Antioxidant activity of phenolic acids and their metabolites: Synthesis and antioxidant properties of the sulfate derivatives of ferulic and caffeic acids and of the acyl glucuronide of ferulic acid

Article abstract of DOI:10.1021/jf304076z

The main metabolites of caffeic and ferulic acids (ferulic acid-4′-O-sulfate, caffeic acid-4′-O-sulfate, and caffeic acid-3′-O-sulfate), the most representative phenolic acids in fruits and vegetables, and the acyl glucuronide of ferulic acid were synthes

Full text of DOI:10.1021/jf304076z

Subscriber access provided by YORK UNIV
Article
ANTIOXIDANT ACTIVITY OF PHENOLIC ACIDS AND THEIR
METABOLITES. SYNTHESIS AND ANTIOXIDANT PROPERTIES
OF THE SULFATE DERIVATIVES OF FERULIC AND CAFFEIC
ACIDS AND OF THE ACYL GLUCURONIDE OF FERULIC ACID.
Alessandro Piazzon, urska Vrhovsek, Domenico Masuero, Fulvio Mattivi, Federica Mandoj, and Mirella Nardini
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf304076z • Publication Date (Web): 18 Nov 2012
Downloaded from http://pubs.acs.org on November 26, 2012
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 46
Journal of Agricultural and Food Chemistry
1
2
ANTIOXIDANT ACTIVITY OF PHENOLIC ACIDS AND THEIR METABOLITES.
SYNTHESIS AND ANTIOXIDANT PROPERTIES OF THE SULFATE DERIVATIVES OF
FERULIC AND CAFFEIC ACIDS AND OF THE ACYL GLUCURONIDE OF FERULIC
ACID
3
4
5
6
Piazzon A., Vrhovsek, U.§, Masuero D.§, Mattivi F.§ , Mandoj F.# and Nardini M.
7
8
Agricultural Research Council (CRAꢀex INRAN), Rome, Italy; §IASMA Research and
Innovation Centre, Edmund Mach Foundation, Food Quality and Nutrition Department,
San Michele all’Adige, Italy; #Department of Chemical Sciences and Technologies,
University of Rome “Tor Vergata”, Rome, Italy.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Running title: Antioxidant activity of phenolic acids and their metabolites.
*Corresponding author:
Dr. Mirella Nardini
Consiglio per la Ricerca e la Sperimentazione in Agricoltura (CRAꢀexꢀINRAN)
Via Ardeatina 546, 00178 Rome, Italy
Phone: +39 06 51494454; Fax: +39 06 51494550
Eꢀmail: nardini@inran.it
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 46
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
ABSTRACT
The main metabolites of caffeic and ferulic acids (ferulic acidꢀ4’ꢀOꢀsulfate, caffeic acidꢀ4’ꢀ
Oꢀsulfate and caffeic acidꢀ3’ꢀOꢀsulfate), the most representative phenolic acids in fruit and
vegetables, and the acyl glucuronide of ferulic acid were synthesized, purified and tested
for their antioxidant activity in comparison with their parent compounds and other related
phenolics. Both the Ferric Reducing Antioxidant Power (FRAP) assay and the 2,2’ꢀazinoꢀ
bis(3ꢀethylbenzothiazolineꢀ6ꢀsulfonic acid) (ABTS) radical scavenging method were used.
Ferulic acidꢀ4’ꢀOꢀsulfate and ferulic acidꢀ4’ꢀOꢀglucuronide exhibited very low antioxidant
activity, while the monosulfate derivatives of caffeic acid were fourꢀfold less efficient as
antioxidant than caffeic acid. The acyl glucuronide of ferulic acid showed strong
antioxidant action. The antioxidant activity of caffeic acidꢀ3’ꢀOꢀglucuronide and caffeic
acidꢀ4’ꢀOꢀglucuronide was also studied. Our results demonstrate that some of the
products of phenolic acids metabolism still retain strong antioxidant properties. Moreover,
we firstly demonstrate the ex vivo synthesis of the acyl glucuronide of ferulic acid by
mouse liver microsomes, in addition to the phenyl glucuronide.
Keywords: caffeic acid, ferulic acid, glucuronide, sulfate, ferulic acid acyl glucuronide
2
ACS Paragon Plus Environment

Page 3 of 46
Journal of Agricultural and Food Chemistry
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
INTRODUCTION
Oxidative stress is involved in many human diseases, such as atherosclerosis, cancer,
neurodegenerative diseases, diabetes, ageing. Dietary antioxidants have been
considered to exert a protective role against the development and progression of these
oxidative stressꢀrelated pathological conditions 1. Among dietary antioxidants, polyphenols
received particular attention in the last decade due to their antioxidant activity and
biological effects. Epidemiological studies have suggested associations between the
consumption of polyphenolsꢀrich food and the prevention of many human diseases
associated with oxidative stress 2ꢀ5. On the basis of their daily intake, which greatly
exceeds that of other antioxidants (vitamin E, vitamin C, betaꢀcarotene), phenolic
compounds may be a major factor in assuring the antioxidant potential of the diet and may
contribute to maintaining the endogeneous redox balance in humans.
Phenolic acids, a major class of polyphenols, are widely distributed in human diet,
particularly in fruits, vegetables and beverages (coffee, beer, wine, fruit juices) 6,7. Daily
intake of phenolic acids has been reported to be in the order of 200 mg/d within a large
range depending on nutritional habits and preferences 2,6,7. Phenolic acids received
special attention because of their relatively high concentrations in food and beverages,
strong antioxidant activity and easy intestinal absorption. Dietary phenolic acids are
extensively metabolized in humans 8ꢀ16. In particular, caffeic acid and ferulic acid, the most
representative phenolic acids in the diet, after their absorption from the gastrointestinal
tract, circulate in human plasma as conjugated forms, mainly glucuronate and sulfate
derivatives 8ꢀ10,12,17ꢀ24. Glucuronidation and sulfation of polyphenols are generally believed
to significantly reduce their antioxidant activity, particularly because both sulfation and
glucuronidation occur at the reducing hydroxyl groups of the phenolic structure, which are
the functions mainly responsible for the antioxidant properties of polyphenols. However,
phenolic acids bear a carboxyl function in addition to the hydroxyl groups in their structure.
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 46
79
80
Consequently, while sulfation (implying an “ether” like bond) can occur only at the
hydroxyl group of the phenolic acid, glucuronidation instead might occur both at the
hydroxyl group (phenylꢀOꢀglucuronides, in which the hydroxyl group on Cꢀ1 of the
glucuronide moiety is bound to the hydroxyl group of phenolic acid by an “ether” type
bond) or at the carboxylic group (acyl glucuronides, in which the hydroxyl group on Cꢀ1 of
the glucuronide moiety is bound to the carboxylic group by an “ester” type bond) of the
phenolic acid. Acyl glucuronides of many substances (xenobiotics, endogeneous
compounds, drugs) bearing a carboxyl group, have been recognized in animal and human
urine, plasma and tissues 25ꢀ27. Noteworthy, acyl glucuronides of phenolic acids, retaining
all the reducing hydroxyl functions of the phenolic acid, might be supposed to possess an
antioxidant activity higher than that of their respective phenyl-Oꢀglucuronides, in which at
least one reducing hydroxyl group of the phenolic acid bind the glucuronate moiety. In
spite of the large data concerning the antioxidant activity and biological effects of phenolic
acids, very few studies deal with the antioxidant activity, chemical and biological properties
of their metabolites 23,24,28ꢀ30, due to the fact that most of these compounds are not
commercially available. Moreover, whereas the occurrence in animals and human beings
of sulfate derivatives of caffeic and ferulic acid has been well described in literature 8,12,22ꢀ
25, for glucuronate derivatives all data focused on the phenylꢀOꢀglucuronides, in which the
glucuronide moiety is bound to the hydroxyl group of phenolic acid by an “ether” type
bond, of caffeic and ferulic acid 12,22,25. The first aim of this study was to assess the
antioxidant properties of the sulfate and glucuronate metabolites of ferulic and caffeic
acids in respect to that of their parent compounds and of other related phenolic acids. Due
to the fact that many of these metabolites are not commercially available, we synthesized
and purified some of the metabolites of caffeic and ferulic acids: the sulfate derivative of
ferulic acid (ferulic acid 4’ꢀOꢀsulfate) and the monoꢀsulfate derivatives of caffeic acid
(caffeic acid 3’ꢀOꢀsulfate and caffeic acid 4’ꢀOꢀsulfate). The second aim of our study was
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
4
ACS Paragon Plus Environment

Page 5 of 46
Journal of Agricultural and Food Chemistry
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
to assess the possible occurrence of the acyl glucuronides of phenolic acids, in addition to
the well known phenylꢀOꢀglucuronides. We firstly synthesized and purified the acyl
glucuronide of ferulic acid (FAG) (Figure 1) and compared its antioxidant activity with that
of the respective phenylꢀOꢀglucuronide and with the parent ferulic acid.
Finally, we demonstrated for the first time the ex vivo synthesis of FAG by mouse liver
microsomes.
MATERIALS AND METHODS
Chemicals. 2,4,6ꢀTriꢀ(2ꢀpyridyl)ꢀSꢀtriazine (TPTZ), βꢀglucuronidase (EC 3.2.1.31 type
IX A, from E. Coli), gallic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, pꢀ
coumaric acid, ferulic acid, trolox, 2,2’ꢀazinoꢀbis(3ꢀethylbenzothiazolineꢀ6ꢀsulfonic acid)
diammonium salt (ABTS), acetobromoꢀαꢀDꢀglucuronic acid methyl ester (2,3,4ꢀtriꢀOꢀacetylꢀ
1ꢀbromoꢀDꢀglucuronic acid methyl ester), uridineꢀ5’ꢀdiphosphoglucuronic acid trisodium
salt (UDPꢀGA), sinapic acid, pyridin, triton Xꢀ100, potassium peroxodisulfate, methanol
LC/MS grade and formic acid were from SigmaꢀAldrich (St. Louis, MO, USA). 4ꢀ
Hydroxyphenylacetic acid and oꢀcoumaric acid were from Extrasynthese (Genay Cedex,
France). Caffeic acid 3’ꢀβꢀDꢀOꢀglucuronide, caffeic acid 4’ꢀβꢀDꢀOꢀglucuronide and ferulic
121
122
123
124
125
126
127
128
129
130
acid 4’ꢀOꢀβꢀDꢀglucuronide (FFG) were from Toronto Research Chemicals (North York,
Ontario, Canada). Supelclean LC18 SPE cartridges (3 ml or 12 ml tubes, as specified)
were from Supelco (Bellefonte, PA, USA). Glacial acetic acid, chlorosulphonic acid,
methanol (MeOH), acetonitrile and diethyl ether were obtained from Carlo Erba (Milano,
Italy). For HPLC analysis, ultrapure water from a MilliꢀQ system (Millipore, Bedford, MA,
USA) was used.
Stock solutions of standard phenolic acids were prepared in methanol (MeOH) (1
mg/ml), stored at – 80 °C and used within 1 week. Standard solutions of glucuronate and
sulfate derivatives of caffeic and ferulic acids were prepared in twiceꢀdistilled water (1
mg/ml), stored at – 80 °C and used within 1 week. Working standard solutions were
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 46
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
prepared daily by dilution in sample buffer (1.25% acetic acid, 7% MeOH in twiceꢀdistilled
water).
Ferulic acid sulfate ester and caffeic acid mono-sulfate esters: synthesis,
purification and identification. Ferulic acid 4’ꢀOꢀsulfate and the two monoꢀsulfate esters
of caffeic acid (caffeic acid 4’ꢀOꢀsulfate and caffeic acid 3’ꢀOꢀsulfate) (Figure 1) were
31
synthesized and purified according to Todd
et al. Purification of the two monoꢀsulfate
esters of caffeic acid gave a mixture of the two isomers (4’ꢀOꢀsulfate and 3’ꢀOꢀsulfate in
the ratio 3:1). Purified ferulic acid sulfate ester and the mixture of the two monoꢀsulfate
1
esters of caffeic acid were identified by HNMR spectra (300 MHz, CD3OD), as already
reported 31 and by UPLCꢀMS analyses, as described in the following 32. Ferulic acid 4’ꢀOꢀ
sulfate showed a purity > 95%; for caffeic acid monosulfate purity was > 90%. Yield was
92% for ferulic acid 4’ꢀOꢀsulfate and 47% for caffeic acid monosulfate (as mixture of the
two isomers 4’ꢀOꢀsulfate and 3’ꢀOꢀsulfate ).
Ferulic acid acyl-glucuronide: synthesis, purification and identification. Ferulic
acid 1ꢀOꢀacylꢀglucuronide (FAG) was synthesized in two steps (Figure 2). First, the
protected form of ferulic acid 1ꢀOꢀacylꢀglucuronide (2,3,4ꢀtriꢀOꢀacetylꢀ1ꢀferuloylꢀDꢀ
glucuronic acid methyl ester, FAGP) was synthesized by reaction of ferulic acid with 2,3,4ꢀ
triꢀOꢀacetylꢀ1ꢀbromoꢀDꢀglucuronic acid methyl ester following the procedure described by
33
Fialova
et al., with minor modification. FAGP was then purified by chromatography on
SPE cartridge. Purified FAGP was finally deprotected by hydrolytic procedure to give
ferulic acid acyl glucuronide (FAG).
Briefly, for FAGP synthesis, to a solution of ferulic acid (1 g) and 2,3,4ꢀtriꢀOꢀacetylꢀ1ꢀ
bromoꢀDꢀglucuronic acid methyl ester (1 g) in 10 ml ACN and 8.7 ml twiceꢀdistilled water,
1.3 ml NaOH 4N were added. The mixture was stirred for 24 h at room temperature, then
the solution was evaporated under reduced pressure at 40°C. To eliminate excess ferulic
acid, the residue was extracted twice with 15 ml CH2Cl2. The organic phase was dried
6
ACS Paragon Plus Environment

Page 7 of 46
Journal of Agricultural and Food Chemistry
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
under reduced pressure at 40°C and about 1 g FAGP was obtained as white powder. For
FAGP purification, 0.5 g FAGP dissolved in absolute MeOH were loaded on a 12 ml
Supelclean LC18 SPE tube equilibrated with water. The tube was washed in the order with
100 ml twiceꢀdistilled water; 100 ml MeOH/H2O, 75/25; 80 ml MeOH/H2O 60/40 (v/v).
FAGP was eluted with 100 ml 50:50 MeOH/H2O, the eluated was evaporated under
reduced pressure at 40°C and about 200 mg purified FAGP as white crystalline powder
were obtained. Purified FAGP was identified by UPLCꢀMS analyses 32, using the same
experimental conditions applied for sulfate derivatives and described in the following.
Purity of FAGP was > 90%. Yield was about 25%.
To obtain FAG, purified FAGP was deprotected following the procedure described by
Gauthier 34 et al. with minor modifications. 100 mg purified FAGP were added with 47 mg
KOH (4.2 equivalents) in 36 ml water. The mixture was stirred at room temperature for 40
min, the pH was brought at about 6.0 with 1N HCl. The solution was evaporated under
reduced pressure at 40°C and the residue purified by solid phase extraction as follows.
The residue was dissolved in 0.5 ml twiceꢀdistilled water, centrifuged 5 min at 18 000 x g,
then the supernatant was loaded on a Supelclean LC 18 SPE tube (3 ml) equilibrated with
water. FAG was eluted with 12 ml twiceꢀdistilled water. The eluate was evaporated under
reduced pressure at 40°C. The residue was dissolved in 0.5 ml 0.22 M acetic acid and
loaded on a Supelclean LC18 SPE tube (3 ml) equilibrated with 0.22 M acetic acid. The
tube was washed with 7 ml 0.22 M acetic acid; FAG was eluted with about 10 ml 0.22 M
acetic acid/MeOH 90/10 (v/v). The eluate was evaporated under reduced pressure at
40°C. The purified FAG obtained was identified by UPLCꢀMS analyses 32, as described in
the following. FAG obtained showed a purity > 85%. Yield was about 20%. FAG was
obtained as a mixture of different positional isomers (see below). To purify and separate
the different FAG isomers, the last purification step was repeated, the eluate fractionated
and each fraction analyzed on HPLCꢀECD.
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 46
32
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
UPLC/TOF-MS analyses. The UPLC/Q/TOF conditions are detailed in Theodoridis
et al. In short: UPLCꢀMS analyses were performed on a Waters Acquity UPLC system
coupled with to a Synapt HDMS QTOFꢀMS (Waters, Manchester, UK) via an electrospray
interface (ESI), operating in Wꢀmode and controlled by MassLynx 4.1 and UVꢀVis (DAD)
detector recording the spectra fro 200 nm to 490 nm. An Aquity UPLC C18 column (2.1 x
150 mm, 1.8 ꢀm) (HSS T3, Waters) thermostated at 30°C was used. The column,
equilibrated with solvent A (0.1% formic acid in twiceꢀdistilled water), was eluted with
increasing concentration of solvent B (0.1% formic acid in methanol) at 0.3 ml/min: 0ꢀ6
min., 100% A; 7ꢀ56 min: from 100% A to 100% B; 57ꢀ60 min, 100% B. The injection
volume was 5 ꢀl. The mass spectrometry data were collected in negative mode (CV 2.5),
in centroid mode (mass to charge ratio (m/z) 100ꢀ1000). For MS2 experiments, the
collision energy in transfer was settled at 30 V. External calibration with sodium formate
and Lock Mass calibration with leucine enkephaline solution (0.5 mg/l, m/z 556.2771 at 0.1
ml/min) was applied.
In our UPLC conditions, the elution times were: ferulic acid 4’ꢀOꢀsulfate, 19.5 min;
caffeic acid 4’ꢀOꢀsulfate, 17.9 min; caffeic acid 3’ꢀOꢀsulfate, 18.6 min; FAGP, 36.2 min.
FAG isomers were eluted at 16.6 min, 18.4 min, 18.7 min and 20.0 min.
Orbitrap. For MS3 experiments Orbitrap XL mass spectrometer (Thermo Fisher
Scientific (Bremen), GmbH, Germany) with a resolution of 100 000 [at m/z 400, full width
half maximum (FWHM)] was employed. Capillary temperatures were set at 270° C. ESI
capillary voltage of 2.7 kV in negative mode. Scan time was set to 3 microseconds with
maximum acquisition time 200 milliseconds. Fourier transform MS (FTMS) full scans from
m/z 50 to m/z 1000 were acquired using the Instrument Control Software (LTQ Tune Plus
2.5.5 SP1) and Xcalibur 2.1. The Orbitrap was calibrated prior to mass analysis by
external calibration using standard peptide mixtures (ProteoMass MALDI calibration kit for
8
ACS Paragon Plus Environment

Page 9 of 46
Journal of Agricultural and Food Chemistry
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
LTQ XL and LTQ hybrids, SigmaꢀAldrich) for the normal mass range. The samples were
diluted with methanol water 1:1 and infused into the instrument by glass syringe.
HPLC instrumentation. The synthesis and purification of the derivatives of caffeic acid
and ferulic acid were followed by HPLC with both electrochemical detection (ECD) and UV
detection. The HPLC consists of a PerkinꢀElmer Liquid Chromatograph (PerkinꢀElmer,
Norwalk, CT, USA) with gradient pump, column thermoregulator, autosampling injector
equipped with electrochemical coulometric detector (Coulochem II, ESA, Bedford, MA,
USA) or diode array detector (PerkinꢀElmer, Norwalk, CT, USA). A cromatography
workstation with Totalchrom software (PerkinꢀElmer, Norwalk, CT, USA) was used for data
processing. Operating conditions were as follows: column temperature, 30°C; flow rate: 1
ml/min; injection volume, 100 ꢁl; electrochemical detection at + 600 mV, sensitivity range
200 nA, filter 2 sec. For UV detector, analyses were performed at 320 nm or 230 nm as
specified. Chromatographic separations were performed on a Supelcosil LCꢀ18 C18
column, 5.0 ꢀm particle size, 250 x 4.6 mm ID (Supelco, Bellefonte, PA, USA) including a
guard column. For gradient elution mobile phases A and B were employed. Solution A was
1.25% glacial acetic acid in twiceꢀdistilled water; solution B was absolute methanol. For the
sulfate derivatives of caffeic acid and ferulic acid, the following gradient was used: 0ꢀ30
min, from 98% A, 2% B to 88% A, 12% B, linear gradient; 31ꢀ60 min, from 88% A, 12%
B% to 80% A, 20% B, linear gradient; 61ꢀ90 min, from 80% A, 20% B to 74% A, 26% B,
linear gradient; 91ꢀ98 min, 55% A, 45% B; 99ꢀ129 min, 98% A, 2% B. Retention times
were 25.3 min for ferulic acid 4’ꢀOꢀsulfate; 15.6 min and 16.5 min for caffeic acid 4’ꢀOꢀ
sulfate and caffeic acid 3’ꢀOꢀsulfate, respectively. For the synthesis, purification and
characterization of FAGP and FAG, the following gradient was used: 0ꢀ50 min, from 98%
A, 2% B to 50% A, 50% B, linear gradient; 51ꢀ60 min, 50% A, 50% B; 61ꢀ90 min, 98% A,
2% B. Retention times were: 20,9 min, 22.5 min, 25.2 min and 26.0 min for the different
positional FAG isomers; 36.9 min for ferulic acid; 61.7 min for FAGP. Prior to HPLC
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 46
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
analysis, all samples were filtered using MillexꢀHV filters (Millipore, Bedford, MA, USA)
with 0.45 ꢀm pore size.
Microsomes preparation. Animal studies were performed under conditions approved
by the National Health Ministry (Department of Food, Nutrition and Public Animal Health).
Balb/c mice (Charles River, Calco (LC), Italy) were anesthetized with pentobarbital
injection (10 mg), and the liver excised and placed in cold phosphateꢀbuffered saline
(PBS). After weighing, livers were homogenized in 4 volumes of 0.1 M TrisꢀHCl buffer (pH
7.4) containing 1 mM EDTA and 0.1 M KCl with an Ultraturrax homogenizer (IKA, Staufen,
Germany) for two 40ꢀs intervals and treated for microsome purification as described by
van der Hoeven 35 et al. The purified microsomal fractions were suspended in 0.01 M Trisꢀ
acetate buffer pH 7.4 containing 20% glycerol and 0.1 mM EDTA and stored at ꢀ80°C until
36
used. Protein concentrations were measured by the method of Lowry
serum albumine as standard.
using bovine
Biosynthesis of ferulic acid glucuronides in liver microsomes. The conjugation
37
reaction was carried out according to Jemnitz
et al. The incubation mixture contained:
0.1 M TrisꢀHCl buffer pH 6.9; 0.05% Triton Xꢀ100; 5 mM UDPꢀGA; 10 mM MgCl2; 4 mg/ml
microsomal protein; 1 mM ferulic acid in a final volume of 1 ml. Microsomes were preꢀ
incubated with Triton Xꢀ100 for 30 min at 0°C, and the reaction started with the addition of
ferulic acid. Blanks without UDPꢀGA or ferulic acid were run. Incubation was performed at
37 °C for 60 min. The reaction was stopped by addition of 0.05 ml 1 N HCl to the
incubation mixture. For phenolic compounds extraction, the pH was brought to 3.0 and
300 mg NaCl were added. Samples were then extracted four times for 10 min with 6
volume excess ethyl acetate, following by 10 min centrifugation at 2 500 g. The pooled
organic supernatant was dried under nitrogen stream and the residue dissolved in 0.5 ml
1.25 % glacial acetic acid containing 7% (v/v) MeOH, and analyzed by HPLC with ECD or
10
ACS Paragon Plus Environment

Page 11 of 46
Journal of Agricultural and Food Chemistry
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
UV detection after appropriate dilution. The extraction procedure allows the quantitative
recovery of ferulic acid, ferulic acid 4’ꢀOꢀglucuronide and FAG (range 87.6% ꢀ 99.4%).
Antioxidant activity. The antioxidant activity on phenolic acids and their metabolites
was measured by both the ferric reducing antioxidant power (FRAP) and the ABTS radical
scavenging assay.
FRAP assay. Ferricꢀreducing antioxidant power was measured as described by Benzie
38
et al. FRAP assay is a simple method of determining the reduction of a ferricꢀ
tripyridyltriazine complex to its ferrous, colored form in the presence of antioxidants.
Phenolic compounds were tested at various concentrations in the range 0ꢀ500 ꢁM, as
specified. Sulfate and glucuronide derivatives were dissolved in twiceꢀdistilled water.
Methanol, used to dissolve most of the phenolic acids, was ≤ 5.0 % in the final text
mixture. Aqueous or methanolic (≤ 5.0 % MeOH in the final text mixture) iron sulfate
solutions were used in the range 10ꢀ100 ꢁM for calibration curves. The working FRAP
reagent was prepared daily as follows: 2.5 ml 10 mM TPTZ in 40 mM HCl and 2.5 ml 20
mM FeCl3 added with 25 ml 0.3 M acetate buffer pH 3.6. FRAP reagent (0.9 ml), preꢀ
warmed at 37°C, was mixed with 100 ꢁl test sample. The reaction was followed for up to 4
min at 593 nm at 37°C. Blanks containing FRAP reagent and water or methanol instead of
phenolic compounds were also run; the difference between sample absorbance and blank
absorbance at 593 nm after 4 min reaction at 37°C was used for calculation. Each sample
was analyzed in triplicate. Simple regression analysis was performed to calculate the
doseꢀresponse relationship of each compound tested.
ABTS assay. The free radical scavenging capacity of phenolic acids and their
metabolites was also studied using the ABTS radical cation decolorization assay 39, which
is based on the reduction of ABTS+° radicals by antioxidants tested. ABTS (7 mM) was
dissolved in twiceꢀdistilled water, and ABTS radical cation (ABTS+°) was produced by
reacting ABTS solution with 2.45 mM potassium persulfate (final concentration) at room
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 46
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
temperature in the dark for 16 h before use. A working solution was diluted to absorbance
values between 1.2 and 1.3 AU at 734 nm with twiceꢀdistilled water and equilibrated at
30°C. ABTS reagent (0.95 ml) preꢀwarmed at 30°C, was mixed with 50 ꢁl test sample. The
absorbance reading was taken at 734 nm, 30°C, at 20 s intervals up to 6 min after initial
mixing (AE). Phenolic compounds were tested at various concentrations in the range 0ꢀ20
ꢁM, as specified. Sulfate and glucuronide derivatives were dissolved in twiceꢀdistilled
water. Methanol, used to dissolve most of the phenolic acids, was ≤ 0.5% in the final text
mixture. Trolox (0ꢀ15 nmol), the waterꢀsoluble analogue of vitamin E, was used as
reference compound. An appropriate solvent blank reading was also taken (AB). The
percentage inhibition of absorbance was calculated using the following formula:
% Inhibition = [(AB – AE)/AB] x 100
where AB= absorbance of the blank sample, and AE= absorbance of the sample tested (at
6 min reaction), and plotted as a function of the concentration of the compound tested. All
solutions were prepared daily. All measurements were made in triplicate. Simple
regression analysis was performed to calculate the doseꢀresponse relationship of each
phenolic compound tested.
Hydrolytic treatments.
β
-Glucuronidase treatments. Enzymatic hydrolysis of FAG was performed as already
described 8, with minor modification. Briefly, FAG was dissolved in twiceꢀdistilled water at 1
mg/ml. Aliquots were incubated in 0.7 ml final volume 0.2 M Kꢀphosphate buffer pH 6.8
with 2 000 U βꢀglucuronidase, dissolved in the same buffer, at 37°C for 2 h. At the end of
incubation, samples were centrifuged 10 min at 18 000 g, filtered and analyzed by HPLC
with ECD or UV detection, after appropriate dilution.
Alkaline hydrolysis. Alkaline hydrolysis of FAG was performed by the alkaline hydrolytic
treatment previously described 10. FAG dissolved in twiceꢀdistilled water was hydrolysed in
the presence of 10 mM EDTA, 1% ascorbic acid, 1.8 N NaOH in 1 ml final volume. After
12
ACS Paragon Plus Environment

Page 13 of 46
Journal of Agricultural and Food Chemistry
311
312
313
314
315
316
317
318
319
320
321
30 min at 30°C, 4 N HCl was added to bring the pH to 3.0. Samples were filtered and
analysed by HPLC with ECD or UV detection, after appropriate dilution.
Statistical analysis. Data presented are means ± standard error. Statistical analysis
was performed using a statistical package running on a PC (KaleidaGraph 4.0, Synergy
Software, Reading, PA, USA). Student’s t test was used for regression analyses. The
probability of p <0.05 was considered statistically significant.
RESULTS
Characterization of sulfate derivatives of ferulic and caffeic acids. Figure 3 shows
the UPLCꢀMS profile of ferulic acid 4’ꢀOꢀsulfate: in panel A, the UPLCꢀMS elution
chromatogram shows a major component eluting at 19.5 min; in panel B, the mass
spectrum of the peak eluting at 19.5 min shows the presence of the negatively charged
322
molecular ion [MꢀH]ꢀ at m/z 273.0071 corresponding to the value expected for ferulic acid
323
324
325
326
327
328
329
330
331
4’ꢀOꢀsulfate, and a major fragment at 193.0486 corresponding to the ferulic acid moiety.
Further confirmation of the compound was obtained by Orbitrap mass spectrometry, which
gave the resulting MS2 fragmentation pattern with ion at m/z 193.0500 and MS3
fragmentation pattern with ions at m/z 134.0369, 149.0603 and 178.0265 (data not
shown). These data are in agreement with the literature 12.
Figure 4 shows the UPLCꢀMS profile of caffeic acid monoꢀsulfate derivatives (caffeic acid
4’ꢀOꢀsulfate and caffeic acid 3’ꢀOꢀsulfate): in panel A, the UPLCꢀMS elution chromatogram
shows two major peaks eluting at 17.9 min and 18.6 min; in panel B and C the mass
spectra of the two peaks eluting at 17.9 min (panel B) and 18.6 min (panel C) show the
332
presence of the negatively charged molecular ion [MꢀH]ꢀ at m/z 258.9904 and 258.9913
333
334
335
336
respectively, corresponding to the value expected for both caffeic acid 4’ꢀOꢀsulfate and
caffeic acid 3’ꢀOꢀsulfate, with a major fragment at 179.0340 for both peaks, corresponding
to the caffeic acid moiety. The resulting MS2 fragmentation pattern gave ions at m/z
135.0449 and 179.0341 for both peaks (data not shown). According to the literature 12,
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 46
337
338
339
340
341
342
343
344
345
peak eluting at 17.9 min min should be attributable to the caffeic acid 4’ꢀOꢀsulfate
derivative, while peak eluting at 18.6 min should be ascribed to caffeic acid 3’ꢀOꢀsulfate.
The ratio of caffeic acid 4’ꢀOꢀsulfate and caffeic acid 3’ꢀOꢀsulfate is about 2.75 : 1.
Characterization of ferulic acid acyl glucuronide and microsomal biosynthesis. In
the present study, we synthesize for the first time the acyl glucuronide of ferulic acid
(FAG). Figure 5 shows the UPLCꢀMS analyses of purified FAG: in panel A, the UPLCꢀMS
elution chromatogram exhibits a main component eluting at 20.0 min and three minor
component eluting at 16.6 min, 18.4 min, 18.7 min. In panels B, C, D, E the mass spectra
of the four peaks in order of elution (16.6 min panel B; 18.4 min, panel C; 18.7 min, panel
346
D; 20.0 min, panel E) show the presence of the negatively charged molecular ion [MꢀH]ꢀ at
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
m/z 369.0822, m/z 369.0830, m/z 369.0831, m/z 369.0830 respectively, corresponding to
the value expected for FAG, and fragment at m/z 175.025 (corresponding to the loss of the
glucuronic acid moiety), and m/z 113.024 for all the four peaks. The resulting MS2
fragmentation patterns show ions at m/z 175.025 and m/z 113.024 for all the four peaks
(data not shown). Fragments at 175 and 113 have been already described for
dihydroferulic acidꢀ4’ꢀOꢀglucuronide 12. In spite of the same molecular ion, the ratio of
fragments in the mass spectra of the four peaks shows somewhat differences, suggesting
that the conjugates of glucuronic acid with ferulic acid are not identical. A similar behaviour
has been described for isomers of the acyl glucuronide of the synthetic flavonoid
ipriflavone 37. These results are in agreement with the isomerisation of the ferulic acid 1ꢀOꢀ
acyl glucuronide (the Cꢀ1 isomer of FAG), due to intramolecular acyl migration from ꢀOH to
the adiacent –OH, generating different positional isomers of FAG, with the same molecular
ion, in which the ferulic acid moiety is attached to either Cꢀ2, ꢀ3 or ꢀ4 position of the
glucuronic acid ring. Intramolecular acyl migration of substituted sugars is a known
process occurring under slightly alkaline conditions and it has been already described for
several acyl glucuronides 25ꢀ27,40,41. Since only C1ꢀisomers are substrates for βꢀ
14
ACS Paragon Plus Environment

Page 15 of 46
Journal of Agricultural and Food Chemistry
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
glucuronidase 25ꢀ27,40,41, we further purified the different FAG isomers and treated them
with βꢀglucuronidase as described in method section. Only the fraction containing the more
retained and abundant FAG isomer (peak at 20.0 min in the figure 5, panel A) was
hydrolyzed by βꢀglucuronidase, giving 69.0 ± 2.5 % of the calculated ferulic acid,
demonstrating that the more retained and abundant FAG isomer, eluting at 20.0 min in
UPLC, is the Cꢀ1 isomer. Alkaline hydrolysis of purified FAG C1ꢀisomer gave 98.4 ± 1.9 %
of the theoretical ferulic acid.
To investigate the biological relevance of the acyl glucuronide of ferulic acid (FAG), we
studied the biosynthesis of FAG by mouse liver microsomes. Upon incubation of ferulic
acid with UDPꢀGA and microsomal proteins from mouse liver as described in method
section, three main components were detected with HPLCꢀUV detector at 320 nm, as
shown in fig. 6, panel B. The first peak at 23.5 min coeluted with standard ferulic acid 4’ꢀOꢀ
glucuronide (FFG, which is the phenyl glucuronide of ferulic acid) (fig. 6 panel C), the
second component at 26.0 min coeluted with FAG (Cꢀ1 isomer) (fig. 6 panel D), the third
component at 36.9 min was residual ferulic acid. UPLC analyses, with both DAD at 320
nm and MS detector, are shown in figure 7, panel A and B respectively. The mass spectra
identified the peaks, in the order of elution, as: panel C, ferulic acid 4ꢀOꢀ glucuronide
(FFG); panel D, ferulic acid 1ꢀOꢀacyl glucuronide (FAG, Cꢀ1 isomer); panel E, ferulic acid.
Interestingly, the mass spectra of FFG and FAG are almost undistinguishable, with the
molecular ion at m/z 369.0818 and m/z 369.0826, respectively, and fragments at m/z
193.050, m/z 175.025 and m/z 113.024. The ratio between FFG and FAG is 2.4: 1 on the
basis of UPLCꢀMS analyses (mean of 3 separate experiments). The identity of FAG and
FFG was further demonstrated in HPLCꢀECD at +600 mV (data not shown). In fact, FAG
molecule possesses an unbound, reducing OHꢀgroup on the phenolic ring, which is
revealed by the electrochemical detector, while FFG was not detected by electrochemical
detector, due to the lack of reducing groups (in FFG the OHꢀ group of ferulic acid moiety is
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 46
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
esterified with glucuronic acid). Overall, these results demonstrate for the first time the
synthesis of the acyl glucuronide of ferulic acid by liver microsomes in ex vivo
experiments. Noteworthy, microsomal proteins from mouse liver seem to synthesize only
the Cꢀ1 isomer of FAG.
Antioxidant activity determined by FRAP assay. The antioxidant activity of
metabolites of phenolic acids, and more generally of polyphenols, has been poorly
investigated 29,30,42, mainly due to the lack of commercially available standards.
In table 1, we compared the ferric reducing ability (FRAP) of the caffeic acid and ferulic
acid metabolites synthesized in this study with that of their respective parent compounds
and with the activity of some commercially available caffeic acid and ferulic acid
metabolites and of other related phenolic acids, as a measure of their antioxidant power.
Iron sulfate was used as reference compound. The slope of the curve reflects the
antioxidant’s reducing capacity, therefore slope values allow for a comparison of the
antioxidant efficiencies of the tested compounds. High slope values denote high ferric
reducing ability. Caffeic and ferulic acid showed a strong ferricꢀreducing activity, in the
same order of that of trolox, the water soluble analogue of vitamin E.
The ferricꢀreducing activity of the sulfate derivatives, caffeic acid monosulfate (a mixture of
caffeic acid 4’ꢀOꢀsulfate and caffeic acid 3’ꢀOꢀsulfate in the ratio 2.75: 1) and ferulic acid 4’ꢀ
Oꢀsulfate was very low, compared to that of the respective parent compounds. Infact the
antioxidant activity of caffeic acid monosulfate was about 6.0% in respect to that of the
parent caffeic acid, while the antioxidant activity of the ferulic acid 4’ꢀOꢀsulfate was 5.5%
of that of the parent ferulic acid.
Among glucuronides, FAG, the acyl glucuronide of ferulic acid, exhibited an antioxidant
activity comparable to that of the parent ferulic acid, being 0.0401 the slope for ferulic acid,
0.0383 and 0.0368 the values obtained for FAG (as a mixture of positional isomers) and
FAGꢀ1 isomer, respectively. On the contrary, ferulic acid 4’ꢀOꢀglucuronide, which is the
16
ACS Paragon Plus Environment

Page 17 of 46
Journal of Agricultural and Food Chemistry
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
phenylꢀOꢀglucuronide of ferulic acid, showed a ferricꢀreducing ability very low (slope
0.0006) in respect not only to that of the parent ferulic acid, but also in respect to FAG.
From our data, the activity of ferulic acid 4’ꢀOꢀglucuronide was about seventy fold lower in
respect to that of ferulic acid and about sixty fold lower in respect to that of the acyl
glucuronide of ferulic acid (FAG). The ferricꢀreducing activity of caffeic acid 3’ꢀO-
glucuronide was high, in the same order of that of iron sulfate, and it was about a half in
respect to that of the parent caffeic acid. On the contrary, the ferricꢀreducing ability of
caffeic acid 4’ꢀOꢀglucuronide was about ten fold lower in respect to that of caffeic acid 3’ꢀ
Oꢀglucuronide. Overall these results suggest that the 4’ꢀhydroxyl group on the aromatic
ring is crucial for the ferricꢀreducing activity.
Among the other phenolic compounds tested, gallic acid with three hydroxyl groups on the
aromatic ring is the strongest antioxidant, on the opposite the monoꢀsubstituted phenolic
acids (pꢀcoumaric acid, oꢀcoumaric acid and 4ꢀOHꢀphenyl acetic acid) showed very low
activity. In general, the presence of the propenoid chain seems to have a positive effect on
the reducing properties of the hydroxyl group, as demonstrated by the higher antioxidant
activity of pꢀcoumaric acid in respect to that of 4ꢀOHꢀphenyl acetic acid, and the higher
antioxidant activity of ferulic acid in respect to that of vanillic acid, due to a stabilizing
resonance effect on the phenoxyl radical 43. Syringic acid and sinapic acid represent an
exception, being the antioxidant activity of syringic acid a little bit higher than that of
sinapic acid. Therefore, beside the presence of the 4’ꢀhydroxyl group, also the number
(degree) of substitutions (hydroxyl or methoxyl groups) on the aromatic ring is determinant
for the ferricꢀreducing antioxidant activity 44,45
.
Antioxidant activity determined by ABTS assay. In table 2, we examined the ability
of the same above reported compounds to reduce the ABTS radical cation, using the
ABTS radical cation decolorization assay. Trolox, the waterꢀsoluble analogue of vitamin E,
was used as reference compound. Again, high slope values denote high antioxidant
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 46
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
activity, therefore slope values allow for a comparison of the antioxidant efficiencies of the
tested compounds.
Caffeic and ferulic acid showed a strong antioxidant activity. The antioxidant activity of
caffeic acid is about a half in respect to that of ferulic acid, and quite similar to the
antioxidant activity of trolox. The antioxidant activity of caffeic acid monosulfate (a mixture
of caffeic acid 4’ꢀOꢀsulfate and caffeic acid 3’ꢀOꢀsulfate, in the ratio 2.75: 1) is four fold
lower in respect to that of the parent caffeic acid, while the antioxidant activity of ferulic
acid 4’ꢀOꢀsulfate is about eleven fold lower in respect to that of the parent ferulic acid.
Among glucuronides, the antioxidant activity of FAG is about a half of that of the parent
ferulic acid and quite close or slightly lower to the antioxidant activity of trolox and caffeic
acid. Noteworthy, the antioxidant activity of ferulic acid 4’ꢀOꢀglucuronide, the phenylꢀOꢀ
glucuronide of ferulic acid, is about thirtyꢀforty fold lower in respect to the antioxidant
activity of FAG, the acyl glucuronide of ferulic acid and about eighty fold lower in respect to
that of ferulic acid. Interestingly, the antioxidant activity of caffeic acid 3’ꢀOꢀglucuronide is
quite similar to that of the parent caffeic acid, while the antioxidant ability of caffeic acid 4’ꢀ
Oꢀglucuronide is about one third of that of caffeic acid and caffeic acid 3’ꢀOꢀglucuronide.
Again, the presence of a 4’ꢀhydroxyl group on the aromatic ring seems to be crucial for the
antioxidant activity, as observed also with FRAP assay. Among the other phenolic
compounds tested, gallic acid with three hydroxyl groups on the aromatic ring is the
strongest antioxidant. The presence of one or two methoxy substitutions in ortho position
to the 4’ꢀhydroxyl group increased the antioxidant efficiency: substitution of the 3’ꢀhydroxyl
group of caffeic acid by a methoxy group (ferulic acid) considerably enhances the
antioxidant activity and syringic acid is more active than vanillic acid 43. Again, as already
observed with the FRAP assay, the presence of the propenoid chain seems to have a
positive effect on the reducing properties of the hydroxyl group, as demonstrated by the
higher antioxidant activity of pꢀcoumaric acid in respect to that of 4ꢀOHꢀphenyl acetic acid,
18
ACS Paragon Plus Environment

Page 19 of 46
Journal of Agricultural and Food Chemistry
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
and the higher antioxidant activity of ferulic acid in respect to that of vanillic acid, due to a
stabilizing resonance effect on the phenoxyl radical 43. Syringic acid and sinapic acid
represent an exception, being the antioxidant activity of syringic acid quite similar to that of
sinapic acid.
DISCUSSION
Dietary phenolic acids, abundant in most common fruits, vegetables and beverages
(coffee, beer, wine, fruit juices) are absorbed and extensively metabolized in humans, and
circulate in plasma mainly as sulfate and glucuronate derivatives 8ꢀ16. Very few data are
present in the literature concerning the antioxidant activity of polyphenols metabolites.
Quercetin glucuronides and quercetin 3ꢀOꢀsulfate have been reported to retain antioxidant
properties, although to a minor extent in respect to quercetin 29. Ferulic acid βꢀglucuronide,
prepared from the plasma of feruloyl arabinose fed rats, has been reported to possess an
antioxidant activity stronger than that of ferulic acid in the LDL oxidation system 30,
however in this study ferulic acid βꢀglucuronide was isolated from plasma and its
concentration was measured by the release of ferulic acid by betaꢀglucuronidase
hydrolysis, which is known not to be completely exhaustive 46. The glucuronidation and
sulfation processes modify the hydrophobicity and the possibilities of electron
delocalization, so that the antioxidant activity of conjugates might be different from that of
the parent compounds. Glucuronidation and sulfation of polyphenols are generally
believed to significantly reduce their antioxidant activity, due to the fact that both sulfation
and glucuronidation occur at the reducing hydroxyl groups of the phenolic structure, which
are the functions mainly responsible for the antioxidant properties of polyphenols. For
phenolic acids, bearing a carboxyl function in addition to the hydroxyl groups,
glucuronidation can occur, other than at the reducing hydroxyl group (phenylꢀOꢀ
glucuronides), also at the carboxylic group (acyl glucuronides) of the phenolic acid.
Therefore, acylꢀglucuronides retain all the free, unbound, reducing hydroxyl functions of
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 46
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
the parent compound, while in the respective phenylꢀOꢀglucuronide at least one hydroxyl
group of the phenolic acid is bound to the glucuronate moiety. In this study, the synthesis
of the sulfate derivatives of ferulic and caffeic acids and of the acyl glucuronide of ferulic
acid (FAG) allowed for the first time the comparison of their antioxidant activity with that of
their parent compounds and of other common phenolic acids. The antioxidant activity of
commercially available Oꢀphenyl glucuronides of ferulic and caffeic acids was also
measured. FRAP and ABTS assays were used to evaluate the antioxidant activity. Both
assays take advantage of electronꢀtransfer reactions, in which the probe itself is an oxidant
that abstracts an electron from the antioxidant. ABTS assay is carried out at neutral pH,
while FRAP assay under acidic conditions. The pH values have an important effect on the
reducing capacity of antioxidants. In particular, at acidic pH the reducing capacity may be
suppressed due to protonation of antioxidants, whereas at basic pH proton dissociation of
phenolic compounds would enhance their reducing 47.
With regard to glucuronide derivatives, our results show that FAG, the acyl glucuronide
of ferulic acid, retain a very good antioxidant activity, similar to that of the parent ferulic
acid with FRAP assay, while with ABTS assay it was about a half in respect to ferulic acid.
On the contrary, the antioxidant activity of ferulic acid 4’ꢀOꢀglucuronide, the phenylꢀOꢀ
glucuronide of ferulic acid, is much lower in respect to that of both FAG (about sixty fold
lower with the FRAP assay and thirtyꢀforty fold lower with the ABTS assay) and ferulic acid
(about seventy fold lower with the FRAP assay and eighty fold lower with the ABTS
assay). Caffeic acid 3’ꢀOꢀglucuronide retains a strong antioxidant activity, comparable to
that of the parent caffeic acid with ABTS assay, while with FRAP assay it was a half in
respect to that of caffeic acid. Caffeic acid 4’ꢀOꢀglucuronide displayed a good antioxidant
activity with ABTS assays, which is about a third in respect to that of caffeic acid, while
with the FRAP assay it was about twentyꢀfold lower than that of caffeic acid. Concerning
the sulfate derivatives of ferulic acid and caffeic acid, the antioxidant activity seems to be
20
ACS Paragon Plus Environment

Page 21 of 46
Journal of Agricultural and Food Chemistry
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
low compared with that of the parent compounds. Ferulic acid 4’ꢀOꢀsulfate shows an
antioxidant activity about elevenꢀfold lower than that of ferulic acid in the ABTS assay, and
about eighteenꢀfold lower in the FRAP assay. Caffeic acid monosulfate (a mixture of
caffeic acid 4’ꢀOꢀsulfate and caffeic acid 3’ꢀOꢀsulfate in the ratio 2.75: 1) displayed an
antioxidant efficiency about fourꢀfold lower in the ABTS assay and approximately
seventeenꢀfold lower than that of the parent caffeic acid in the FRAP assay.
Overall, these results underline the role of the reducing hydroxyl functions in the
antioxidant activity of phenolic acids and their metabolites.
Among all the metabolites tested, the acyl glucuronide of ferulic acid and the phenyl 3’ꢀ
Oꢀglucuronide of caffeic acid retained a strong antioxidant activity comparable or about a
half in respect to that of their parent compounds. This could be explained by the fact that
all these derivatives retained the 4’ꢀhydroxyl function on the aromatic ring, which is
determinant for the antioxidant activity 44,45. Noteworthy, for most of the phenolic acids
tested, the lowest effective concentrations used in this study to evaluate their antioxidant
activity are quite close to the physiological reachable concentrations 8,10,12,28. The order of
reactivity for gallic acid, caffeic acid, ferulic acid and trolox (the waterꢀsoluble analogue of
vitamin E) obtained with both FRAP and ABTS assays is in agreement with the results of
previous studies 45,48,49, however the concentrations used in these studies were very high.
Summarizing, our results demonstrate that, although ferulic and caffeic acids are
extensively metabolized upon absorption, some of these metabolites retain a strong
antioxidant activity and might still exert a significant antioxidant action in vivo. These
results are particularly remarkable for human health, due to the fact that both ferulic and
caffeic acids are the most representative phenolic acids in human diet and, upon
absorption, they circulate in human plasma almost exclusively as conjugated forms
(glucuronates and sulfates), while only traces, if any, of the free, unbound forms were
detected8ꢀ10, 12, 17ꢀ24. Therefore, the strong antioxidant activity exhibited by some of these
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 46
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
metabolites might account for the increase in plasma antioxidant potential measured upon
ingestion of phenolic acidsꢀrich beverages and food, reported by many authors1, 50ꢀ53
.
Moreover, it should be taken into consideration that the simultaneous presence of different
metabolites might produce additional synergistic effects in vivo. Further, an accumulation
of phenolic acids and their metabolites might occur in many tissues (lung, liver, heart) as
described for 3ꢀpalmitoylcatechin, epigallocatechin gallate and resveratrol 54ꢀ56
.
Whereas the occurrence of phenyl glucuronides and sulfate derivatives of caffeic and
ferulic acids in human plasma and urine has been widely reported 8ꢀ10,12,17ꢀ24, little or no
attention has been paid on acyl glucuronides of phenolic compounds. The acyl
glucuronide of ipriflavone, an isoflavon derived from genistein, has been identified in dog,
rat and human liver microsomes 37. In this study, in addition to the chemical synthesis of
the acyl glucuronide of ferulic acid (FAG), we firstly reported evidences demonstrating the
biosynthesis of FAG by mouse liver microsomes, in addition to the ferulic acid phenyl 4’ꢀOꢀ
glucuronide. The ability of liver tissue to synthesize FAG and the strong antioxidant activity
exhibited by FAG, much higher than that of the respective ferulic acid phenyl 4’ꢀOꢀ
glucuronide, might suggest a role of this metabolite in contributing to the plasma
antioxidant potential. Although acyl glucuronides had been known as mammalian
metabolites for half a century 57, 58, with the development of LCꢀMS it became evident that
many carboxylic acidꢀcontaining compounds were metabolized extensively to their acyl
glucuronides 25. Acyl glucuronides react with sulfhydryl and hydroxyl groups and can be
hydrolyzed back to the aglycone under physiological conditions 25. They undergo covalent
binding to plasma proteins 59, can react with glutathione and transacylate cellular
macromolecules 27. Further, acyl glucuronides undergo rearrangement due to
intramolecular acyl migration from –OH to the adjacent –OH, generating different positional
isomers. The potential rearrangement of glucuronides from the biosynthetic Cꢀ1 isomers to
other positional isomers is important, since only 1ꢀOꢀsubstituted acyl glucuronides are
22
ACS Paragon Plus Environment

Page 23 of 46
Journal of Agricultural and Food Chemistry
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
substrates of βꢀglucuronidase, which is commonly used to identify such conjugates 40 and
must be taken into account when measuring the concentrations of phenolic acids present
in biological fluids as glucuronic acid esters 27. Acyl group migration can occur in plasma,
bile and urine 27, therefore the concentrations of glucuronic acid conjugates of carboxylic
acid might be underestimated if βꢀglucuronidase hydrolysis is used to liberate the
aglycones in sample containing the rearranged isomers, and this will be particularly
important in bioavailability studies 27.
Our results demonstrate the biological relevance of the acyl glucuronide of ferulic acid and
its strong antioxidant activity. Additional studies will be necessary to evaluate the in vivo
role of acyl glucuronides of phenolic acids.
ABBREVIATIONS USED: FAG: ferulic acid acyl glucuronode; FAGP: protected form of
ferulic acid acyl glucuronide; FFG, ferulic acid 4’ꢀOꢀβꢀDꢀglucuronide; UDPꢀGA: uridineꢀ5’ꢀ
diphosphoglucuronic acid trisodium salt; ABTS: 2,2’ꢀazinoꢀbis(3ꢀethylbenzothiazolineꢀ6ꢀ
sulfonic acid) diammonium salt; TPTZ: 2,4,6ꢀTriꢀ(2ꢀpyridyl)ꢀSꢀtriazine; EDTA,
ethylenediamineꢀtetraacetic acid; FRAP: ferric reducing antioxidant activity.
SUPPORTING INFORMATION AVAILABLE:
Figure A Nardini: chemical structures of the most representative phenolic acids.
This material is available free of charge via the Internet at http://pubs.acs.org.
REFERENCES
(1) Pulido, R; HernandezꢀGarcia, M.; SauraꢀCalixto, F. Contribution of beverages to the
intake of lipophilic and hydrophilic antioxidants in the Spanish diet. Eur. J. Clin. Nutr.
2003, 57, 1275ꢀ1282.
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 46
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
(2) Scalbert, A.; Williamson, G. Dietary intake and bioavailability of polyphenols. J. Nutr.
2000, 130, 2073Sꢀ2085S.
(3) Block, G.; Patterson, B.; Subar, A. Fruit, vegetables and cancer prevention: a review of
the epidemiological evidence. Nutr. Cancer 1992, 18, 1ꢀ30.
(4) Fuhrman, B.; Lavy, M.; Aviram, M. Consumption of red wine with meals reduces the
susceptibility of human plasma and lowꢀdensity lipoprotein to lipid peroxidation. Am. J.
Clin. Nutr. 1995, 61, 549ꢀ554.
(5) Renaud, S.; de Lorgeril, M. Wine, alcohol, platelets and the French paradox for
coronary heart disease. Lancet 1992, 339, 1523ꢀ1526.
(6) Clifford, M.N. Chlorogenic acids and other cinnamates – nature, occurrence and dietary
burden. J. Sci. Food Agric. 1999, 79, 362ꢀ372.
(7) Herrmann, K. Occurrence and content of hydroxycinnamic and hydroxybenzoic acid
compounds in foods. Crit. Rev. Food Sci. Nutr. 1989, 28, 315ꢀ347.
(8) Nardini, M.; Natella, F.; Scaccini, C.; Ghiselli, A. Phenolic acids from beer are absorbed
and extensively metabolized in humans. J. Nutr. Biochem. 2006, 17, 14ꢀ22.
(9) Nardini, M.; Forte, M.; Vrhovsek, U.; Mattivi, F.; Viola, R.; Scaccini, C. White wine
phenolics are absorbed and extensively metabolized in humans. J. Agric. Food Chem.
2009, 57, 2711ꢀ2718.
(10) Nardini, M.; Cirillo, E.; Natella, F.; Scaccini, C. Absorption of phenolic acids in humans
after coffee consumption. J. Agric. Food Chem. 2002, 50, 5735ꢀ5741.
(11) Scalbert, A.; Williamson G. Dietary intake and bioavailability of polyphenols. J. Nutr.
2000, 130, 2073Sꢀ2085S.
(12) Stalmach, A.; Mullen, W.; Barron, D.; Uchida, K.; Yokota, T.; Cavin, C.; Steiling, H.;
Williamson, G.; Crozier, A. Metabolite profiling of hydroxycinnamate derivatives in
plasma and urine after the ingestion of coffee by humans: identification of biomarkers
of coffee consumption. Drug. Metab. Dispos. 2009, 37, 1749ꢀ1758.
24
ACS Paragon Plus Environment

Page 25 of 46
Journal of Agricultural and Food Chemistry
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
(13) Manach, C.; Scalbert, A.; Morand, C.; Remesy, C.; Jiemenez, L. Polyphenols: food
sources and bioavailability. Am. J. Clin. Nutr. 2004, 79, 727ꢀ747.
(14) Rechner, A. R.; Kuhnle, G.; Bremner, P.; Hubbard, G.P.; Moore, K.P.; RiceꢀEvans,
C.A. The metabolic fate of dietary polyphenols in humans. Free Rad. Biol. Med. 2002,
33, 220ꢀ235.
(15) Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C. Bioavailability and
bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am. J.
Clin. Nutr. 2005, 81, 230Sꢀ242S.
(16) Jimenez, J.P.; Hubert, J.; Hooper, L.; Cassidy, A.; Manach, C.; Williamson, G.;
Scalbert, A. Urinary metabolites as biomarkers of polyphenol intake in humans: a
systemic review. Am. J. Cln. Nutr. 2010, 92, 801ꢀ809.
(17) Rondini, L.; PeyratꢀMaillard, MꢀN.; MarssetꢀBaglieri, A.; Berset, C. Sulfated ferulic acid
is the main in vivo metabolite found after shortꢀterm ingestion of free ferulic acid in
rats. J. Agric. Food Chem. 2002, 50, 3037ꢀ3041.
(18) Azuma, K.; Ippoushi, K.; Nakayama, M.; Ito, H.; Higashio, H.; Terao, J. Absorption of
chlorogenic acid and caffeic acid in rats after oral administration. J. Agric. Food
Chem. 2000, 48, 5496ꢀ5500.
(19) Wittemer, S.M.; Ploch, M.; Windeck, T.; Muller, S.C.; Drewelow, B.; Derendorf, H.;
Veit, M. Bioavailability and pharmacokinetics of caffeoylquinic acids and flavonoids
after oral administration of artichoke leaf extracts in humans. Phytomedicine 2005, 12,
28ꢀ38.
(20) Baba, S.; Osakabe, N.; Natsume, M.; Terao, J. Orally administered rosmarinic acid is
present as the conjugated and/or methylated forms in plasma, and is degraded and
metabolized to conjugated forms of caffeic acid, ferulic acid, and mꢀcoumaric acid. Life
Sci. 2004, 75, 165ꢀ178.
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 46
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
(21) Choudhury, R.; Srai, S.K.; Debnam, E.; RiceꢀEvans, C.A. Urinary excretion of
hydroxycinnamates and flavonoids after oral and intravenous administration. Free
Rad. Biol. Chem. 1999, 27, 278ꢀ286.
(22) Omar, M.H.; Mullen, W.; Stalmach, A.; Auger, C.; Rouanet, J.M.; Teissedre, P.L.;
Caldwell, S.T.; Hartley, R.C.; Crozier, A. Absorption, disposition, metabolism and
excretion of [3ꢀ14C]caffeic acid in rats. J. Agric. Food Chem. 2012, 60, 5205ꢀ5214.
(23) Fumeaux, R.; MenozziꢀSmarrito, C.; Stalmach, A.; Munari, C.; Kraehenbuehl, K.;
Steiling, H.; Crozier, A.; Williamson, G.; Barron, D. First synthesis, characterization,
and evidence for the presence of hydroxycinnamic acid sulfate and glucuronide
conjugates in human biological fluids as results of coffee consumption. Org. Biomol.
Chem. 2010, 8, 5199ꢀ5211.
(24) Rondini, L.; PeyratꢀMaillard, MN; MarssetꢀBaglieri, A.; Berset ,C. Sulfated ferulic acid
is the main in vivo metabolite found after shortꢀterm ingestion of free ferulic acid in
rats. J. Agric. Food Chem. 2002, 50, 3037ꢀ41.
(25) SpahnꢀLangguth, H. and Benet, L.Z. Acyl glucuronides revisited: is the
glucuronidation process a toxification as well as detoxification mechanism? Drug
Metab. Reviews 1992, 24, 5ꢀ48.
(26) Stachulski, A.V.; Harding, J.R.; Lindon, J.C.; Maggs, J.L.; Park, B.K., Wilson, J.D.
Acyl glucuronides: biological activity, chemical reactivity and chemical synthesis. J.
Med. Chem. 2006, 49, 6931ꢀ6945.
(27) Faed, E.M. Properties of acyl glucuronides: implications for studies of the
pharmacokinetics and metabolism of acidic drugs. Drug Metab. Rev. 1984, 15, 1213ꢀ
1249.
(28) Zhao, Z.; Egashira, Y.; Sanada, H. Ferulic acid sugars are recovered in rat plasma
and urine mainly as the sulfoglucuronide of ferulic acid. J. Nutr. 2003, 133, 1355ꢀ1361.
26
ACS Paragon Plus Environment

Page 27 of 46
Journal of Agricultural and Food Chemistry
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
(29) Manach, C.; Morand, C.; Crespy, V.; Demigné, C.; Texier, O.; Régérat, F.; Rémésy,
C. Quercetin is recovered in human plasma as conjugated derivatives which retain
antioxidant activity. FEBS Lett. 1998, 426, 331ꢀ336.
(30) Ohta, T.; Nakano, T.; Egashira, Y.; Sanada, H. Antioxidant activity of ferulic acid betaꢀ
glucuronide in the LDL oxidation system. Biosci. Biotechnol. Biochem. 1997, 61, 1942ꢀ
1943.
(31) Todd, J.S.; Zimmerman, R.C.; Crews, P. and Alberte, R.S. The antifouling activity of
natural and synthetic phenolic acid sulphate esters. Phytochemistry, 1993, 34, 401ꢀ
404.
(32) Theodoridis, G.; Gika, H;. Franceschi, P.; Caputi, L.; Arapitsas, P.; Scholz, M.;
Masuero, D; Wehrens, R; Vrhovsek, U; Mattivi, F. LCꢀMS based global metabolite
profiling of grapes: solvent extraction protocol optimisation. Metabolomics, 2012, 8,
175ꢀ185.
(33) Fialova, P.; Weignerova, L.; Rauvolfova, J.; Prikrylova, V.; Pisvejcova, A.; Ettrich, R;
Kuzma, M.; Sedmera, P.; Kren, V. Hydrolytic and transglycosylation reactions of Nꢀ
acyl modified substrates catalysed by βꢀNꢀacetylhexosaminidases. Tetrahedron 2010,
60, 693ꢀ701.
(34) Gauthier, C.; Legault, J.; Rondeau, S.; Pichette, A. Synthesis of betulinic acid acyl
glucuronide for application in anticancer prodrug monotherapy. Tetrahedron Lett.
2009, 50, 988ꢀ991.
(35) van der Hoeven, T. and Coon, M.J. Preparation and properties of partially purified
cytochrome Pꢀ450 and reduced nicotinamide adenine dinucleotide phosphateꢀ
cytochrome Pꢀ450 reductase from rabbit liver microsomes. J. Biol. Chem. 1974, 249,
6302ꢀ6310.
(36) Lowry, O.H.; Rasebrough, N.J.; Fare, A.L.; Randall, R.J. Protein measurements with
the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265ꢀ271.
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 46
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
(37) Jemnitz, K.; Levai, F.; Monostory, K.; Szatmari, I.; Vereczkey, L. The in vitro
biosynthesis and stability measurement with acylꢀglucuronide isoformes of the main
metabolite ipriflavone. Eur. J. Drug Metab. Pharmacokin. 2000, 25, 153ꢀ160.
(38) Benzie, I.F.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure
of “antioxidant power”: the FRAP assay. Anal. Biochem. 1996, 239, 70ꢀ76.
(39) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; RiceꢀEvans, C.
Antioxidant activity applying an improved ABTS radical cation decolorization assay.
Free Radical. Biol. Med. 1999, 26, 1231ꢀ1237.
(40) Sinclair, K.A. and Cardwell, J. The formation of βꢀglucuronidase resistant
glucuronides by the intramolecular rearrangement of glucuronide acid conjugates at
mild alkaline pH. Biochem. Pharmacol. 1982, 31, 953ꢀ957.
(41) Skordi, E.; Wilson, I.D.; Lindon, J.C.; Nicholson, J.K. Kinetic studies on the
intramolecular acyl migration of βꢀ1ꢀOꢀacyl glucuronides: application to the
glucuronides of (R)ꢀ and (S)ꢀ ketoprofen, (R)ꢀ and (S)ꢀ hydroxyꢀketoprofen metabolites,
and tolmetin by 1HꢀNMR spectroscopy. Xenobiotica, 2005, 35, 715ꢀ725.
(42) Cano, A.; Arnao, M.B.; Williamson, G.; GarciaꢀConesa, M.T. Superoxide scavenging
by polyphenols: effect of conjugation and dimerization. Redox Rep. 2002, 7, 379ꢀ383.
(43) RiceꢀEvans, C.; Miller, N.J.; Paganga, G. Structureꢀantioxidant activity relationships of
flavonoids and phenolic acids. Free Rad. Biol. Med. 1996, 20: 933ꢀ956.
(44) Hodnick, W.F.; Milosevljevic, E.B.; Nelson, J.H.; Pardini, R.S. Electrochemistry of
flavonoids. Biochem. Pharmacol. 1988, 37, 2606ꢀ2611.
(45) Pulido, R.; Bravo, L.; SauraꢀCalixto, F. Antioxidant activity of dietary polyphenols as
determined by a modified ferric reducing/antioxidant power assay. J. Agric. Food
Chem. 2000, 48, 3396ꢀ3402.
(46) Guy, F.; Renouf, M.; Barron, D.; Cavin, C.; Dionisi, F.; Kochhar, S.; Rezzi, S.;
Williamson, G.; Steiling, H. Quantitative analysis of plasma caffeic and ferulic acid
28
ACS Paragon Plus Environment

Page 29 of 46
Journal of Agricultural and Food Chemistry
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
equivalents by liquid chromatography tandem mass spectrometry. J. Chromatogr. B
2009, 877, 3965ꢀ3974.
(47) Huang D, Ou B., Prior R.L. The chemistry behind antioxidant capacity assays. J.
Agric. Food Chem. 2005, 53, 1841ꢀ1856.
(48) Nenadis, N.; Lazaridou, O.; Tsimidou, M.Z. Use of reference compounds in
antioxidant activity assessment J. Agric. Food Chem. 2007, 55, 5452ꢀ5460.
(49) Villano, D.; FernandezꢀPachon, M.S.; Troncoso, A.M.; GarciaꢀParrilla, M.C.
Comparison of antioxidant activity of wine phenolic compounds and metabolites in
vitro. Analytica Chimica Acta 2005, 538, 391ꢀ398.
(50) Ghiselli, A., Natella, F., Guidi, A., Montanari, L., Fantozzi, P., Scaccini, C. Beer
increases plasma antioxidant capacity in humans. J. Nutr. Biochem. 2000, 11, 76ꢀ80.
(51) Natella, F., Nardini, M., Giannetti, I., Dattilo, C., Scaccini, C. Coffee drinking
influences plasma antioxidant capacity in humans. J. Agric. Food Chem. 2002, 50,
6211ꢀ6216.
(52) Serafini, M., Bugianesi, R., Salucci, M., Azzini, E., Raguzzini, A., Maiani, G. Effect of
acute ingestion of fresh and stored lettuce (Lactuca sativa) on plasma total antioxidant
capacity and antioxidant levels in human subjects. Br. J. Nutr. 2002, 88, 615ꢀ623.
(53) Simonetti, P., Gardana, C., Pietta, P. Plasma levels of caffeic acid and antioxidant
status after red wine intake. J. Agric. Food Chem. 2001, 49, 5964ꢀ5968.
(54) Das, N.P.; Griffiths, L.A. Studies of flavonoid metabolism. Metabolism of (+)ꢀ[14C]
catechin in rat and guinea pig. Biochem. J. 1969, 115, 831ꢀ836.
(55) Suganuma, M.; Okabe, S.; Oniyama, M.; Tada, Y.; Ito, H.; Fujiki, H. Wide distribution
of [3H](ꢀ)epigallocatechin gallate, a cancer preventive tea polyphenol, in mouse tissue.
Carcinogenesis 1998, 19, 1771ꢀ1776.
29
ACS Paragon Plus Environment