In Vitro Comparative Study of the Inhibitory Effects of Mangiferin and Its Aglycone Norathyriol towards UDP-Glucuronosyl Transferase (UGT) Isoforms

Mangiferin (MGF), the predominant constituent of extracts of the mango plant Mangifera Indica L., has been investigated extensively because of its remarkable pharmacological effects. In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was used to investigate the inhibition of mangiferin and aglycone norathyriol towards various isoforms of UGTs in our study, which evaluated the inhibitory capacity of MGF and its aglycone norathyriol (NTR) towards UDP-glucuronosyltransferase (UGT) isoforms. Initial screening experiment showed that deglycosylation of MGF into NTR strongly increased the inhibitory effects towards almost all the tested UGT isoforms at a concentration of 100 μM. Kinetic experiments were performed to further characterize the inhibition of UGT1A3, UGT1A7 and UGT1A9 by NTR. NTR competitively inhibited UGT1A3, UGT1A7 and UGT1A9, with an IC50 value of 8.2, 4.4, and 12.3 μM, and a Ki value of 1.6, 2.0, and 2.8 μM, respectively. In silico docking showed that only NTR could dock into the activity cavity of UGT1A3, UGT1A7 and UGT1A9. The binding free energy of NTR to UGT1A3, 1A7, 1A9 were −7.4, −7.9 and −4.0 kcal/mol, respectively. Based on the inhibition evaluation standard ([I]/Ki < 0.1, low possibility; 0.1 < [I]/Ki < 1, medium possibility; [I]/Ki > 1, high possibility), an in vivo herb–drug interaction between MGF/NTR and drugs mainly undergoing UGT1A3-, UGT1A7- or UGT1A9-catalyzed metabolism might occur when the plasma concentration of NTR is above 1.6, 2.0 and 2.8 μM, respectively.


Introduction
Mangiferin (1,3,6,7-tetrahydroxyxanthone-C-2-β-D-glucoside, MGF, Figure 1), the predominant constituent of extracts of the mango plant Mangifera Indica L., has been investigated extensively because Although many studies have been carried on the in vitro and in vivo metabolism profiles of MGF and NTR, only a few articles that deal with metabolism enzymes have elucidated the potential toxicity and herb-drug interactions. Goud et al. found that mangiferin showed modulation of certain P450 enzymes, the most important phase I metabolizing enzyme [25]. Nevertheless, compared with phase I metabolizing enzymes, no research had been carried out on the interaction of MGF and NTR with phase II conjugating enzymes.
UGTs (EC 2.4.1.17) are a family of conjugating enzymes that play important roles in the metabolism of endogenous and exogenous compounds [26]. UGT-catalyzed glucuronidation reactions are responsible for about 35% of all drugs metabolized by phase II enzymes [27]. A glucuronidation reaction catalyzed by UGT enzymes serves as an integral step in transforming lipophilic substrates into hydrophilic glucuronides, a process that increases their ability to partition into the aqueous intraand extra-cellular compartments of the body, facilitating the transport to excretory organs and subsequent elimination through the bile and urine [27,28]. Thus, glucuronidation serves as an essential clearance and detoxification mechanism for endogenous and exogenous compounds [29,30].
Herb-drug interactions caused by UGT enzymes have received increasing attention over the past few decades. For example, sorafenib-induced UGT1A1 inhibition might also be an important reason for the elevation of serum bilirubin [31]. Fluconazole itself is not metabolized by UGT but alter pharmacokinetic parameters of co-administrated zidovudine in AIDS patients, because the glucuronidation of zidovudine by UGT2B7 is strongly inhibited by fluconazole [32]. Cycloastragenol has been proven to specifically inhibit UGT1A8 and UGT2B7, thus affecting drugs undergoing UGT1A8-and UGT2B7-catalyzed metabolism [33].
In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was used to investigate the inhibition of mangiferin and aglycone norathyriol towards various isoforms of UGTs in our study. The aim of this research was to investigate the inhibitory effects of mangiferin and aglycone norathyriol towards UGT isoforms, thus indicating potential UGT inhibition-related Although many studies have been carried on the in vitro and in vivo metabolism profiles of MGF and NTR, only a few articles that deal with metabolism enzymes have elucidated the potential toxicity and herb-drug interactions. Goud et al. found that mangiferin showed modulation of certain P450 enzymes, the most important phase I metabolizing enzyme [25]. Nevertheless, compared with phase I metabolizing enzymes, no research had been carried out on the interaction of MGF and NTR with phase II conjugating enzymes.
UGTs (EC 2.4.1.17) are a family of conjugating enzymes that play important roles in the metabolism of endogenous and exogenous compounds [26]. UGT-catalyzed glucuronidation reactions are responsible for about 35% of all drugs metabolized by phase II enzymes [27]. A glucuronidation reaction catalyzed by UGT enzymes serves as an integral step in transforming lipophilic substrates into hydrophilic glucuronides, a process that increases their ability to partition into the aqueous intra-and extra-cellular compartments of the body, facilitating the transport to excretory organs and subsequent elimination through the bile and urine [27,28]. Thus, glucuronidation serves as an essential clearance and detoxification mechanism for endogenous and exogenous compounds [29,30].
Herb-drug interactions caused by UGT enzymes have received increasing attention over the past few decades. For example, sorafenib-induced UGT1A1 inhibition might also be an important reason for the elevation of serum bilirubin [31]. Fluconazole itself is not metabolized by UGT but alter pharmacokinetic parameters of co-administrated zidovudine in AIDS patients, because the glucuronidation of zidovudine by UGT2B7 is strongly inhibited by fluconazole [32]. Cycloastragenol has been proven to specifically inhibit UGT1A8 and UGT2B7, thus affecting drugs undergoing UGT1A8and UGT2B7-catalyzed metabolism [33].
In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was used to investigate the inhibition of mangiferin and aglycone norathyriol towards various isoforms of UGTs in our study. The aim of this research was to investigate the inhibitory effects of mangiferin and aglycone norathyriol towards UGT isoforms, thus indicating potential UGT inhibition-related herb-drug interaction. Furthermore, molecular docking was used to elucidate the interactions between ligand and enzyme.

Inhibition Type and Kinetics of NTR towards UGT1A3, UGT1A7 and UGT1A9
As the inhibitory capacity by NTR towards UGT1A3, UGT1A7 and UGT1A9 was above 80%, kinetic experiments were performed to further characterize the inhibition of these three UGT isoforms by NTR. The dose-dependent inhibition of NTR towards UGT1A3, UGT1A7 and UGT1A9 was shown in Figures

Inhibition Type and Kinetics of NTR towards UGT1A3, UGT1A7 and UGT1A9
As the inhibitory capacity by NTR towards UGT1A3, UGT1A7 and UGT1A9 was above 80%, kinetic experiments were performed to further characterize the inhibition of these three UGT isoforms by NTR. The dose-dependent inhibition of NTR towards UGT1A3, UGT1A7 and UGT1A9 was shown in Figures  herb-drug interaction. Furthermore, molecular docking was used to elucidate the interactions between ligand and enzyme.

Inhibition Type and Kinetics of NTR towards UGT1A3, UGT1A7 and UGT1A9
As the inhibitory capacity by NTR towards UGT1A3, UGT1A7 and UGT1A9 was above 80%, kinetic experiments were performed to further characterize the inhibition of these three UGT isoforms by NTR. The dose-dependent inhibition of NTR towards UGT1A3, UGT1A7 and UGT1A9 was shown in Figures

Discussion
In our research, the inhibitory effects of MGF and NTR on UGT isoforms were studied and led to the conclusion that deglycosylation of MGF to NTR can strongly increase the inhibitory effects towards almost all tested UGT isoforms. Exogenous compounds have two ways of regulating enzymes, namely inhibition and activation. In our experiments, it was found that MGF and NTR activated UGT 1A10 and deglycosylation of MGF into NTR strongly increased the inhibitory effects towards others tested UGT isoforms except UGT1A10. The deglycosylation of saponin to aglycone always exhibited a different inhibition profile, such as astragaloside IV and cycloastragenol, glucoaurantio-obtusin and aurantio-obtusin, liquiritin and liquiritigenin, icariin and its intestinal metabolites (icariside I, icariside II and icaritin), arctiin and arctigenin, scutellarin and scutellarein, and in most situations, aglycone showed stronger inhibitory effects than saponin [33][34][35][36][37][38][39][40]. In our study, in silico docking method was used to explain why the inhibition potential of NTR was stronger than MGF on the activity of UGT1A3, UGT1A7 and UGT1A9. As shown in Figures 6-8, several amino acid residues of these three UGT isoforms made hydrogen bonds or hydrophobic interactions to NTR. The binding free energy of NTR to UGT1A3, 1A7, 1A9 were −7.4, −7.9 and −4.0 kcal/mol, respectively. However, MGF could not dock into the activity cavity between N-terminal and C-terminal of UGT1A3, UGT1A7 and UGT1A9. The 1-OH and 3-OH groups in NTR played important role in making interactions to these three UGT isoforms, while MGF was the 2-C -glucosides of NTR, which generated stereohindrance effect compared with NTR, thus hindering the interactions between MGF and protein.
As exhibited in Figure 2, 100 μM NTR strongly inhibited the activity of UGT1A3, UGT1A7 and UGT1A9 by 87.6%, 94.9% and 94.2%, respectively. UGT1A3 is found in the liver, kidney, and prostate, and throughout the gastrointestinal tract [41]. UGT1A3 metabolizes endobiotic substances such as bile acid (CDCA, LCA, HDCA) and xenobiotic substances such as polyaromatic hydrocarbons amines, non-steroidal anti-inflammatory drugs (NSAIDs) and statins [42][43][44][45][46]. UGT1A7 represents one of several UGTs that were shown to be active on chemical carcinogens. UGT1A7 is an important extrahepatic UGT, and only presents in the esophagus, stomach, and lung [47]. UGT1A9 is one of the major enzymes responsible for the conjugation of carboxylic acids. UGT1A9 shows a wide spectrum of substrate specificity including bulky phenols, dietary constituents, steroids, and fatty acids, as well as prescribed drugs including anticancer agents, fibrates, NSAIDs, and antiarrhythmic agents [48].
In practice, the [I]/Ki ratio is used to predict the likelihood inhibitory drug-drug interactions. When assessing in vivo interaction potential, [I] represents the mean steady-state Cmax value following the administration of the highest proposed clinical dose [49]. Extensive research into the pharmacokinetics of MGF in rat had been investigated. The maximum concentrations in the plasma were 8.13 ± 1.96 μg/mL (19.2 ± 4.64 μM) and 0.19 μg/mL (0.45 μM) after oral administration of MGF at a dose of 120 mg/kg

Discussion
In our research, the inhibitory effects of MGF and NTR on UGT isoforms were studied and led to the conclusion that deglycosylation of MGF to NTR can strongly increase the inhibitory effects towards almost all tested UGT isoforms. Exogenous compounds have two ways of regulating enzymes, namely inhibition and activation. In our experiments, it was found that MGF and NTR activated UGT 1A10 and deglycosylation of MGF into NTR strongly increased the inhibitory effects towards others tested UGT isoforms except UGT1A10. The deglycosylation of saponin to aglycone always exhibited a different inhibition profile, such as astragaloside IV and cycloastragenol, glucoaurantio-obtusin and aurantio-obtusin, liquiritin and liquiritigenin, icariin and its intestinal metabolites (icariside I, icariside II and icaritin), arctiin and arctigenin, scutellarin and scutellarein, and in most situations, aglycone showed stronger inhibitory effects than saponin [33][34][35][36][37][38][39][40]. In our study, in silico docking method was used to explain why the inhibition potential of NTR was stronger than MGF on the activity of UGT1A3, UGT1A7 and UGT1A9. As shown in Figures 6-8, several amino acid residues of these three UGT isoforms made hydrogen bonds or hydrophobic interactions to NTR. The binding free energy of NTR to UGT1A3, 1A7, 1A9 were −7.4, −7.9 and −4.0 kcal/mol, respectively. However, MGF could not dock into the activity cavity between N-terminal and C-terminal of UGT1A3, UGT1A7 and UGT1A9. The 1-OH and 3-OH groups in NTR played important role in making interactions to these three UGT isoforms, while MGF was the 2-C -glucosides of NTR, which generated stereo-hindrance effect compared with NTR, thus hindering the interactions between MGF and protein.
As exhibited in Figure 2, 100 µM NTR strongly inhibited the activity of UGT1A3, UGT1A7 and UGT1A9 by 87.6%, 94.9% and 94.2%, respectively. UGT1A3 is found in the liver, kidney, and prostate, and throughout the gastrointestinal tract [41]. UGT1A3 metabolizes endobiotic substances such as bile acid (CDCA, LCA, HDCA) and xenobiotic substances such as polyaromatic hydrocarbons amines, non-steroidal anti-inflammatory drugs (NSAIDs) and statins [42][43][44][45][46]. UGT1A7 represents one of several UGTs that were shown to be active on chemical carcinogens. UGT1A7 is an important extrahepatic UGT, and only presents in the esophagus, stomach, and lung [47]. UGT1A9 is one of the major enzymes responsible for the conjugation of carboxylic acids. UGT1A9 shows a wide spectrum of substrate specificity including bulky phenols, dietary constituents, steroids, and fatty acids, as well as prescribed drugs including anticancer agents, fibrates, NSAIDs, and antiarrhythmic agents [48].
In practice, the [I]/Ki ratio is used to predict the likelihood inhibitory drug-drug interactions. When assessing in vivo interaction potential, [I] represents the mean steady-state C max value following the administration of the highest proposed clinical dose [49]. Extensive research into the pharmacokinetics of MGF in rat had been investigated. The maximum concentrations in the plasma were 8.13 ± 1.96 µg/mL (19.2 ± 4.64 µM) and 0.19 µg/mL (0.45 µM) after oral administration of MGF at a dose of 120 mg/kg and 70mg/kg in rat, respectively [12,50]. To the best of our knowledge, no one research on the pharmacokinetic study for mangiferin in human has been done. The concentration of mangiferin in plasma reached 38.64 ± 6.75 ng/mL (0.091 ± 0.016 µM) about 1 h after oral administration of 0.9 g mangiferin in human [51]. As demonstrated in Figures 6-8, the inhibition constant (Ki) were calculated to be 1.6, 2.0, and 2.8 µM for the inhibition of NTR towards UGT1A3, UGT1A7 and UGT1A9, respectively. However, we cannot accurately predict in vivo inhibition magnitude only based on the above experimental results. Multiple factors should be taken into account when predict the likelihood inhibitory drug-drug interactions. First, the drug dose should be the highest proposed clinical dose. Secondly, the aglycone of MGF, rather than MGF, had the inhibition effect on UGTs, so the metabolism of MGF to NTR should also be considered. In fact, the NTR exposure after oral administration of MGF was extremely slow, and after administration of 200 mg/kg MGF, the concentration and exposure of NTR was quite low (C max < 3 ng/mL), which was lower than that of the parent drug (C max : 202.58 ng/mL) by roughly two orders of magnitude (AUCNTR/AUCMGF < 3%) [52]. However, given of its slow generation and elimination rates, possible accumulation of NTR in the long term of MGF treatment and exert effects should be considered. Thirdly, the species difference has prevented their results from being directly applied to humans. Although complicated, study on the inhibition potential of MGF/NTR applied us more information for the MGF/NTR-drug interactions in clinical application.
In conclusion, the specific inhibition of MGF and its aglycone NTR was demonstrated in this manuscript. The deglycosylation of MGF into NTR strongly increased the inhibitory effects towards others tested UGT isoforms except UGT1A10. In silico prediction model was successfully employed to explain the different inhibition effects of MGF and NTR on UGT1A3, UGT1A7 and UGT1A9. The inhibition kinetic type and behavior were determined for the inhibition of NTR towards UGT1A3, UGT1A7 and UGT1A9, and using IVIVE to clarify the possibility of herb-drug interaction between MGF/NTR and drugs. Based on these results, clinical close monitoring the utilization of norathyriol is very important and necessary.

Investigation of the Inhibition Potential of MGF and NTR on UGT Isoforms
4-MU, a nonspecific probe substrate for all the UGT isoforms, was employed to investigate the inhibition of MEF and NIR towards the activity of UGT isoforms. The incubation and analytical methods have been previously described [53][54][55]. A typical 200 µL incubation mixture, contained various recombinant UGT isoforms (0.125 mg/mL for UGT1A1, 0.05 mg/mL for UGT1A3, UGT1A7, UGT1A9, UGT1A10 and UGT2B7, 0.025 mg/mL for UGT1A6 and UGT1A8, 0.25 mg/mL for UGT2B4, 0.2 mg/mL for UGT2B15, 0.5 mg/mL for UGT2B17), 5 mM UDPGA, 5 mM MgCl2, 50 mM Tris-HCl buffer (pH = 7.4), and various concentrations of 4-MU (110 µM for UGT1A1 and UGT1A6, 1200 µM for UGT1A3, 30 µM for UGT1A7, UGT1A9 and UGT1A10, 750 µM for UGT1A8, 1 000 µM for UGT2B4, 350 µM for UGT2B7, 250 µM for UGT2B15 and 2 000 µM for UGT2B17) in different concentration of MGF or NTR. MEF and NIR were dissolved in the DMSO to make a stock solution of 20 mM, and various concentrations of working solutions were prepared through dilution with DMSO. 4-MU was used as a non-selective substrate of UGTs. There was a 5-min pre-incubation step at 37 • C before the reaction was initiated by the addition of UDPGA. 4-MU and inhibitors were previously dissolved in DMSO, and the total concentration of DMSO was 1%. The reactions were continued at 37 • C for 120 min for UGT1A1, UGT1A3, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, 30 min for UGT1A6, UGT1A7, UGT1A8, and UGT1A9, respectively. Reactions were terminated by the addition of 100 µL acetonitrile with 7-hydroxycoumarin (100 µM) as internal standard. The incubation mixtures were then centrifuged at 12,000 r/min for 10 min. 2 µL supernatant was injected into the UPLC system for analysis. An ACQUITY UPLC System (Waters, Milford, MA, USA) equipped with UV detector was used to analyze the samples, and the separation of all the compounds were carried out using a ACQUITY UPLC ® BEH C18 (2.1 × 100 mm, 1.7 µm Waters) at a flow rate of 0.2 mL/min and UV detector at 316 nm. The mobile phase was consisted of ultrapure water containing 0.5% formic acid (A) and acetonitrile (B). The following gradient condition was used: 0-3.5 min, 10-65% B; 3.5-4.0 min, 65% B; 4.0-9.0 min, 10% B. To calculate the standard curve 0.1-100 µM of 4-MUG was used through drawing the peak area ration of 4-MUG/internal standard towards the concentration range of 4-MUG. The curve was linear over this concentration range, with an r 2 value > 0.99.

In Silico Docking to Explain the Inhibition of NTR towards UGT1A3, UGT1A7 and UGT1A9
Comparative homology modeling with MODELLER9v14 program was carried out to elucidate the three-dimensional structure of UGT1A, UGT1A7 and UGT1A9, respectively [56,57]. The amino acid sequence of UGT1A3, UGT1A7 and UGT1A9 was downloaded from National Center for Biotechnology Information with the code NP_061966.1, NP_061950.2 and NP_066307.1, respectively. The crystal structure of human glucosyltransferase UGT78G1 (PDB code: 3hbf) has 33% identity in amino acid sequence with the C-terminal of UGT1A3, UGT1A7 and UGT1A9. Twenty comparative models of target sequence were built by MODELLER program, and the best model was selected based on the Modeller objective function and Discrete Optimization Protein Energy score. Auto-dock version 4.2 program was employed to perform the interaction between NTR and UGT1A3, UGT1A7 and UGT1A9, respectively [58,59]. Polar hydrogen atoms were added to UGT1A3, UGT1A7 and UGT1A9, and nonpolar hydrogenatoms were merged. AutoDock tool was used to add the Kollman charges to UGT1A3, UGT1A7 and UGT1A9. The grid box was generated with a dimension of 40 × 40 × 40. The grid spacing was set to 0.375 Å. The Lamarckian geneticalgorithm was utilized to deal with the protein-fixed ligand-flexible docking calculations. After docking calculation, the ligands were ranked according to the docked energy, and the best conformation with the lowest docked energy was selected to analyze the interactions between inhibitor and protein.

Inhibition Kinetic Analysis and In Vitro-In Vivo Extrapolation (IVIVE)
The inhibition type and Kinetics were determined for the inhibition of NTR towards UGT1A3, UGT1A7 and UGT1A9. The glucuronidation velocity of 4-MU was determined at multiple concentrations of 4-MU and NTR. Dixon and Lineweaver-Burk plots were employed to determine the inhibition type, and the second plot of the slopes from the Lineweaver-Burk plot versus the compound concentrations was utilized to calculate the Ki value. The determination of IC 50 was calculated using Probit analysis in SPSS11.5 (SPSS, Chicago, IL, USA). The Student's t-test was adopted at a significance level of p < 0.05 to determine statistically significant differences among experimental groups. In vitro-in vivo extrapolation (IVIVE) was performed using the following equation: AUCi/AUC = 1 + [I] in vivo /Ki The terms are defined as follows: AUCi/AUC is the predicted ratio of in vivo exposure of xenobiotics or endogenous substances with or without the co-exposure of CAG.
[I] in vivo is the in vivo exposure concentration of CAG, and the Ki value was in vitro inhibition constant. The evaluation standard was as follows: [I]/Ki < 0.1, low possibility; 0.1 < [I]/Ki < 1, medium possibility; [I]/Ki > 1, high possibility.