Formation of (2 R)- And (2 S)-8-Prenylnaringenin Glucuronides by Human UDP-Glucuronosyltransferases

Article abstract of DOI:10.1021/acs.jafc.9b04657

Occurring in hops (Humulus lupulus) and beer as a racemic mixture, (2R,2S)-8-prenylnaringenin (8-PN) is a potent phytoestrogen in hop dietary supplements used by women as alternatives to conventional hormone therapy. With a half-life exceeding 20 h, 8-PN is excreted primarily as 8-PN-7-O-glucuronide or 8-PN-4′-O-glucuronide. Human liver microsomes and 11 recombinant human UDP-glucuronosyltransferases (UGTs) were used to catalyze the formation of the two oxygen-linked glucuronides of purified (2R)-8-PN and (2S)-8-PN, which were subsequently identified using mass spectrometry and nuclear magnetic resonance spectroscopy. Formation of (2R)- and (2S)-8-PN-7-O-glucuronides predominated over the 8-PN-4′-O-glucuronides except for intestinal UGT1A10, which formed more (2S)-8-PN-4′-O-glucuronide. (2R)-8-PN was a better substrate for all 11 UGTs except for UGT1A1, which formed more of both (2S)-8-PN glucuronides than (2R)-8-PN glucuronides. Although several UGTs conjugated both enantiomers of 8-PN, some conjugated just one enantiomer, suggesting that human phenotypic variation might affect the routes of metabolism of this chiral estrogenic constituent of hops.

Full text of DOI:10.1021/acs.jafc.9b04657

Article
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/JAFC
Formation of (2R)- and (2S)‑8-Prenylnaringenin Glucuronides by
Human UDP-Glucuronosyltransferases
†
,‡
‡
David C. Lankin, Charlotte Simmler, Shao-Nong Chen,‡
‡
‡
Jin-Bo Fang, Dejan Nikolic,
́
‡
‡
‡
,‡,§
Rene F. Ramos Alvarenga, Yang Liu, Guido F. Pauli, and Richard B. van Breemen*
†School of Pharmacy, Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430030, China
‡
UIC/NIH Center for Botanical Dietary Supplements Research, PCRPS and Department of Pharmaceutical Sciences, University of
Illinois College of Pharmacy, 833 S. Wood Street, Chicago, Illinois 60612, United States
§
Linus Pauling Institute, Oregon State University, 305 Linus Pauling Science Center, Corvallis, Oregon 97331, United States
*
S Supporting Information
ABSTRACT: Occurring in hops (Humulus lupulus) and beer as a racemic mixture, (2R,2S)-8-prenylnaringenin (8-PN) is a
potent phytoestrogen in hop dietary supplements used by women as alternatives to conventional hormone therapy. With a half-
life exceeding 20 h, 8-PN is excreted primarily as 8-PN-7-O-glucuronide or 8-PN-4′-O-glucuronide. Human liver microsomes
and 11 recombinant human UDP-glucuronosyltransferases (UGTs) were used to catalyze the formation of the two oxygen-
linked glucuronides of purified (2R)-8-PN and (2S)-8-PN, which were subsequently identified using mass spectrometry and
nuclear magnetic resonance spectroscopy. Formation of (2R)- and (2S)-8-PN-7-O-glucuronides predominated over the 8-PN-
4
′-O-glucuronides except for intestinal UGT1A10, which formed more (2S)-8-PN-4′-O-glucuronide. (2R)-8-PN was a better
substrate for all 11 UGTs except for UGT1A1, which formed more of both (2S)-8-PN glucuronides than (2R)-8-PN
glucuronides. Although several UGTs conjugated both enantiomers of 8-PN, some conjugated just one enantiomer, suggesting
that human phenotypic variation might affect the routes of metabolism of this chiral estrogenic constituent of hops.
KEYWORDS: hops, Humulus lupulus, glucuronide, 8-prenylnaringenin, enantiomer
INTRODUCTION
■
Hops (Humulus lupulus L.) are used primarily to preserve and
1
2,3
flavor beer. Exhibiting considerable estrogenic activity,
extracts of hops are also being developed as dietary
supplements for the management of menopausal symptoms
4
,5
in women. 8-Prenylnaringenin (8-PN) (Figure 1) is the
most estrogenic constituent of hops and among the most
6
,7
potent of all phytoestrogens. A chiral prenylated flavonoid,
8
8
-PN occurs in hops as a racemate probably due to the
nonenzymatic intramolecular cyclization of the corresponding
chalcone, desmethylxanthohumol. Both 8-PN enantiomers are
9
estrogenic have a higher affinity for estrogen receptor-α than
for estrogen receptor-β and some reports suggest that (2S)-8-
PN and (2R)-8-PN might have different estrogen receptor
10
affinities and estrogenicities.
8
-PN is well-absorbed in humans following oral admin-
11
istration but is conjugated rapidly with glucuronic acid.
UDP-glucuronosyltranferases (UGTs) are expressed in many
organs but most abundantly in the liver and intestine.
Incubations of 8-PN with human Caco-2 intestinal epithelial
cell monolayers formed primarily 8-PN-4′-O-glucuronide
Figure 1. Chemical structures of (2R)-8-PN, (2S)-8-PN, and all
theoretical monoglucuronide metabolites. Note that glucuronidation
occurred only at the 7-O and 4′-O positions.
(
Figure 1), while incubations with human hepatocytes formed
12
8
-PN-7-O-glucuronide as the most abundant metabolite. The
isozymes UGT1A1, UGT1A6, UGT1A8, and UGT1A9 were
12
reported to be most responsible for glucuronidation of 8-PN.
Received: July 23, 2019
Although human hepatic metabolism can form a variety of
phase I 8-PN metabolites, phase II glucuronidation predom-
inates.
Revised: September 20, 2019
Accepted: September 26, 2019
Published: September 26, 2019
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.9b04657
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Metabolism of 8-PN enantiomers by human enzymes has
not been reported previously. However, Martinez and Davies
administered each enantiomer of the structurally related hop
flavonoid isoxanthohumol to rats and observed (2R)-8-PN or
To determine which UGT enzymes were responsible for
glucuronidation, 1, 4, 10, 40, or 100 μM (2R,2S)-8-PN, (2R)-8-PN,
or (2S)-8-PN were incubated individually with each of 11
recombinant human UGT isoenzymes (1A1, 1A3, 1A4, 1A6,1A8,
13
1
A9, 1A10, 2B4, 2B7, 2B15, and 2B17). In addition to 8-PN, each
incubation mixture contained 125 μg of UGT, 25 μg/mL alamethicin,
mM MgCl , 50 mM Tris−HCl (pH 7.5), and 2 mM UDPGA in a
(
2S)-8-PN and their glucuronides in rat serum and urine. O-
Demethylation of isoxanthohumol by gut microbiota in the rat,
8
2
14
as reported by Bolca et al., had resulted in the formation of 8-
total volume of 500 μL. Note that 8-PN was dissolved in methanol
before being added to the incubation mixture such that the final
concentration of methanol was 1% of the reaction volume. Each
mixture was incubated at 37 °C for 60 min. Control incubations were
identical, except for the omission of UDPGA. Reactions were
terminated by the addition of 50 μL of ice-cold methanol. After 10
min on ice, the samples were centrifuged at 12 000g and 4 °C for 15
min. Supernatants were analyzed by using HPLC−UV and LC−MS/
MS.
1
3
PN. Martinez and Davies
reported that (2S)-8-PN
glucuronides predominated almost 6-fold over (2R)-8-PN
glucuronides in rat urine following oral administration of (S)-
or (R)-isoxanthohumol, respectively, but they did not identify
which glucuronides were formed for each 8-PN enantiomer.
Because women are consuming hop dietary supplements
3
containing estrogenic 8-PN and because of the importance of
11−13
glucuronidation in 8-PN metabolism and excretion,
we
Semipreparative HPLC Isolation of 8-PN-glucuronides. For
the isolation of 8-PN glucuronides, 100 μL of aliquots of the
reconstituted incubation mixture was injected onto a Shimadzu
investigated the glucuronidation of each 8-PN enantiomer by
human UGTs. The structures of the regioisomers of (2R)-8-
PN glucuronides and (2S)-8-PN glucuronides were deter-
mined using high-performance liquid chromatography−
tandem mass spectrometry (LC−MS/MS), accurate mass
measurement, and one-dimensional (1D) and two-dimensional
(
Kyoto, Japan) Prominence HPLC-XR system equipped with a UV
diode array detector set at 290 nm. Semipreparative separations were
carried out using a YMC (Wilmington, NC) YMC-Pack ODS-A
reversed-phase HPLC column (10 × 250 mm , 5 μm) with a linear
2
gradient from 10 to 70% methanol in 0.1% aqueous formic acid as
follows: 0−10 min hold at 10% methanol, 10−30 min gradient from
10 to 70% methanol, and 30−40 min hold at 70% methanol. The flow
rate was 5 mL/min.
1
13
(
2D) H and C NMR spectra. Using recombinant human
enzymes, the relative contributions of 11 UGT isoforms were
determined for the formation of glucuronic acid conjugates of
2R)-8-PN and (2S)-8PN. Significant differences were
observed between UGT isoforms concerning the glucuronida-
tion of (2R)-8-PN and (2S)-8-PN.
LC−UV−MS/MS, LC−MS/MS, and HPLC−UV Analyses. For
(
analytical-scale analyses using LC−UV−MS/MS and LC−MS/MS,
10 μL of aliquots of the incubation mixture was injected onto a
Shimadzu Prominence HPLC-XR system equipped with an Agilent
2
(
Santa Clara, CA, USA) XDB C18 column (3.0 × 150 mm , 3.5 μm)
MATERIALS AND METHODS
maintained at 40 °C in a column oven. 8-PN glucuronides were eluted
with a 25 min linear gradient from 5 to 60% acetonitrile in water
containing 0.1% formic acid at a flow rate of 0.2 mL/min. UV
absorbance detection was carried out at 290 nm, and mass
spectrometric analyses were carried out on a Shimadzu IT-ToF
high-resolution ion trap/time-of-flight hybrid mass spectrometer
using electrospray and polarity switching. Shimadzu LabSolutions
software was used for instrument control and data processing. During
mass spectrometric analysis, the positive-ion and negative-ion
electrospray conditions were as follows: desolvation line temperature
■
Reagents. Alamethicin and uridine 5′-diphosphoglucuronic acid
trisodium salt (UDPGA) were purchased from Sigma-Aldrich (St.
Louis, MO). Deuterated dimethyl sulfoxide (DMSO) and methanol
were purchased from Cambridge Isotope Laboratories (Tewksbury,
MA). UGT Reaction Mix, Solution B, pooled human liver
microsomes, and recombinant human UGTs 1A1, 1A3, 1A4, 1A6,
1
(
A8-10, 2B4, 2B7, 2B15, and 2B17 were purchased from BD Gentest
Woburn, MA). High-performance liquid chromatography-grade
solvents and analytical-grade reagents were used in all experiments.
2
+
00 °C; heat block temperature 200 °C; electrospray interface voltage
Isolation of (2R)-8-PN and (2S)-8-PN. Racemic 8-PN was
4.5 and −3.5 kV; nitrogen nebulizer gas flow 1.5 L/min; ion
2
prepared as described previously, and the purity was determined to
accumulation time 30 ms; collision energy 50% for MS/MS; and scan
be 95.57% using quantitative NMR. (2R)-8-PN and (2S)-8-PN were
separated from racemic 8-PN by chiral chromatography with UV
range m/z 100−800.
HPLC−UV data were also acquired using a YMC AQ column (2.0
detection at 275 nm. A semipreparative ChiralPak IA column (10 ×
2
×
100 mm , 3 μm) with a mobile phase consisting of a 15 min linear
2
2
50 mm , 5 μM) was eluted with a mobile phase consisting of
gradient from 40 to 60% methanol in 0.1% aqueous formic acid. The
isocratic n-hexane/ethanol (90:10; v/v) at a flow rate of 2 mL/min.
Each 100 μL injection contained 7 mg/mL of racemic 8-PN. Under
these conditions, (2R)-8-PN eluted before (2S)-8-PN as determined
by circular dichroism polarimetry (Jasco J 710 polarimeter) of each
purified enantiomer prepared at 12.5 μM in acetonitrile (see the
flow rate was 0.25 mL/min.
One-Dimensional NMR. All one-dimensional 1H and 13C nuclear
magnetic resonance (NMR) spectra were acquired at 900 and 225
MHz, respectively, on a Bruker (Karlsruhe, Germany) Avance-900
NMR spectrometer equipped with a 5 mm triple resonance inverse
8,15
Supporting Information).
The purities of isolated (2R)-8-PN and
1
13
15
detection ( H, C, N) TCI cryoprobe. The ambient sample
temperature at the probe was regulated at 25 °C (298 K) for all
experiments. Samples of 8-PN and metabolites were dissolved in
(
2S)-8-PN were >99.0% based on high-performance liquid
chromatography-ultra violet (HPLC−UV) and LC−MS analyses.
Enzymatic Glucuronidation of 8-PN. To obtain 8-PN
glucuronides in ample quantities for NMR analysis, 20 μM (2R,2S)-
1
50−175 μL [d ]-DMSO (Cambridge Isotopes; Tewsbury, MA)
6
contained in 3 mm NMR tubes. The measured NMR chemical shifts
1
13
8
-PN was incubated with pooled human liver microsomes (1 mg/mL)
(
H and C) are expressed in ppm (δ), relative to the solvent (δ
1
13
in 50 mM Tris−HCl (pH 7.5) containing 8 mM MgCl , 25 μg/mL
2
DMSO = 2.500 ppm, H; and δ DMSO = 39.50 ppm, C; which are
both computer referenced with respect to tetramethylsilane, TMS =
0.00 ppm).
alamethicin, and 2 mM UDPGA as the UDP-glucuronyltransferase
cofactor at 37 °C for 60 min with continuous shaking as described
16,17
Survey 1D 1H NMR spectra for 8-PN and metabolites were
acquired under quantitative proton NMR (qHNMR) conditions:
relaxation delay (d1) 60 s; acquisition time (aq) 4.0 s; spectral
window (sw) 30 ppm; delay after pulse excitation (de) 26.26 us;
previously.
Negative control incubations were identical except for
the omission of UDPGA or human liver microsomes. Reactions were
terminated by adding a 3-fold excess (v/v) of ice-cold methanol,
followed by centrifugation at 12 000g at 4 °C for 15 min. The
supernatants were removed, evaporated to dryness under a stream of
nitrogen gas, and redissolved in methanol (1/50th of the incubation
volume) prior to HPLC−UV separation or LC−MS/MS analysis.
1
3
dummy scans (ds) 4; zero-filling (si) = 512 k. All 1D C data were
acquired using DEPT-Q-135 as implemented on Bruker spectrom-
eters, which produced a spectrum in which the phase of the signals
B
DOI: 10.1021/acs.jafc.9b04657
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Journal of Agricultural and Food Chemistry
Article
reflects the number of protons bonded to each of the carbons: C
(
nonprotonated), CH down, CH; CH up. Key acquisition
2
3
parameters included the relaxation delay (d1) = 1.5 s; acquisition
time (aq) 1.0 s; and spectral window (sw) 200 ppm.
1
One-dimensional H NMR spectra of the metabolites were
additionally acquired with water suppression, which was necessary
to remove exchange-broadened signal intensities arising from
dissolved water, and the carbohydrate and phenolic −OH resonances
in the sample that overlapped with the signal regions of interest (δ
5
.90−7.50 ppm). Because of the relatively broad nature of the signals
in the metabolite samples, these experiments used the Water
Attenuation by Transverse (T ) Relaxation (WATR)
18,19
technique
2
and were performed using the Carr−Purcell−Meiboom−Gill experi-
ment as implemented on the Bruker NMR spectrometers using the
pulse sequence “cpmg1d”. The variable echo delay (d20) contained in
the sequence was adjusted to suppress the exchange-broadened
resonances in the spectral regions of interest, which provided crucial
information about the glucuronidation sites but was kept as short as
possible to avoid or minimize baseline modulation arising from
1
1
H, H−J-coupling. An echo delay (d20) of 6.53 ms for WATR in the
experiments described here was found to afford satisfactory
suppression in the spectral region of interest.
Two-Dimensional NMR. A full complement of 2D experiments
correlation spectroscopy (COSY), heteronuclear single quantum
(
coherence (HSQC), and heteronuclear multiple bond correlation)
was acquired on a sample of 8-PN (1.0 mg in 170 μL [d ]-DMSO)
6
1
and each of the 8-PN glucuronides. For 8-PN, confirmation of the H
and 13C NMR assignments was crucial for the subsequent work on the
8
-PN metabolites. The original assignments for 8-PN (360 MHz,
2
[d4]-MeOH) have previously been reported, but the assignments
were reconfirmed for the present work (900 MHz, [d ]-DMSO). For
6
the glucuronides, a similar complement of 2D data was acquired. The
COSY experiments for 8-PN and 8-PN glucuronides facilitated the
confirmation of the proton assignments in all cases, which was
important for the subsequent determination of the sites of
glucuronidation. However, the HSQC/heteronuclear multiple bond
correlation data for both of the metabolites were disappointing in that
they were difficult to acquire with good signal-to-noise ratio due to
the limited sample availability. For 8-PN-7-O-glucuronide, which was
the more abundant (100−200 μg) of the two glucuronides, the data
did allow for the assignment of structure of all protons and carbons in
the molecule. For the 8-PN-4′-O-glucuronide, even with limited
sample availability (<∼100 μg), all protons could be assigned from
the COSY data, especially the protons used for the glucuronidation
site determination. The DEPT-Q-135 data for both metabolites were
used for the assignment of all carbons (protonated and non-
protonated) for 8-PN-7-O-glucuronide by analogy with the data
obtained for 8-PN, but only the protonated carbons in 8-PN-4′-O-
glucuronide could be observed and assigned. Thus, for the purposes
Figure 2. LC−UV−MS/MS analysis of 8-PN-glucuronides formed by
the incubation of human liver microsomes and cofactor UDPGA with
(A) racemic 8-PN; (B) (2R)-8-PN; or (C) (2S)-8-PN. Retention
times are longer for the mass chromatogram because it was recorded
in tandem to UV detection.
is determined to correspond to 8-PN-4′-O-glucuronide by
using high-resolution tandem mass spectrometry (Figure 3)
1
and H NMR (Table 1). No 8-PN-5-O-glucuronide was
12
observed, which is consistent with previous reports,
presumably due to hydrogen bonding between the the
hydroxyl group at carbon-5 and the carbonyl group at position
4
. Positive and negative ion electrospray accurate mass
measurements confirmed the elemental composition of each
glucuronide to be C H O (measured [M + H] m/z
+
2
6
28 11
517.1704; theoretical 517.1710; ΔM = −1 ppm; and measured
1
−
of determining the sites of glucuronidation of 8-PN, H NMR was
used exclusively.
[
M − H] m/z 515.1559; theoretical 515.1553; ΔM = −1
ppm).
The base peak of each positive ion tandem mass spectrum
RESULTS AND DISCUSSION
■
was observed at m/z 341, which represents the protonated
aglycone 8-PN formed by the elimination of dehydrated
glucuronic acid (Figure 3). The regioisomers 8-PN-7-O-
glucuronide and 8-PN-4′-O-glucuronide were distinguished
by the abundant fragment ions of m/z 397 and m/z 285 in the
product ion tandem mass spectra of 8-PN-7-O-glucuronide.
The product ion of m/z 397 corresponded to the retro Diels
Alder cleavage of the central C-ring with retention of
glucuronic acid on the A-ring (Figure 3). The ion of m/z
285 was formed by loss of isobutene from protonated 8-PN.
HPLC−UV analysis of the glucuronides formed during the
incubation of racemic 8-PN with human liver microsomes
showed two abundant peaks eluting at 12.8 and 13.1 min and a
smaller peak at 15.3 min (Figure 2A). Almost no 8-PN
remained in the mixture (retention time 17.8 min). Partial
resolution of the first two peaks suggested that they
corresponded to a mixture of (R)-8-PN-glucuronide and (S)-
8
-PN-β-D-glucuronide. All three peaks were collected during
semipreparative HPLC−UV (the first two partially resolved
peaks were combined), freeze-dried, and identified as 8-PN-7-
1
The sites of 8-PN glucuronidation were confirmed using H
O-glucuronide (t = 12.8/13.1 min; ∼200 μg) and 8-PN-4′-O-
NMR analysis of the diasteromers of 8-PN-7-O-glucuronide as
well as 8-PN-4′-O-glucuronide in a self-consistent manner. The
assignments of all proton and carbon-13 signals for 8-PN and
both metabolites are shown in Tables 1 and 2, respectively.
R
glucuronide (t = 15.3 min; ∼150 μg). The purity of each
R
regioisomer was >90.0% by HPLC−DAD and LC−MS.
The peaks eluting at 12.8/13.1 min were identified as 8-PN-
-O-glucuronide, and the peak eluting at 15.4 min in Figure 2
1
7
Table 3 summarizes a subset of the key H chemical shift data
C
DOI: 10.1021/acs.jafc.9b04657
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Journal of Agricultural and Food Chemistry
Article
1
Table 1. H NMR (900 MHz) Chemical Shifts for Racemic
a
8
-PN and its Glucuronides
8
-PN-4′-O-
8
-PN-7-O-glucuronide
R
Figure 3) δ [ppm],
mult (J [Hz])
glucuronide (
8
-PN δ
(t = 12.8/13.1 min;
t = 15.4 min; Figure
3) δ [ppm], mult (J
R
[
ppm], mult
hydrogen
(J [Hz])
[Hz])
H-2
5.416, dd
5.478, dd (3.26,
5.494, dd
(3.20,13.15)
b
(
3.06,12.56)
13.03)
5
.453, dd (3.16, 13.32)
c
H-3a
H-3b
3.203, dd
3.04−3.08, br m
3.199, dd (13.07,−
(
12.53,
17.7)
−
16.96)
2.713, dd
3.06,
16.96)
2.763, dd (3.06,
2.752, dd (3.45,−
(
−17.57)
17.7)
−
2
.796, dd (3.63,
17.76)
−
H-6
5.961, s
6.260, s
.245, s
7.317 (8.49)
.306 (8.49)
6.793 (8.49)
.798 (8.49)
3.062, br d (7.65)
.048, br d (6.81)
5.116, t.sept.
.106, t.sept (7.50,
.49)
6.052, s
6
d
d
H-2′/6′ 7.306 (8.75)
H-3′/5′ 6.786 (8.75)
7.441 (8.70)
7.055 (8.70)
3.082, br d (7.51)
7
6
H-1″
H-2″
3.071, br d
7.31)
(
3
5.078, t.sept
7.31, 2.34)
5.093, t. sept.
(7.52,1.36)
(
5
1
H-4″
H-5″
1.581, br s
1.531, br s
12.106, s
1.573, br s
1.539, br s
12.07, br s
1.580, br s
1.532, br s
12.08, s
5
-OH
1
2.05, br s
e
e
e
7
4
-OH
′-OH
10.766, br s
9.573, s
e
H-1‴
H-2‴
H-3‴
H-4‴
H-5‴
5.085, d (7.38)
3.287, t (8.17)
3.314, t (8.87)
3.361, t (9.44)
5.075, d(7.70)
3.268, t(8.66)
3.319, t(9.10)
3.418, t(9.41)
3.902, t(10.16)
Figure 3. Positive ion electrospray product ion tandem mass spectra
of 8-PN-glucuronides, [M + H] m/z 517.1704, obtained using a
+
high-resolution IT-ToF mass spectrometer. (A) (2R)-8-PN-7-O-
glucuronide (tR = 13.1 min in Figure 2); (B) (2S)-8-PN-7-O-
glucuronide (t = 12.8 min); (C) (2R)-8-PN-4′-O-glucuronide (t =
3.936, t (9.51)
R
R
a
b
Obtained in [d ]-DMSO at 298 K. The presence of two values
corresponds to the 2R- and 2S-isomers of 8-PN-glucuronides. Signals
heavily overlapped for both diastereomers. Assignment confirmed
from the H− H COSY spectrum. Displays an AA′MM′/AA′XX′
spin-coupling system. The OH resonances are exchange broadened
by water + OH groups from the glucuronic acid residue and were not
individually observable.
1
5.3 min); and (D) (2S)-8-PN-4′-O-glucuronide (t = 15.2 min).
6
R
c
1
1
d
used for determining the sites of glucuronidation of 8-PN.
e
1
Examination of the H chemical shifts of 8-PN with those of
the 8-PN-glucuronide regioisomers indicates that the broad
high-frequency singlets for 5-OH remain largely unchanged,
clearly indicating that glucuronidation had not occurred at this
position (Table 3). The signals in the 1D spectrum of 8-PN
and those of the analogous water suppression spectra (spectral
region δ 5.90−7.50 ppm) of the 7-O- and 4′-O-glucuronides
are shown in Figure 4. The telltale chemical shifts used for
assigning the sites of glucuronidation are the 5-OH (noted
earlier), H-2′/H-6′, H-3′/H-5′, and H-6. The large changes
7-O-glucuronide and the (2R)-glucuronide and (2S)-8-PN-4′-
O-glucuronide showed different retention times during HPLC
(Figure 2). Although (2R)-8-PN-7-O-glucuronide and (2S)-8-
PN-7-O-glucuronide were observed as partially resolved
chromatographic peaks following glucuronidation of racemic
8-PN by human liver microsomes, only one peak was detected
for 8-PN-4′-O-glucuronide. This isomer was determined to be
(2R)-8-PN-4′-O-glucuronide, based on the incubation of pure
(2R)-8-PN with human liver microsomes (Figure 2B).
(
δΔ) calculated for the specific chemical shifts of H-3′/H′5′,
H-2′/H-6′, and H-6 serve to unambiguously confirm the sites
1
of glucuronidation. For example, in the H NMR spectra of the
(
2R)-8-PN-7-O-glucuronide and (2S)-8-PN-7-O-glucuronide,
The ratio of (2R)-8-PN-7-O-glucuronide to (2S)-8-PN-7-O-
glucuronide resulting from the incubation of racemic 8-PN
with human liver microsomes was determined using
quantitative H NMR. For the H NMR signals at H-6, the
ratio of (2R)-8-PN-7-O-glucuronide (6.245 ppm) to (2S)-8-
PN-O-glucuronide (6.260 ppm) was 1.37/1.00. This finding is
consistent with the ratio of the corresponding HPLC−UV
peak areas (1.33/1.00) eluting at 13.1 and 12.8 min,
respectively (Figure 2B,C).
the signals for H-6 were shifted 0.284 and 0.299 ppm
downfield, respectively, compared with the analogous proton
in 8-PN. In the case of (2R)-8-PN-4′-O-glucuronide,
glucuronidation of (2R)-8-PN shifted both the H-3′/5′ signal
δΔ = +0.269 ppm and the H-2′/6′ signal δΔ = +0.135 ppm
downfield (Table 3).
1
1
In addition to the chiral center at C-2 of 8-PN, glucuronic
acid is chiral. Therefore, the (2R)-glucuronide and (2S)-8-PN-
D
DOI: 10.1021/acs.jafc.9b04657
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Journal of Agricultural and Food Chemistry
Article
Table 2. 13C NMR (225 MHz) Chemical Shifts for Racemic
a,b
8
-PN and Its Glucuronides
carbon
8-PN
7-O-8-PN-glucuronide
4′-O-8-PN-glucuronide
c
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
C-1′
C-2′/6′
C-3′/5′
C-4′
C-1″
C-2″
C-3″
C-4″
C-5″
G-1
78.19
41.89
196.73
101.76
159.68
95.25
161.14
106.89
164.34
129.20
128.05
115.12
157.55
21.25
78.45/78.37
76.76
42.03
42.17/41.94
197.72/197.68
103.28/103.20
159.18
94.95/94.89
161.09/161.03
108.97/108.94
162.60/162.54
129.00/128.94
128.11
115.18
157.63
21.47/21.42
122.43/122.41
130.45
101.73
127.80
116.04
21.29
122.65
130.24
122.65
130.19
25.53
1
Figure 4. Expanded region (δ 5.90−7.50 ppm) of the 900 MHz H
25.55
17.63
99.48/99.65
73.0
NMR spectra (d -DMSO) of 8-PN (bottom), the WATR spectrum of
6
17.57
diastereomeric 8-PN-7-O-glucuronide (center), and the WATR
spectrum of 8-PN-4′-O-glucuronide (top). Note that for 8-PN-7-O-
glucuronide (center), the signal for H-6 shifted to a higher frequency
relative to that for H-6 in 8-PN, and the pairs of proton signals H-2′/
H-6′ and H-3′/H-5′ were essentially unaffected by glucuronidation.
In contrast, glucuronidation at the 4′-OH position of 8-PN (top
spectrum) shifted the H-6 signal only slightly to a higher frequency,
but the proton signals for H-2′/H6′ and H-3′/H-5′ were significantly
shifted to a higher frequency.
99.77
G-2
G-3
G-4
G-5
G-6
75.86
71.32
75.13
170.32
a
b
Obtained in [d ]-DMSO at 298 K. Assignments were confirmed by
DEPT-Q-135 and COSY. The presence of two chemical shifts for a
6
c
particular carbon corresponds to the 2R- and 2S- isomers of 8-PN
glucuronide, respectively.
substrate, and the results of these incubations are summarized
in Figure 5.
Comparison of the relative amounts of glucuronides formed
from each UGT isoform incubation shows that (2R)-8-PN
formed more abundant glucuronides than did (2S)-8-PN.
Furthermore, 8-PN-7-O-glucuronide predominated over 8-PN-
4′-O-glucuronide for both 8-PN enantiomers (Figure 5).
Table 3. Key NMR Chemical Shifts (δ, ppm) for Assigning
Sites of Glucuronidation of 8-PN
C-
3
compound
5-OH
H-2′/H-6′ H-3′/H5′
H-6
(CH )
2
a
8
-PN
12.106
7.306
6.786
5.961
1.581
.532
1.580
.532
−0.001
1
8
-PN-4′-O-
12.085
-0.021
12.070
7.441
7.055
6.052
b
GlcA
1
c
Δδ
+0.135
+0.269
+0.091
+
0.001
1.573
1.539
−0.008
+0.008
8
-PN-7-O-
7.317
7.306
6.793
6.798
6.260
6.245
+0.299
+0.284
d
GlcA
1
2.050
−0.036
0.056
c
e
e
Δδ
+0.011
0.000
+0.007
+0.012
−
a
b
1
.0 mg/170 μL DMSO-d . 0.1 mg (100 μg)/170 μL DMSO-d6..
− δ . 0.1−0.2 mg (100−200 μg)/170 μL DMSO-
8‑PN
d . The diastereomeric ratio was determined by qHNMR: H-6
6
.
c
d
Δδ = δ
metabolite
e
6
.
(6.245 ppm)/H-6 (6.260 ppm) = 1.37/1.00.
After racemic 8-PN was used to prepare larger quantities of
glucuronides for NMR analysis, purified (2R)-8-PN and (2S)-
-PN were incubated in small quantities with individual human
8
recombinant UGT isozymes. The incubation mixtures were
analyzed using HPLC−UV or LC−MS/MS to determine
which glucuronides were formed and their relative amounts.
During preliminary studies, incubations were carried out using
a constant amount of UGT enzyme with 5 different
concentrations of (2R)-8-PN or (2S)-8-PN. The highest yields
of glucuronide metabolites were obtained using 40 μM
Figure 5. Glucuronidation of (2R)-8-PN and (2S)-8-PN (40 μM
initial concentration) by recombinant human UGT enzymes. (R.S.D.
of mean values, 5−15%).
E
DOI: 10.1021/acs.jafc.9b04657
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Journal of Agricultural and Food Chemistry
Article
Abundant in both liver and intestine, UGT1A1 rapidly
catalyzed the formation of 7-O-glucuronide of both 8-PN
enantiomers in addition to a small amount of 4′-O-
glucuronide. Also occurring in both liver and intestine but
conjugating only (2R)-8-PN, UGT1A6 was the only enzyme
tested to form more (2R)-8-PN-4′-O-glucuronide than (2R)-8-
PN-7-O-glucuronide. UGT1A3 and UGT1A9 formed 7-O-
glucuronides of (2R)-8-PN and (2S)-8-PN, whereas
UGTA1A4 formed only (2S)-8-PN-7-O-glucuronide.
UGT1A8 and UGT2B4 conjugated both 8-PN enantiomers
to form 7-O-glucuronides but only formed 4′-O-glucuronide of
ORCID
Dejan Nikolic: 0000-0003-3569-8069
́
David C. Lankin: 0000-0003-3166-5980
Charlotte Simmler: 0000-0002-6923-2630
Shao-Nong Chen: 0000-0003-0748-0863
Yang Liu: 0000-0002-8064-7108
Guido F. Pauli: 0000-0003-1022-4326
Richard B. van Breemen: 0000-0003-2016-0063
Funding
This research was supported by NIH grants P50 AT000155
from the Office of Dietary Supplements and the National
Center for Complementary and Alternative Medicine. Co-
support of Dr. Jinbo Fang was provided by China Scholarship
Council (CSC 2011616519) and HUST (Huazhong Uni-
versity of Science and Technology). Acquisition of the 900-
MHz NMR spectrometer and construction of the Center for
Structural Biology were funded by NIH grant P41 GM068944,
and acquisition of the IT-ToF mass spectrometer was funded
by NIH grant 1 S10 RR023785 with support from Shimadzu
Scientific Instruments.
(
2R)-8-PN. The enzymes UGT2B7, UGT2B15, and
UGT2B17 did not form any 8-PN glucuronides.
UGT1A1, UGT1A3, and UGT1A6 are expressed in both
liver and intestine; UGT1A4, UGT1A9, and UGT2B4 are
found in the liver but not the intestine; and UGT1A8 and
20,21
UGT1A10 occur in the intestine but not in the liver.
Because UGT1A1 was the only source of (2S)-8-PN-4′-O-
glucuronide in the liver and formed just a small amount of this
metabolite relative to (2S)-u8-PN-7-O-glucuronide, only a
trace amount of (2S)-8-PN-4′-O-glucuronide was detected by
HPLC−UV following the incubation of (2S)-8-PN with
human liver microsomes (Figure 2) and none was detected
by NMR. In contrast, UGT1A10, which is expressed in the
intestine and colon but not in the liver, formed not only (2R)-
Notes
The authors declare no competing financial interest.
Raw NMR data for 8-prenylnaringenin and its glucuronides
and mass spectra for 8-prenylnaringenin glucuronides are
available free of charge at https://doi.org/10.7910/DVN/
HEOV91 (Harvard Dataverse).
8
-PN-O-glucuronide but more (2S)-8-PN-4′-O-glucuronide
than (2S)-8-PN-7-O-glucuronide. Although the liver can also
form (2R)-8-PN-4′-O-glucuronide, an important source of
ACKNOWLEDGMENTS
The authors acknowledge Dr. Benjamin Ramirez for his
assistance with the NMR spectrometers.
(
2S)-8-PN-4′-O-glucuronide in vivo is probably intestinal
■
metabolism during absorption.
Most UGTs catalyzed the formation of 8-PN-7-O-glucur-
onide, while fewer formed 8-PN-4′-O-glucuronide. Some
UGTs conjugated both enantiomers of 8-PN with glucuronic
acid, others conjugated just (2R)-8-PN, and a few formed no
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