, Huang T, Chen JX, Zeng J, Fan XR, Xu-Zhu, Yu ZW, Sun XY, Hong M, Sun HZ. Arbidol exhibits strong inhibition towards UDP-glucuronosyltransferase (UGT) 1A9 and 2B7. Pharmazie, 68, 945–950 (2013).
• 29) Ma HY, Sun DX, Cao YF, Ai CZ, Qu YQ, Hu CM, Jiang C, Dong PP, Sun XY, Hong M, Tanaka N, Gonzalez FJ, Ma XC, Fang ZZ. Herb-drug interaction prediction based on the high specific inhibition of andrographolide derivatives towards UDP-glucuronosyltransferase (UGT) 2B7. Toxicol. Appl. Pharmacol., 277, 86–94 (2014).
• 30) Fang Z-Z, Cao Y-F, Hu C-M, Hong M, Sun XY, Ge GB, Liu Y, Zhang YY, Yang L, Sun HZ. Structure–inhibition relationship of ginsenosides towards UDP-glucuronosyltransferases (UGTs). Toxicol. Appl. Pharmacol., 267, 149–154 (2013).
• 31) Dong RH, Fang ZZ, Zhu LL, Liang SC, Ge GB, Yang L, Liu ZY. Investigation of UDP-glucuronosyltransferases (UGT) inhibitory properties of carvacrol. Phytother. Res., 26, 86–90 (2012).
• 32) Fang ZZ, Zhang YY, Wang XL, Cao YF, Huo H, Yang L. Bioactivation of herbal constituents: simple alerts in the complex system. Expert Opin. Drug Metab. Toxicol., 7, 989–1007 (2011).
• 33) Erlund I, Meririnne E, Alfthan G, Aro A. Plasma kinetics and urinary excretion of the flavanones naringenin and hesperetin in humans after ingestion of orange juice and grapefruit juice. J. Nutr., 131, 235–241 (2001).