Regioselective Glucuronidation of Flavones at C5, C7, and C4′ Positions in Human Liver and Intestinal Microsomes: Comparison among Apigenin, Acacetin, and Genkwanin

Abstract

Flavones, which are distributed in a variety of plants and foods in nature, possess significant biological activities, including antitumor and anti-inflammatory effects, and are metabolized into glucuronides by uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes in humans. In this study, apigenin, acacetin, and genkwanin, flavones having hydroxyl groups at C5, C7, and/or C4′positions were focused on, and the regioselective glucuronidation in human liver and intestinal microsomes was examined. Two glucuronides (namely, AP-7G and AP-4′G for apigenin, AC-5G and AC-7G for acacetin, and GE-5G and GE-4′G for genkwanin) were formed from each flavone by liver and intestinal microsomes, except for only GE-4′G formation from genkwanin by intestinal microsomes. The order of total glucuronidation activities was liver microsomes > intestinal microsomes for apigenin and acacetin, and liver microsomes < intestinal microsomes for genkwanin. The order of CL_int values (x-intercept) based on v versus V/[S] plots for apigenin glucuronidation was AP-7G > AP-4′G in liver microsomes and AP-7G < AP-4′G in intestinal microsomes. The order of CL_int values was AC-5G < AC-7G for acacetin and GE-5G < GE-4′G genkwanin glucuronidation in both liver and intestinal microsomes. This suggests that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical structure of flavones.

INTRODUCTION

Flavonoids are a class of plant secondary metabolites having a polyphenolic structure, and are widely found in the human diet such as vegetables, fruits, seeds, and medicinal herbs.^1–3) Flavones are one of the main classes of flavonoids, and the typical variations in chemical structure are include hydroxy- and/or methoxy-substitutions, mainly in the A- and B-rings of the 2-phenyl-1,4-benzopyrone skeleton.¹⁾ For example, apigenin has three hydroxyl groups at C5, C7, and C4′ positions, whereas the 4′-position in acacetin and 7-position in genkwanin are substituted with a methoxy group from the respective positions of apigenin (Fig. 1).

Fig. 1. Chemical Structures of Apigenin, Acacetin, and Genkwanin

Flavones exhibit several biological properties, including antimicrobial, antitumor, anti-allergic, antioxidant, and anti-inflammatory activities, in in vitro and in vivo systems.^3–7) Regardless of these promising bioactivities, flavonoids possess poor oral bioavailability (generally less than 5%) because they are easily metabolized to their conjugates (glucuronides and sulfates) in the intestines and liver.^6,8–11) In the studies on the main Scutellariae radix flavones, such as baicalein, wogonin, and oroxylin A, following oral administration of the extracts to rats, their glucuronides were identified in the plasma and urine, and underwent enterohepatic recycling due to β-glucuronidase and the subsequent intestinal reabsorption of flavones.^10,12–15)

In addition to in vivo studies, a few in vitro studies on rats and humans reported that apigenin and its O-methylated derivatives, acacetin and genkwanin, in addition to baicalein, wogonin, and oroxylin A, are predominantly conjugated to their glucuronides by uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes in the liver and intestines in humans.^16–19) Furthermore, we recently found that the abilities of hepatic and intestinal glucuronidation toward wogonin differ extensively between humans and experimental animals, such as monkeys, dogs, rats, and mice, in vitro using microsomal fractions.²⁰⁾ Thus, the bioactivities of flavones are considered to be closely associated with glucuronidation in the body.

Although several studies have flavone glucuronidation activities in liver and intestinal microsomes of humans,^16–20) there has been little focus on how base structure of flavones affects the regioselectivity. To predict and insight of regioselective and tissue-dependent glucuronidation of flavones represent an important aspect of pharmaceutical, pharmacological and nutritional researches for flavonoids contained in vegetables, fruits, and medicinal herbs. In this study, we focused on apigenin, acacetin, and genkwanin, flavones having hydroxyl groups at C5, C7, and/or C4′ positions, and the regioselective glucuronidation in human liver and intestinal microsomes was investigated.

MATERIALS AND METHODS

Materials

Apigenin (purity, > 99%), acacetin (purity, > 99%), genkwanin (purity, > 99%), luteolin (purity, > 99%), and diosmetin (purity, > 99%) were obtained from MedChemExpress (Monmouth Junction, NJ, U.S.A.). Apigenin 5-, 7-, and 4′-glucuronides (purity, > 98%) were purchased from Toronto Research Chemicals (Toronto, ON, Canada). Alamethicin was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). UDP-glucuronic acid and BCIP-nitro blue tetrazolium (NBT) Solution Kit were obtained from Nacalai Tesque (Kyoto, Japan). Pooled microsomes of human livers (race, Caucasian and Hispanic; age, 20–78 years old) and human intestines (race, Caucasian and Hispanic; age, 18–55 years old) were obtained from Sekisui XenoTech (Lenexa, KS, U.S.A.). Recombinant human UGT1A1 expressed in baculovirus- infected insect cells was purchased from Corning (Corning, NY, U.S.A.). Rabbit anti-human UGT1A peptide polyclonal antibody recognizing all UGT1A isoforms was prepared previously.²¹⁾ All other chemicals were purchased from Nacalai Tesque or Fujifilm Wako Pure Chemical Corporation (Osaka, Japan).

Assay for Glucuronidation Activities of Apigenin, Acacetin, and Genkwanin

Glucuronidation activities of apigenin, acacetin, and genkwanin in the human liver and intestinal microsomes were measured using the glucuronides formed from each flavone by HPLC. The standard incubation mixture contained substrate (apigenin, acacetin, and genkwanin), liver or intestinal microsomes, alamethicin (25 µg/mL), 10 mM MgCl₂, and 2.0 mM UDP-glucuronic acid in a final volume of 200 µL of 50 mM Tris–HCl buffer (pH 7.4). Substrate concentrations were 0.1–100 µM for apigenin and acacetin, and 0.05–50 µM for genkwanin. Substrates were dissolved in methanol/dimethyl sulfoxide (50 : 50, v/v) and stored at −20 °C with protection from light. The final concentration of the organic solvent (methanol and dimethyl sulfoxide) in the incubation mixture was 1% (v/v). The microsomal protein concentrations in the liver and intestines were 6.0 and 20 µg protein/mL for apigenin glucuronidation, 5.0 and 20 µg protein/mL for acacetin, and 50 and 5.0 µg protein/mL for genkwanin glucuronidation, respectively.

After preincubation for 2 min at 37 °C, the reactions were initiated by the addition of UDP-glucuronic acid. Incubation was performed for 10 min at 37 °C. The reactions were terminated by the addition of 200 µL of methanol containing 1% phosphoric acid, spiked with 300 pmol of luteolin for apigenin glucuronidation, and 300 and 150 pmol of diosmetin for acacetin and genkwanin glucuronidation as the internal standards, respectively. Samples were centrifuged at 12000 × g for 20 min at 4 °C, and a 10-µL aliquot was subjected to HPLC after filtration through a polytetrafluoroethylene membrane filter (0.45 µm).

An Inertsil ODS-SP column (5 µm, 3.0 mm i.d. × 150 mm; GL Sciences, Tokyo, Japan) was used in the determination of glucuronides of apigenin, acacetin, and genkwanin. The column temperature was maintained at 40 °C. Glucuronides, the substrate, and internal standard were isocratically eluted with the mobile phase composed of 0.1% phosphoric acid, acetonitrile, and methanol at a flow rate of 0.5 mL/min. The ratio of 0.1% phosphoric acid–acetonitrile–methanol (v/v/v) was 69 : 10 : 21 for apigenin glucuronidation, 67 : 25 : 8 acacetin glucuronidation, and 67 : 26 : 7 for genkwanin glucuronidation. UV detection was performed at 338 nm for apigenin and genkwanin glucuronidation, and at 333 nm for acacetin glucuronidation. The calibration curves for glucuronides formed from each substrate were preliminarily constructed by the decomposition with β-glucuronidase and a decrease in the substrate peak on HPLC. The rates of glucuronides formed from each flavone were estimated based on the peak areas.

Immunoblot Analysis of UGT1A Enzymes

Human liver and intestinal microsomes (each 20 µg protein/lane) and recombinant human UGT1A1 (20 µg protein/lane) as a positive control were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred electrophoretically to nitrocellulose membrane. The membrane was incubated with anti-human UGT1A antibody (1 : 4000 dilution) as the primary antibody and then with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgG) (1 : 4000 dilution) as the secondary antibody. Immunoreactive proteins were stained using BCIP-NBT Solution Kit, and band densities were relatively determined with ImageJ v1.53k (National Institute of Health Sciences, Bethesda, MD, U.S.A.).

Data Analysis

Kinetic parameter values (K_m, V_max, and K_si) for glucuronidation of apigenin, acacetin, and genkwanin in the human liver and intestinal microsomes were calculated by constructing v versus V/[S] plots using SigmaPlot v14.5 software (Systat Software, San Jose, CA, U.S.A.). The kinetic profile was evaluated from the respective coefficient of determination and/or Akaike’s information criterion values for the Michaelis–Menten, isoenzyme, substrate inhibition, and Hill equations. In vitro clearance values are presented as CL_int (V_max/K_m). All values are expressed as the mean ± standard deviation (S.D.) of three separate experiments.

RESULTS

General Properties of Apigenin, Acacetin, and Genkwanin Glucuronidation in Human Liver and Intestinal Microsomes

Glucuronidation activities of apigenin, acacetin, and genkwanin in human liver and intestinal microsomes were initially examined at a substrate concentration of 10 µM for apigenin and acacetin, and 5.0 µM for genkwanin. The typical HPLC chromatograms of apigenin, acacetin, and genkwanin glucuronidation are shown in Figs. 2–4. Two glucuronides (namely, AP-7G and AP-4′G for apigenin, AC-5G and AC-7G for acacetin, and GE-5G and GE-4′G for genkwanin) were formed from each flavone in the incubation with liver and intestinal microsomes, except for only GE-4′G formation from genkwanin by intestinal microsomes. The metabolites formed from apigenin at retention times of 12.8 and 13.7 min were identified to 7-glucuronide (AP-7G) and 4′-glucuronide (AP-4′G) by their authentic standards, respectively, whereas the peak of 5-glucuronide was not detected at a retention time of 10.0 min by liver and intestinal microsomes. Although the authentic standards of 5- and 7- or 4′-glucuronides of acacetin and genkwanin were not commercially available, the metabolites were identified or assumed from the chemical structures of flavones and HPLC chromatogram for apigenin to be as follows. The peaks at retention times of 7.8 and 9.5 min in acacetin glucuronidation were 5-glucuronide (AC-5G) and 7-glucuronide (AC-5G), and the peaks at 4.7 and 10.2 min in genkwanin glucuronidation were 5-glucuronide (GE-5G) and 4′-glucuronide (GE-4′G), respectively.

Fig. 2. HPLC Analysis of Apigenin Glucuronidation in Human Liver and Intestinal Microsomes

The substrate concentration used was 10 µM. The light line indicates the blank sample in the absence of an internal standard. Panels: A, liver microsomes; B, intestinal microsomes. IS, internal standard (luteolin).

Fig. 3. HPLC Analysis of Acacetin Glucuronidation in Human Liver and Intestinal Microsomes

The substrate concentration used was 10 µM. The light line indicates the blank sample in the absence of an internal standard. Panels: A, liver microsomes; B, intestinal microsomes. IS, internal standard (diosmetin).

Fig. 4. HPLC Analysis of Genkwanin Glucuronidation in Human Liver and Intestinal Microsomes

The substrate concentration used was 5.0 µM. The light line indicates the blank sample in the absence of an internal standard. Panels: A, liver microsomes; B, intestinal microsomes. IS, internal standard (diosmetin).

The glucuronidation activities of apigenin, acacetin and genkwanin in human liver and intestinal microsomes are summarized in Table 1. The formation activities of AP-7G and AP-4′G of apigenin in liver microsomes were 11.0 and 1.01 nmol/min/mg protein, respectively. The activities in intestinal microsomes were 12% and 3.2-fold those in liver microsomes for AP-7G and AP-4′G, respectively. The formation activities AC-5G and AC-7G of acacetin in liver microsomes were 0.97 and 14.5 nmol/min/mg protein, respectively. The levels of AC-5G and AC-7G in intestinal microsomes were 15 and 29% those in liver microsomes, respectively. In genkwanin, the formation activities of GE-5G and GE-4′G in liver microsomes were 0.57 and 1.49 nmol/min/mg protein, respectively. The activity of GE-4′G in intestinal microsomes was 2.6-fold higher than that in liver microsomes. In the total formation of two glucuronides, the activities of apigenin and acacetin glucuronidation in liver microsomes were 2.6- and 3.5-fold higher than those in intestinal microsomes, respectively, whereas the activity of genkwanin glucuronidation in intestinal microsomes was 1.9-fold higher than that in liver microsomes.

Table 1. Glucuronidation Activities of Apigenin, Acacetin, and Genkwanin in Human Liver and Intestinal Microsomes

	Apigenin (nmol/min/mg protein)		Acacetin (nmol/min/mg protein)		Genkwanin (nmol/min/mg protein)
	AP-7G	AP-4′G	AC-5G	AC-7G	GE-5G	GE-4′G
Liver microsomes	11.0 ± 1.2	1.01 ± 0.08	0.97 ± 0.08	14.5 ± 0.3	0.57 ± 0.02	1.49 ± 0.05
Intestinal microsomes	1.35 ± 0.06	3.19 ± 0.15	0.15 ± 0.01	4.23 ± 0.16	ND	3.90 ± 0.14

The substrate concentrations used were 10 µM for apigenin and acacetin, and 5.0 µM for genkwanin. Each value represents the mean ± S.D. of three separate experiments. ND, not detectable.

Kinetics of Apigenin, Acacetin, and Genkwanin Glucuronidation in Human Liver and Intestinal Microsomes

Kinetic analyses of apigenin, acacetin, and genkwanin glucuronidation in human liver and intestinal microsomes were performed at substrate concentrations of 0.1–100 µM for apigenin and acacetin, and 0.05–50 µM for genkwanin (19 points in each assay). The plots (v versus [S] and v versus V/[S]) and parameters of kinetics are shown in Figs. 5–7, and the calculated kinetic parameters are summarized in Tables 2–4.

Fig. 5. Kinetics of Apigenin Glucuronidation in Human Liver and Intestinal Microsomes

Substrate concentrations were 0.1–100 µM. Each point represents the mean ± S.D. of three separate experiments. Panels: A, v versus [S] plots for liver microsomes; B, v versus [S] plots for intestinal microsomes; C, v versus V/[S] plots for liver microsomes; D, v versus V/[S] plots for intestinal microsomes. Symbols: open circle, AP-7G; filled circle, AP-4′G.

Fig. 6. Kinetics of Acacetin Glucuronidation in Human Liver and Intestinal Microsomes

Substrate concentrations were 0.1–100 µM. Each point represents the mean ± S.D. of three separate experiments. Panels: A, v versus [S] plots for liver microsomes; B, v versus [S] plots for intestinal microsomes; C, v versus V/[S] plots for liver microsomes; D, v versus V/[S] plots for intestinal microsomes. Symbols: open circle, AC-5G; filled circle, AC-7G.

Fig. 7. Kinetics of Genkwanin Glucuronidation in Human Liver and Intestinal Microsomes

Substrate concentrations were 0.05–50 µM. Each point represents the mean ± S.D. of three separate experiments. Panels: A, v versus [S] plots for liver microsomes; B, v versus [S] plots for intestinal microsomes; C, v versus V/[S] plots for liver microsomes; D, v versus V/[S] plots for intestinal microsomes. Symbols: open circle, GE-5G; filled circle, GE-4′G.

Table 2. Kinetic Parameters for Apigenin Glucuronidation in Human Liver Microsomes

	Km (µM)	Vmax (nmol/min/mg protein)	CLint (mL/min/mg protein)	Ksi (µM)	Kinetic model	R2
Liver microsomes
AP-7G					Biphasic	0.98 ± 0.01
High-affinity phase	0.60 ± 0.13	6.90 ± 0.57	11.7 ± 1.4
Low-affinity phase	15.1 ± 2.7	12.9 ± 0.9	0.88 ± 0.23
AP-4′G	0.17 ± 0.04	1.22 ± 0.11	7.61 ± 1.53		Atypical	0.70 ± 0.08
Intestinal microsomes
AP-7G	1.19 ± 0.01	1.55 ± 0.07	1.31 ± 0.06	501 ± 96	Substrate inhibition	0.99 ± 0.00
AP-4′G	0.83 ± 0.09	3.65 ± 0.21	4.43 ± 0.40	209 ± 18	Substrate inhibition	0.98 ± 0.00

Each value represents the mean ± S.D. of three separate experiments.

Table 3. Kinetic Parameters for Acacetin Glucuronidation in Human Liver Microsomes

	Km (µM)	Vmax (nmol/min/mg protein)	CLint (mL/min/mg protein)	Kinetic model	R2
Liver microsomes
AC-5G	5.81 ± 0.35	1.53 ± 0.02	0.26 ± 0.02	Michaelis–Menten	0.97 ± 0.01
AC-7G				Biphasic	0.99 ± 0.00
High-affinity phase	0.49 ± 0.03	6.55 ± 0.39	13.4 ± 0.8
Low-affinity phase	14.2 ± 0.8	21.6 ± 0.7	1.53 ± 0.11
Intestinal microsomes
AC-5G	1.37 ± 0.45	0.17 ± 0.01	0.13 ± 0.03	Atypical	0.87 ± 0.04
AC-7G	1.44 ± 0.01	4.81 ± 0.14	3.35 ± 0.07	Michaelis–Menten	1.00 ± 0.00

Each value represents the mean ± S.D. of three separate experiments.

Table 4. Kinetic Parameters for Genkwanin Glucuronidation in Human Liver Microsomes

	Km (µM)	Vmax (nmol/min/mg protein)	CLint (mL/min/mg protein)	Ksi (µM)	Kinetic model	R2
Liver microsomes
GE-5G	0.47 ± 0.02	0.55 ± 0.02	1.19 ± 0.04		Atypical	0.89 ± 0.02
GE-4′G	0.54 ± 0.03	1.35 ± 0.03	2.49 ± 0.12		Atypical	0.83 ± 0.02
Intestinal microsomes
GE-4′G	0.19 ± 0.01	4.33 ± 0.10	23.3 ± 1.0	166 ± 58	Substrate inhibition	0.96 ± 0.01

Each value represents the mean ± S.D. of three separate experiments.

The kinetics of AP-7G formation of apigenin in human liver microsomes followed the biphasic model. The K_m, V_max, and CL_int values were 0.60 µM, 6.90 nmol/min/mg protein, and 11.7 mL/min/mg protein in the high-affinity phase, respectively, and 15.1 µM, 12.9 nmol/min/mg protein, and 0.88 mL/min/mg protein in the low-affinity phase, respectively. The kinetics of AP-4′G formation exhibited an atypical profile, and the K_m, V_max, and CL_int values calculated by the Michaelis–Menten equation were 0.17 µM, 1.22 nmol/min/mg protein, and 7.61 mL/min/mg protein, respectively. The kinetics of AP-7G and AP-4′G formation in intestinal microsomes fit the substrate inhibition model with K_si values of 501 and 209 µM, respectively. The K_m, V_max, and CL_int values for AP-7G formation compared with those in liver microsomes were 2.0-fold, 22, and 11% in the high-affinity phase, and 7.9, 12%, and 1.5-fold in the low-affinity phase, respectively. The formation of K_m, V_max, and CL_int values for AP-4′G were 4.9- and 3.0-fold and 58% those by liver microsomes, respectively.

The kinetics of AC-5G and AC-7G formation of acacetin in human liver microsomes followed the Michaelis–Menten and biphasic models, respectively. The K_m, V_max, and CL_int values of AC-5G formation were 5.81 µM, 1.53 nmol/min/mg protein, and 0.26 mL/min/mg protein, respectively. The K_m, V_max, and CL_int values for AC-7G formation were 0.49 µM, 6.55 nmol/min/mg protein, and 13.4 mL/min/mg protein in the high-affinity phase, respectively, and 14.2 µM, 21.6 nmol/min/mg protein, and 1.53 mL/min/mg protein in the low-affinity phase, respectively. The kinetics of AC-5G formation in intestinal microsomes followed the Michaelis–Menten model, and the K_m, V_max, and CL_int values were 24, 11, and 50% those in liver microsomes, respectively. The kinetics of AP-4′G formation exhibited an atypical profile. The K_m, V_max, and CL_int values compared with those in liver microsomes were 2.9-fold, 73, and 25% in the high-affinity phase, and 10 and 22% and 2.2-fold in the low-affinity phase, respectively.

The kinetic profiles of GE-5G and GE-4′G formation of genkwanin in human liver microsomes were atypical. The K_m, V_max, and CL_int values calculated by the Michaelis–Menten equation were 0.47 µM, 0.55 nmol/min/mg protein, and 1.19 mL/min/mg protein for GE-5G formation, and 0.54 µM, 1.35 nmol/min/mg protein, and 2.49 mL/min/mg protein for GE-4′G formation, respectively. The kinetics of GE-4′G formation in intestinal microsomes fit the substrate inhibition model with K_si values of 166 µM, and the K_m, V_max and CL_int values were 35% and 3.2- and 9.4-fold those in liver microsomes, respectively.

Expression of UGT1A Enzymes in Human Liver and Intestinal Microsomes

The expression levels of UGT1A enzymes in human liver and intestinal microsomes were assessed by immunoblot analysis using anti-human UGT1A antibody (Fig. 8). Both microsomes yielded immunodetectable UGT1A proteins. The relative intensities of staining bands were liver microsomes (100) and intestinal microsomes (17).

Fig. 8. Immunoblot Analysis of UGT1A Enzymes in Human Liver and Intestinal Microsomes

Protein levels applied were 20 µg protein/lane for human liver and intestinal microsomes, and 1.0 µg protein/lane for recombinant human UGT1A1. Lanes: M, marker; 1A1, recombinant human UGT1A1; HLM, human liver microsomes; HIM, human intestinal microsomes.

DISCUSSION

Flavones, such as apigenin, acacetin, and genkwanin, possess beneficial bioactivities, including antimicrobial, anti-cancer, and anti-allergic effects.^3–7) These flavones are metabolized to glucuronide(s) at positions of C5, C7, and/or C4′ by UGT enzymes.^17–19) Glucuronidation is important for the detoxification and elimination of a large number of xenobiotics in mammals^22–25); therefore, the biotransformation is considered to reduce the bioactivity of flavones. However, information on the regioselective glucuronidation depending on tissues of flavones in humans limited. In this study, the hepatic and intestinal glucuronidation of apigenin, acacetin, and genkwanin in humans using an in vitro system with microsomal fractions was examined.

In order to obtain general information on the regioselective glucuronidation of apigenin, acacetin, and genkwanin, UGT activities toward each flavone in human liver and intestinal microsomes were initially assessed at a single substrate concentration. The formation activities of AP-7G and AC-7G exhibited a contrasting profile between liver microsomes (AP-7G > AC-7G) and intestinal microsomes (AP-7G < AC-7G). In acacetin glucuronidation, two metabolites of AC-5G and AC-7G were formed in both liver and intestinal microsomes, and the activity of AC-5G was higher than that of AC-7G. Regarding genkwanin glucuronidation, two metabolites of GE-5G and GE-4′G were formed in liver microsomes, whereas only GE-4′G was formed in intestinal microsomes. The formation activity of GE-4′G in intestinal microsomes was higher than that in liver microsomes. In terms of the total formation of two glucuronides, the order of activities was liver microsomes > intestinal microsomes for apigenin and acacetin, and liver microsomes < intestinal microsomes for genkwanin, suggesting that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical properties of flavones. Considering the low bioavailability of flavones, the glucuronidation ability in the intestines in vivo system may be higher than in in vitro system.

Kinetic analyses of apigenin, acacetin, and genkwanin glucuronidation in human liver and intestinal microsomes were subsequently performed at a broad range of substrate concentrations. The kinetics of the formation of AP-7G and AC-7G, which were identified or assumed to be 7-glucuronides, followed the biphasic model in liver microsomes and the Michaelis–Menten or substrate inhibition model in intestinal microsomes. Of note, the respective values of kinetic parameters, such as K_m, V_max and CL_int, in both liver and intestinal microsomes were generally comparable between apigenin and acacetin glucuronidation. For AP-4′G and GE-4′G, which were identified or assumed to be 4′-glucuronides, the formation kinetics fit the Michaelis–Menten for AP-4′G in liver microsomes, and the substrate inhibition model for AP-4′G in intestinal microsomes and GE-4′G in liver and intestinal microsomes. Although the V_max values of AP-4′G and GE-4′G had similar values in liver and intestinal microsomes, the orders of K_m values were AP-4′G > GE-4′G in liver microsomes and AP-4′G < GE-4′G in intestinal microsomes. Consequently, the orders of CL_int values for 4′-glucuridation were liver microsomes > intestinal microsomes for apigenin and liver microsomes < intestinal microsomes for genkwanin. In particular, the CL_int value for GE-4′G formation in intestinal microsomes was more than 10-fold that in liver microsomes. The kinetics of the formation of AC-5G in liver and intestinal microsomes and GE-5G in intestinal microsomes, which were assumed to be 5-glucuronides, followed the Michaelis–Menten model. The V_max and CL_int values for AC-5G in liver microsomes were markedly higher than those in intestinal microsomes, and the formation of GE-5G was not detectable in intestinal microsomes. Thus, the kinetic profiles for flavone glucuronidation differed extensively among apigenin, acacetin, and genkwanin, and it was suggested that the C5 and C7, and C4′ positions of flavones in humans are mainly glucuronidated by hepatic and intestinal UGT isoforms, respectively, and that the functions, such as the affinity, catalytic activity, and regioselectivity, toward substrates of each UGT isoform are closely associated with the metabolism and bioactivities of flavones.

Many UGT isoforms have been reported to be expressed in the hepatic and extrahepatic tissues of humans, and their enzymatic properties have been analyzed.^26–30) The role of UGT isoforms in the glucuronidation of apigenin, acacetin, and genkwanin in humans has been relatively or semi-quantitatively identified in studies using recombinant enzymes.^17–19) The major UGT isoforms catalyzing glucuronidation were UGT1A1 and UGT1A9 for apigenin (C7 position), UGT1A8, UGT1A9, and UGT1A10 for acacetin (total of C5 and C7 positions), and UGT1A1, UGT1A9, and UGT1A10 for genkwanin (C4′ position). UGT1A1 and UGT1A9 are abundantly expressed in the liver. These UGT isoforms are also expressed in the small intestine and colon,^26–29) the protein expression levels of UGT1A1 and UGT1A9 in intestinal microsomes were estimated to be approximately 40 and 25% those in liver microsomes, respectively.²⁷⁾ On the other hand, UGT1A8 and UGT1A10 are expressed in the extrahepatic tissues, such as small intestine and colon, and adrenal glands, but not in the liver.^26,28) We also performed immunoblot analysis to determine UGT1A protein levels in human liver and intestinal microsomes. The levels of liver microsomes were approximately 6-fold higher than that of intestinal microsomes. Therefore, the results of catalytic and immunoblot analyses in the present study support that at least UGT1A1 and UGT1A9 play important roles in the glucuronidation of flavones at C7 position, as suggested previously.^17,18)

Although it is impossible to clearly explain the relationship between regioselectivity and UGT isoforms in the glucuronidation of flavones at present, this study revealed that multiple isoforms of the UGT1A subfamily function in the glucuronidation of apigenin, acacetin, and genkwanin in humans, and the predominant UGT isoforms and their contribution ratios depend on the chemical structures based on the positions of hydroxyl groups of flavones. Further studies are needed to clarify the roles of hepatic and intestinal UGT isoforms in the metabolism and bioactive mechanisms of flavones using recombinant enzymes.

CONCLUSION

The hepatic and intestinal glucuronidation of apigenin, acacetin, and genkwanin in human liver and intestinal microsomes was examined. Two glucuronides were formed from each flavone in liver and intestinal microsomes, except for only GE-4′G formation from genkwanin in intestinal microsomes. The orders of CL_int values for apigenin glucuronidation were AP-7G > AP-4′G in liver microsomes and AP-7G < AP-4′G in intestinal microsomes. The orders of CL_int values were AC-5G < AC-7G for acacetin and GE-5G < GE-4′G genkwanin glucuronidation in both liver and intestinal microsomes. This suggests that the abilities and roles of UGT enzymes in the glucuronidation of apigenin, acacetin, and genkwanin in humans differ depending on the chemical properties of flavones, and that the functions, such as the affinity, catalytic activity, and regioselectivity toward substrates of each UGT isoform, are closely associated with the metabolism and bioactivities of flavones.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (19H04295) from the Japan Society for the Promotion of Science.

Conflict of Interest

The authors declare no conflict of interest.

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