Methylated metabolites of chicoric acid ameliorate hydrogen peroxide (H2O2)?induced oxidative stress in HepG2 cells

Article abstract of DOI:10.1021/acs.jafc.0c07521

Chicoric acid (CA) can display health benefits as a dietary polyphenol. However, as CA is widely metabolized in vivo, the actual compounds responsible for its bioactivities are not entirely known. Herein, the major methylated metabolites of CA were isolated from an in vitro co-incubation system, and their structures were elucidated. The antioxidant activities of the monomethylated metabolites (M1) and dimethylated metabolites (M2) of CA were evaluated against H₂O₂-induced oxidative stress damage in HepG2 cells and compared to CA. The results indicated that both M1 and M2 had better antioxidant capacities than CA by increasing cell viability, improving mitochondrial function, and balancing cellular redox status. These compounds also prevented oxidative stress by mediating the Keap1/Nrf2 transcriptional pathway and downregulating enzyme activity. The current research indicates that the methylated metabolites of CA could potentially be the candidates that are responsible for the biological efficacies attributed to CA.

Full text of DOI:10.1021/acs.jafc.0c07521

pubs.acs.org/JAFC
Article
Methylated Metabolites of Chicoric Acid Ameliorate Hydrogen
Peroxide (H2O2)‑Induced Oxidative Stress in HepG2 Cells
Xiaowen Chang, Shan Dong, Wenliang Bai, Yan Di, Ruijuan Gu, Fuguo Liu, Beita Zhao, Yutang Wang,*
and Xuebo Liu
Cite This: J. Agric. Food Chem. 2021, 69, 2179−2189
Read Online
Metrics & More
Article Recommendations
ACCESS
ABSTRACT: Chicoric acid (CA) can display health benefits as a dietary polyphenol. However, as CA is widely metabolized in vivo,
the actual compounds responsible for its bioactivities are not entirely known. Herein, the major methylated metabolites of CA were
isolated from an in vitro co-incubation system, and their structures were elucidated. The antioxidant activities of the monomethylated
metabolites (M1) and dimethylated metabolites (M2) of CA were evaluated against H2O2-induced oxidative stress damage in
HepG2 cells and compared to CA. The results indicated that both M1 and M2 had better antioxidant capacities than CA by
increasing cell viability, improving mitochondrial function, and balancing cellular redox status. These compounds also prevented
oxidative stress by mediating the Keap1/Nrf2 transcriptional pathway and downregulating enzyme activity. The current research
indicates that the methylated metabolites of CA could potentially be the candidates that are responsible for the biological efficacies
attributed to CA.
KEYWORDS: chicoric acid, methylated metabolites, oxidative stress, antioxidant activity, Keap1/Nrf2
acid A have been found to exhibit fairly high antioxidant
capacities and could ameliorate liver lipid peroxidation in
rats.13 Evidence has been reported that 3,4′-dihydroxy-5-
methoxystilbene (R3), a methylated resveratrol derivative,
increased the hydrophobicity and the cellular uptake of R3 and
possessed better neuroprotective effects compared to resver-
atrol.14 Moreover, several polyphenol-derived metabolites were
found in low concentrations in animal brains.15,16 Studies have
demonstrated that methyl-urolithin B, a metabolite of
ellagitannins produced by gut microbiota, is a compound
that can be potentially absorbed by the brain, which could
conduce the anti-Alzheimer’s disease (AD) effects of
pomegranates.17 Thus, we hypothesized that the methylated
metabolites of CA may exhibit critical roles in demonstrating
antioxidative and other biological activities.
Previous studies have shown that the imbalance between the
generation and elimination of reactive oxygen species (ROS)
and/or the impairment of the antioxidant defense system
induces oxidative stress.18 Oxidative stress manifests harmful
effects on biomolecules and eventually causes many
pathophysiological damages, such as cancer, cardiovascular
disease, and neurodegenerative diseases.19−21 The pharmaco-
logical activity of CA was achieved by alleviating oxidative
stress through the activation of the antioxidant defense system.
INTRODUCTION
■
Chicoric acid (CA), also known as dicaffeoyltartaric acid, is the
most representative phenolic compound found in Echinacea
purpurea.1 It has also been identified in many plants, including
dandelions, chicories, iceberg lettuce, and basil.2−4 As a
naturally occurring compound, its health-promoting effects
are largely due to its biological and pharmacological properties,
including antioxidant, antiobesity, immunostimulatory, anti-
viral, and anti-inflammatory properties.5−7 Hence, CA has been
widely used as a food antioxidant and immune system
enhancer.8
However, due to gastrointestinal hydrolysis and extensive
modifications of CA in phase II metabolism by the liver, as well
as the interactions with colonic microbiota, polyphenols show
poor bioavailability, with polyphenols often being partially
converted into various metabolites after entering the human
body.9,10 Therefore, the compounds that are actually
responsible for the biological activities of polyphenols in the
body have not been fully understood yet. Our recent research
elucidated that the absorbed CA was subjected to metabolism
in the liver, kidney, and small intestine, forming methylated,
glucuronidated, acetylated, and sulfated metabolites.11 In
particular, the methylated metabolites of CA were detected
in the bloodstream in much higher concentrations than CA.11
These methylated metabolites could be responsible for the
biological activity associated with CA.
Received: November 28, 2020
Revised: January 16, 2021
Accepted: January 18, 2021
Published: February 12, 2021
Indeed, accumulating evidence has indicated that the
methylated derivatives of polyphenols have better biological
activities compared to their parent compounds and have a
positive influence on the bioactivities and bioavailabilities of
the polyphenols.12 The methylated metabolites of salvianolic
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189
© 2021 American Chemical Society
2179

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 1. Diagrammatic illustration for preparation of the methylated metabolites of CA from the rat liver homogenate.
Signaling Technology (Shanghai, China). All other reagents were of
analytical grade.
Recently, it was revealed that CA could improve cell
inflammation via balancing redox homeostasis and inhibiting
the release of inflammatory factors in BV-2 microglial cells.22
Our previous research found that CA reduced cell viability and
induced apoptosis in 3T3-L1 preadipocytes through the PI3K/
Akt and MAPK signaling pathways.7 In addition, in vitro cell
studies are used to determine the mechanisms of action of CA
in vivo.23 Therefore, we aimed to determine whether the
methylated metabolites of CA contribute to the bioactivity of
CA in vivo, and we explored the molecular mechanisms of their
antioxidant effects in HepG2 cells.
In the present study, the major methylated metabolites of
CA were isolated from an in vitro co-incubation system by
preparative high-performance liquid chromatography (HPLC)
and identified their structures by liquid chromatography
quadrupole time-of-flight mass spectrometry (LC-QTOF-
MS/MS). The antioxidant activities of the methylated
metabolites of CA were evaluated after H2O2-induced
oxidative stress in HepG2 cells by determining their effects
on cell viability, mitochondrial function, redox status, and the
phosphorylation levels of MAPK/p38, MAPK/JNK, and NF-
κB/p65. Furthermore, we also explored whether the
methylated metabolites of CA could play an active role in
the regulation of the Keap1/Nrf2 antioxidant defense signaling
pathway. The results may provide a valuable reference for the
potential preventative effects of CA against oxidative stress-
related diseases.
Treatment of Animals. Rats (male, aged 6 weeks) were acquired
from Xi’an Jiaotong University (Xi’an, Shaanxi, China). All rats were
fed with standard feeds (AIN-93M) without natural polyphenols in
standard animal rooms (temperature: 25 2 °C; humidity: 50 5%;
12 h light/dark cycle) and were adaptively fed for a week before the
test. Afterward, the rats were sacrificed, and their livers were quickly
removed and washed with ice-cold saline solution. Finally, their livers
were added with a 10 mM sodium phosphate buffer (pH 7.4)
containing 10 mM MgCl2 at a rate of 1:4 (v/v), and the homogenate
was prepared by a high-speed homogenizer at 0−4 °C. All rats (n =
10) passed the quality inspection of experimental animals in Shaanxi
province, certificate no. 61001700003429. The animal protocol was
approved by the animal ethics committee of Xi’an Jiaotong University.
Methylation of CA in the Liver Homogenate. The method of
in vitro co-incubation was used as previously described.24 The
diagrammatic illustration for the methylation of CA is displayed in
Figure 1. Briefly, a solution of CA (400 mg) in methanol was added to
100 mL of rat liver homogenate for incubation. After adding SAM (60
mg), the reaction solution was incubated at 37 °C for 3 h, and 100
μM of ice-cold methanol containing 1% (w/v) ascorbic acid was
added to quench the reaction. Then, the reaction mixture was
centrifuged (8000 rpm, 4 °C) for 10 min to remove the solid particles.
The methanol in the supernatant was removed using a rotary
evaporator at 45 °C, and the resulting residue was reconstituted in
deionized water (pH 2.4). The metabolites were extracted three times
with ethyl acetate, and the ethyl acetate layer was evaporated to
dryness to afford a crude residue containing the mixed metabolites of
CA. Finally, the mixed metabolites were dissolved in 50% (v/v)
solution of methanol in water and injected into preparative HPLC.
Isolation of the Methylated Metabolites of CA. Isolation of
the methylated metabolites was performed using a preparative HPLC
system (Shimadzu LC-8A) equipped with an ODS column (250 mm
× 20 mm i.d., Shimadzu), a SPD-M10AVP detector (Shimadzu,
Tokyo, Japan) and an autosampler combined with an automated
fraction collector (Waters 2767). Mobile phase A consisted of 0.2%
trifluoroacetic acid in water, and mobile phase B was acetonitrile. The
run conditions were as follows: from 0 to 10 min, the concentration of
B was 10% isocratic, followed by a gradient elution ramp B to 50%
from 10 to 15 min, after which B was 50% isocratic from 15 to 25
min, recovering to 10% to equilibrate the column for the analysis of
the next sample. The elution flow rate was set to 1.0 mL/min, and the
fraction corresponding to each main peak was collected while eluting
from the column. The methanol and ethyl acetate in the collected
eluents were removed using a rotary evaporator at 45 °C, and the
resulting residues were reconstituted in 10 mL of deionized water and
shaken at room temperature for 60 s. Finally, the solution was
MATERIALS AND METHODS
■
Reagents and Antibodies. CA (purity 98%) was acquired from
Chengdu Pufei De Biotech Co., Ltd. (Chengdu, Sichuan, China). S-
Adenosyl-L-methionine (SAM), 3-(4,5-dimethylthiazol-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT), and 2′,7′-dichloro-
fluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). The JC-1 dye was obtained from the Beyotime
Institute of Biotechnology (Haimen, Jiangsu, China). HPLC-grade
acetonitrile and methanol were purchased from Fisher Scientific
(Waltham, MA, USA). Deionized water was prepared by the Milli-Q
water purification system (Millipore, Bedford, MA, USA). Antibodies
against Nrf2 (SC-722), Lamin B (SC-6217), NQO-1 (SC-16464),
HO-1 (SC-1796), β-actin (SC-47778), NF-κB/p65 (8242), NF-κB/
p-p65 (8242), MAPK/p38 (9212), MAPK/JNK (9252), MAPK/p-
JNK (9251), and MAPK/p-p38 (9211) were acquired from Cell
2180
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 2. Preparation and identification of the methylated metabolites of CA. (A) HPLC chromatograms of the rat liver homogenate group, before
the incubation group, and after the incubation group. (B) MS2 spectra of methylated metabolites of CA. (C) Structural characterization of main
methylated metabolites of CA, proposed fragmentation pathway of CA in negative ion mode with m/z 473.0741 as the precursor ion and
conversion mechanism into M1 and M2.
2181
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
lyophilized in the vacuum freeze dryer for 48 h to yield the pure
compounds.
Measurement of Intracellular ROS. The fluorescent dye
DCFH-DA assay kit (Beyotime Institute of Biotechnology) was
used to measure intracellular ROS. HepG2 cells were seeded into 6-
well plates (1 × 106 cells/well) for 24 h at 37 °C and then treated
with CA, M1, and M2 (25 μM) for 24 h followed by H2O2 (200 μM)
for 24 h. The treated cells were stained with 10 μM DCFH-DA for 30
min at 37 °C in the dark. After the treated cells were washed twice
with PBS solution, intracellular ROS were evaluated by acquiring
images with a Leica inverted fluorescence microscope and measuring
the fluorescence using a multimode microplate reader with excitation
at 485 nm and emission at 538 nm.
Measurement of Antioxidant Enzymes Activities. To
measure the glutathione peroxidase (GPX) activity, glutathione
reductase (GR) activity, catalase (CAT) activity, and MDA levels,
HepG2 cells were seeded into 6-well plates (1 × 106 cells/well) for 24
h at 37 °C and incubated with CA, M1, and M2 (25 μM) for 24 h
followed by H2O2 (200 μM) for 24 h. After the treated cells were
washed twice with PBS solution, the cells were lysed in lysis buffer
(Thermo). A BCA protein assay kit (Thermo) was used to measure
the intracellular protein content, and the activities of GPX, GR, and
CAT were determined according to the kit instructions (Jiancheng
Bioengineering Institute, Nanjing, China). MDA levels were
determined using a Lipid Peroxidation MDA Assay kit (Beyotime
Institute of Biotechnology, Nanjing, China).
Western Blot Analysis. HepG2 cells were seeded into 6-well
plates (1 × 106 cells/well) for 24 h at 37 °C, incubated with CA, M1,
and M2 (25 μM) for 24 h, and then incubated with H2O2 (200 μM)
for 24 h. The intervened cells were collected and treated with cell
lysate buffer (Beyotime Institute of Biotechnology, Jiangsu, China),
and the cytoplasm and nucleus were separated. To measure the total
protein concentration, the proteins were separated by sodium dodecyl
sulphate/polyacrylamide gel electrophoresis and transferred to an
activated polyvinylidene fluoride (PVDF) membrane. After the
transfer was completed, the PVDF membrane was placed into a
suspension of 5% skimmed milk powder in PBST and blocked for 1.5
h at room temperature. The membranes were incubated overnight at
4 °C. After incubation with a secondary antibody for 1.5 h at room
temperature, chemiluminescence was added, and the images of the
bands were collected and analyzed by the Quantity One 4.6.2 software
(Bio-Rad Co., Hercules, CA, USA).
LC-QTOF-MS/MS Identification of the Methylated Metabo-
lites of CA. LC-QTOF-MS/MS was used to qualitatively analyze the
methylated metabolites of CA to obtain accurate mass and
fragmentation patterns of the analytes. Mass spectrometry analysis
was performed on a Triple TOF 5600+ System (AB SCIEX, USA).
The experiment was performed in negative-ion mode. The parameters
of the electrospray mass spectrometer were optimized, such that the
atomizing gas (GS1) pressure was 50 psi, the curtain gas (CUR)
pressure was 10 psi, the heating gas (GS2) pressure was 50 psi, and
the heater temperature (TEM) was 500 °C. The full scan method was
used to obtain the relative molecular mass of the parent compound
and its metabolites. Daughter scans of the [M − H]− ions from CA
and its metabolites were used to obtain their second order mass
(MS2) spectra.
Cell Culture Experiments. HepG2 cells were acquired from the
Cell Resource Center at Peking Union Medical College (CRC/
PUMC). The medium used was minimum essential medium
(Hyclone) containing 10% (v/v) fetal bovine serum (Thermo), 100
IU/mL penicillin, and 100 μg/mL streptomycin, and the cells were
cultured in a humidified incubator at 37 °C (95% air/5% CO2). The
cell line was analyzed and declared free of mycoplasma contamination.
Cell Viability Assay. An MTT assay was used to evaluate cell
viability.25 The HepG2 cells had undergone the following different
treatments: (i) HepG2 cells were cultured in a 96-well plate with a
density of 2 × 104 cells/well in a humidified incubator overnight and
treated with a gradient concentration of H2O2 (0, 50, 100, 200, 300,
and 400 μM) for 24 h to induce oxidative stress response. (ii) HepG2
cells were incubated with different concentrations of CA, M1, and M2
(0, 10, 25, 50, and 100 μM) for 24 h. (iii) HepG2 cells were
pretreated with CA, M1, and M2 (25 μM) for 24 h followed by H2O2
(200 μM) for 24 h. After incubation, 100 μL of MMT (0.5 mg/mL)
was added to each well; the plate was incubated for 4 h in the
incubator; the medium was discarded, and 100 μL of DMSO was
added to each well. After the purple crystals were fully dissolved, a
multifunctional microplate reader (Bio-Rad Hercules, China) was
used to determine the optical density (OD) at 560 nm. This assay was
performed in triplicates, and the cell survival rate was calculated as the
ratio of the absorbances between the treatment group and the control
group.
Statistical Analysis. All experiments were performed at least
Analysis of Mitochondrial Membrane Potential (MMP). JC-1
is a fluorescent probe used to examine mitochondrial membrane
potential (MMP) and can also be employed to monitor the level of
early cell apoptosis.26 Briefly, HepG2 cells were inoculated in a 6-well
plate (1 × 106 cells/well), and the cells were pretreated with CA, M1,
and M2 (25 μM) for 24 h followed by H2O2 (200 μM) for 24 h. The
treated cells were then incubated with JC-1 (10 mg/mL) in an
incubator for 1 h at 37 °C. After washing with PBS, a Leica inverted
fluorescence microscope was used to acquire images of the cells.
Fluorescence emission was measured using a multimode microplate
reader (Molecular Devices Co., Sunnyvale, CA, USA) by excitation at
485 nm and emission at 585 and 538 nm. The fluorescence values
were expressed as the ratio of OD585/OD538. The fluorescence was
normalized by protein levels and expressed as a percentage of the
control group.
Apoptosis Assay and Flow Cytometry. Cell apoptosis was
measured in accordance with a previous method.27 HepG2 cells were
seeded into 6-well plates (1 × 106 cells/well) for 24 h at 37 °C and
incubated with CA, M1, and M2 (25 μM) for 24 h followed by H2O2
(200 μM) for 24 h. After digestion with trypsin without EDTA
(Beyotime Institute of Biotechnology, Nanjing, China), the cells were
collected by centrifugation at 300g for 5 min at 4 °C. The cells were
washed three times with precooled PBS solution, and 100 μL of
binding buffer was added to resuspend the cells. After 5 μL of
Annexin V-FITC and 10 μL of PI staining solution were added, the
cell suspension was mixed gently and left to reacted at room
temperature for 15 min in the dark. Then, 400 μL of binding buffer
was added, and the suspension was mixed well and placed on ice. The
samples were analyzed by flow cytometry (PARTEC flow cytometer).
three times, and the data were expressed as the mean
standard
deviation (SD). The cell apoptosis data were analyzed using the Flow
Jo software (Tree Star Inc., Ashland, Oregon). All statistical analyses
were performed using the GraphPad Prism 6.0 software package, and
the statistical significance was calculated using Duncan’s test, with p <
0.05 being considered significant.
RESULTS
■
Preparation and Identification of the Methylated
Metabolites of CA. The methylated metabolites of CA were
prepared by the in vitro co-incubation of CA and rat liver
homogenate, as illustrated in the preparation flow chart in
Figure 1. Preparative HPLC analysis showed that three
products had formed after the co-incubation, and their
retention times were 7.5, 9.3, and 11.5 min, respectively.
Based on the retention times, products were referred to as M0,
M1, and M2, where M0 had the same retention time as CA
(Figure 2A). The individual compounds were obtained from
the fractions collected over the durations of 7.5−8.75, 9.45−
10.75, and 11.75−13.0 min. These results indicated that CA
was readily methylated in the presence of SAM by rat liver
cytosolic catechol-O-methyltransferase (COMT).
LC-QTOF-MS/MS was employed to identify these
compounds. The data acquired from the full-scan MS revealed
that the molecular ion had an m/z ratio of 473.0741 (M0),
which is in agreement with the molecular weight of the control
2182
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 3. Effects of CA, M1, and M2 on the H2O2-induced cell viability. (A−C) CA, M1, and M2 (0, 10, 25, 50, and 100 μM) for 24 h. (D) H2O2
(0, 50, 100, 200, 300, and 400 μM) for 24 h. (E) HepG2 cells were pretreated with CA, M1, and M2 (25 μM) for 24 h and then treated with or
without H2O2 (200 μM) for 24 h, and cell viability was determined by using the MTT assay. Data were presented as the mean SD, n ≥ 3; *p <
#
0.05 and **p < 0.01, vs control group; p < 0.05 and ##p < 0.01, vs H2O2 group; Δp < 0.05 and ΔΔp < 0.01, vs CA + H2O2 group.
Figure 4. Effects of CA, M1, and M2 on the H2O2-induced apoptosis and mitochondrial function. (A) Annexin V-FITC/PI staining by flow
cytometry. Left: Representative image of fluorescence-activated cell sorting analyses. Right: Quantitative analysis of apoptotic HepG2 cells. (B)
Mitochondrial membrane potential by JC-1 staining under H2O2 treatment. Left: Representative image of mitochondrial membrane potential
analyses. Right: Quantitative analysis of JC-1 staining. HepG2 cells were pretreated with CA, M1, and M2 (25 μM) for 24 h and then treated with
or without H2O2 (200 μM) for 24 h. Data were presented as the mean SD, n ≥ 3; *p < 0.05 and **p < 0.01, vs control group; #p < 0.05 and ##p <
0.01, vs H2O2 group; Δp < 0.05 and ΔΔp < 0.01, vs CA + H2O2 group.
CA. Thus, M0 was the parent compound. As expected, the
molecular ion of M1 with an m/z ratio of 487.0894 was 14 Da
higher than the molecular ion (m/z 473.0741) of the parent
compound, and the molecular ion of M2 was 28 Da higher,
with a m/z ratio of 501.1051. These results suggested the
sequential methylation of CA to M1 first and then to M2,
resulting in the formation of the monomethylated product
followed by the dimethylated product of CA. To further
confirm the structures of M1 and M2, we performed a second-
order scan of the products. Under high-energy collision, the
2183
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 5. Effects of CA, M1, and M2 on the H2O2-induced the intracellular redox status. (A) Cellular oxidation status by H2DCFDA staining under
H2O2 treatment. Left: Representative image of ROS analyses. Right: Quantitative analysis of H2DCFDA staining. (B) Analysis of GPX levels in
HepG2 cells. (C) Analysis of GR levels in HepG2 cells. (D) Analysis of CAT levels in HepG2 cells. (E) MDA activity in HepG2 cells examined
with a MDA assay kit. HepG2 cells were pretreated with CA, M1, and M2 (25 μM) for 24 h and then treated with or without H2O2 (200 μM) for
24 h. Data were presented as the mean SD, n ≥ 3; *p < 0.05 and **p < 0.01, vs control group; #p < 0.05 and ##p < 0.01, vs H2O2 group; Δp < 0.05
and ΔΔp < 0.01, vs CA + H2O2 group.
MS/MS data revealed that all fragment ions generated by M0
were the same as generated by the parent compound. M1 and
M2 produced four identical fragment ions (m/z 193.0526,
233.0479, 307.0492, and 325.0588), which were 14 Da higher
than the fragment ions generated by the parent compound (m/
z 179.0364, 219.0315, 293.0320, and 311.0427) (Figure 2B).
These fragmentation patterns indicated that the phenolic
hydroxyl groups of CA were methylated. When single phenolic
hydroxyl groups were substituted, three possible monomethy-
lated metabolites were formed, while four possible dimethy-
lated products were formed when two phenolic hydroxyl
groups were substituted (Figure 2C).
Effects of the Methylated Metabolites of CA on the
H2O2-Induced Cell Viability and Mitochondrial Dysfunc-
tion in HepG2 Cells. To evaluate the antioxidant activities of
the methylated metabolites of CA, the cell viability and
mitochondrial function of CA were compared to its methylated
metabolites M1 and M2 after H2O2-induced oxidative stress in
HepG2 cells. The HepG2 cells used herein represented an in
vitro model of hepatic cells. We measured the cell viability of
HepG2 cells after treatment with the compounds with
increasing concentrations (0−100 μM). The results showed
that after a 24 h incubation, 25 μM was the highest
concentration of the compounds to demonstrate no significant
cytotoxicity (Figure 3A−C). Therefore, 25 μM was the chosen
concentration of CA, M1, and M2 used in the current study.
Then, an oxidative stress model was established by adding
H2O2 alone. We found that 200 μM H2O2 showed 40−60%
inhibition compared to the control group (Figure 3D), so 200
μM H2O2 was chosen as the optimal concentration for
subsequent experiments. Moreover, the MTT assay confirmed
that the pretreatment of cells with CA, M1, and M2 resulted in
a decrease in the inhibition of cell viability (p < 0.05)
compared to that of the H2O2-treated group. Surprisingly,
pretreatments of M1 and M2 were more sensitive to inhibiting
H2O2-induced cell death compared to the CA treatment, and
the most effective compound was M2. Compared to the
control group, CA and M1 significantly restored cell viability to
81 and 83% of the normal value, respectively, but the
treatment of M2 restored cell viability to 93%, which was
very close to the viability of normal cells (Figure 3E).
To determine the effect of M1 and M2 on the H2O2-induced
apoptosis, the Annexin V/PI flow cytometry method was
employed. The results indicated that H2O2-induced cell
apoptosis was significantly improved compared with the
control group (p < 0.01). Although CA, M1, and M2 affected
2184
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
Figure 6. Effects of CA, M1 and M2 on the protein expressions measured by western blots. (A) Phosphorylation level of MAPK/p38, MAPK/JNK,
and NF-κB/p65 in H2O2-induced HepG2 Cells. (B) Keap1/Nrf2 antioxidant defense pathway in H2O2-induced HepG2 Cells. Lamin B and β-actin
were used as loading control. HepG2 cells were pretreated with CA, M1, and M2 (25 μM) for 24 h and then treated with or without H2O2 (200
μM) for 24 h. Data were presented as the mean SD, n ≥ 3; *p < 0.05 and **p < 0.01, vs control group; #p < 0.05 and ##p < 0.01, vs H2O2 group;
Δp < 0.05 and ΔΔp < 0.01, vs CA + H2O2 group.
the rate of apoptosis compared to the H2O2-treated group, the
apoptosis levels of the M2 treatment group were still
considerably lower than those of the CA-pretreated cells (p
< 0.01) (Figure 4A). The early symbolic indication of cell
apoptosis was the loss of MMP, which is a marker of
mitochondrial dysfunction.28 JC-1 staining results indicated
that H2O2 elicited the loss of MMP, which was reversed by the
pretreatment of M2 (Figure 4B). Taken together, M1 and M2
pretreatment was found to significantly increase cell viability,
reduction in H2O2-induced cell death, and mitochondrial
dysfunction compared to CA.
Moreover, as one of the biomarkers that indicates the oxidized
status within the cell, the CAT levels showed a similar result in
HepG2 cells (Figure 5D). H2O2 was found to stimulate the
production of MDA in cells, and the MDA levels increased to
1.63 times that of the control group (p < 0.01), while the CA,
M1, and M2 treatment led to a significant downregulation of
MDA (p < 0.01). Interestingly, M2 significantly reduced the
levels of MDA in cells compared to the CA treatment group (p
< 0.01) (Figure 5E). These results suggested that the
methylated metabolites of CA play an important role in
regulating the intracellular redox state.
Effects of the Methylated Metabolites of CA on the
H2O2-Induced Intracellular Redox Status. To determine
the effects of M1 and M2 on the H2O2-induced cellular redox
status, we measured the levels of intracellular ROS. As shown
in Figure 5A, H2O2 treatment significantly stimulated the
generation of ROS compared to the control group. However,
pretreatments with CA, M1, and M2 significantly decreased
the generation of ROS to 138.8, 117.2, and 109.2%,
respectively, compared to the H2O2-treated group (p <
0.01). Obviously, M2 significantly suppressed the generation
of ROS (p < 0.01), which also indicated that M1 and M2
protected the integrity of the cells from the H2O2-induced
injury.
The activities of antioxidative enzymes reflect the cell redox
state.29 GPX plays a pivotal role in protecting cells from free
radical damage, and GR is responsible for the regeneration of
oxidized glutathione.30 As expected, the exposure to H2O2 for
24 h led to a significant reduction in the activities of GPX and
GR by 26.6 and 48.5%, respectively, compared to the control
group (p < 0.01). However, M1 and M2 pretreatment
significantly inhibited the decrease in the GPX and GR
activities (p < 0.01). Although CA increased the activities of
GPX and GR, there was no significant difference compared to
the H2O2 treatment group (p < 0.05) (Figure 5B,C).
Effects of the Methylated Metabolites of CA on the
Phosphorylation Levels of MAPK/p38, MAPK/JNK, and
NF-κB/p65 in H2O2-Induced HepG2 Cells. MAPK and NF-
κB are heavily involved in cellular responses such as the
regulation of cell apoptosis, inflammation, and oxidative stress
responses.31 Previous studies demonstrated that dietary CA
downregulated phosphorylation levels of MAPK/p38, MAPK/
JNK, and NF-κB/p65.7,23 In this study, western blot analysis
was used to assess the effects of the methylated metabolites of
CA on the phosphorylation states of the three kinases. As
shown in Figure 6A, H2O2 increased the expressions of
phospho-p38 MAPK and phospho-JNK MAPK in the HepG2
cells. Pretreatment of the cells with CA, M1, and M2 led to
pronounced attenuation of their phosphorylation levels (p <
0.01). As expected, it was found that M2 had a greater
inhibition effect on the phosphorylation levels of MAPK/p38
and MAPK/JNK compared to CA (p < 0.05). Phosphorylated
p65 participates in NF-κB activation. After H2O2 treatment,
the phosphorylation of p65 was significantly increased in the
HepG2 cells compared to the control group (p < 0.01).
Pretreatment with CA, M1, and M2 led to a reduction in the
H2O2-induced phosphorylation of p65 in the HepG2 cells (p <
0.05). As expected, M1 and M2 stimulation significantly
2185
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
increased p65 phosphorylation compared to the CA group (p
< 0.05) (Figure 6A).
attention has been directed toward studying methylated
metabolites of polyphenol, which may beneficially influence
the neuronal activity.40 It has been found that 3-methyl-4-
glucuronate-resveratrol could improve the LPS-induced
inflammation status in macrophages via inhibiting the
production of IL-6 and NO, and it was also found to prevent
neuronal death by increasing the redox activity and attenuating
the level of ROS in SH-SY5Y cells.41 Recent reports have
shown that 3′-O-methyl-epicatechin-5-O-β-glucuronide, one of
the proanthocyanidin metabolites, could reduce pathological
changes in the AD brain and restore neuronal functions related
to learning and memory.42 According to the above
information, polyphenol metabolites demonstrated better in
vitro neuroprotective and anti-inflammatory properties.
Regulation Effects of the Methylated Metabolites of
CA on the Keap1/Nrf2 Antioxidant Defense Pathway in
H2O2-Induced HepG2 Cells. As nuclear factor-erythroid 2-
related factor 2 (Nrf2) is widely accepted to be a key
transcription factor for the regulation of the antioxidant
defense system,32 we questioned whether the antioxidant
function of the methylated metabolites of CA could be
mediated through the Nrf2 signaling pathway. Therefore, the
translocation of Nrf2 to the nucleus was measured to
determine the effects of M1 and M2 on the activation of the
Nrf2 pathway. The results in Figure 6B illustrated that M1 and
M2 treatments effectively promoted the accumulation of Nrf2
in the nucleus. Moreover, the results revealed that M1 and M2
promoted the expression of downstream enzymes [NAD(P)H:
quinone oxidoreductase 1 (NQO1) and hemeoxygenase 1
(HO-1)]. These data confirmed that the primary protective
mechanism of M1 and M2 was through Nrf2 activation.
CA is a natural nutritive fortifier and is widely used in
nutritional products due to its excellent antioxidant properties.
Our previous study reported that CA exerted a stronger free
radical scavenging capability compared to its hydrolysis
metabolites, caffeic acid, and caftaric acid, by inhibiting
DPPH•, OH, and ABTS•+ free radicals.43 Pharmacokinetic
•
DISCUSSION
studies revealed that CA was distributed throughout various
tissues and organs, and the concentration of CA in the liver
was the highest compared to other tissues. The ability of CA to
cross the blood brain barrier (BBB) has also been observed.44
In the present work, the methylated metabolites of CA not
only showed stronger antioxidant activities but also signifi-
cantly increased cell viability, lowered the apoptosis rate of
cells, and restored MMP in H2O2-induced cells. M1 and M2
could also ameliorate H2O2-induced cell oxidative stress by
suppressing the generation of ROS and enhancing the activities
of antioxidant enzymes.
■
In this study, we found that the different monomethylated and
dimethylated products of CA were formed in the liver cytosolic
homogenate, and their structures were identified by LC-
QTOF-MS/MS. These results demonstrated that M1 and M2
had beneficial effects on H2O2-induced oxidative stress
compared to CA by increasing cell viability, alleviating the
production of ROS, and suppressing the phosphorylation of
MAPK/p38, MAPK/JNK, and NF-κB/p65. Furthermore, the
results also showed that M1 and M2 exerted potent
antioxidative potential through the activation of the Nrf2
signaling pathway.
Several hypotheses may explain the observed results. First,
these results may be associated with the increased lipophilicity
of the methoxy polyphenols and their increased uptake across
the cell membrane. It has been found that lipophilic tea
polyphenols display significant antioxidant effects on the
DEN/PB-induced liver damage and hepatocarcinogenesis
compared to the water-soluble green tea polyphenols, which
was due to its enhanced cellular absorption in vivo.45 Similarly,
it has been reported that methylation enhanced the hydro-
phobicity and the cellular uptake of R3 compared to
resveratrol.14 Thus, we inferred that M1 and M2 had more
potential as antioxidants because their hydrophobicities were
significantly greater compared to CA. Through careful
comparison of the structures between M1 and M2, the two
hydroxyl groups in M2 were replaced by methyl groups, which
might be important for the antioxidant functions. Therefore,
the methylation modifications improved lipid solubility by
increasing the number of methyl groups, allowing M2 to more
easily enter the cytoplasm and reach the intracellular targets to
exert its protective effects compared to the other two
compounds. Second, the most likely reason for the observed
results is related to the half-life of CA. The two methoxy
groups of M2 enabled the compound to display its protective
effects for longer periods of time compared to M1 until it was
enzymatically demethylated to regenerate CA in vivo, thereby
acting as a prodrug and increasing the half-life of the parent
compound.
As the activity of rat liver cytosolic COMT was higher than
that of the mouse, rat liver homogenates were used as the
source of the methyltransferase to form the methylated
polyphenols in the presence of SAM as the methyl donor.33
The methylation of polyphenols by rat liver homogenates is a
very effective method for inducing metabolism in vitro.34 Tea
catechins have been shown to undergo methylation by rat liver
enzymes, and the structures of the reaction products were
identified by MS and NMR. The results demonstrated that 4′-
O-methyl-EGC, 4″-O-methyl-ECG, and 4″-O-methyl-EGCG
were the methylated products of EGC, ECG, and EGCG,
respectively.35 In the current study, we observed that the
retention times of M1 and M2 were longer than CA on the
HPLC chromatogram after co-incubation with the rat liver
homogenate. According to the mass accuracy and fragmenta-
tion patterns generated by the LC-QTOF-MS/MS system, M1
was putatively identified with three or more isomers, and M2
was putatively identified with four or more isomers due to the
complexity of the polyphenol structures and high possibility for
positional isomers. These results showed that LC-QTOF-MS/
MS was effective for identifying the methylated metabolites of
CA but could not elucidate the molecular structures of M1 and
M2.
Accumulating evidence has demonstrated that dietary
polyphenols have a potential role in disease prevention and
treatment as natural antioxidants.36−38 However, polyphenols
undergo extensive metabolism in the body, which means they
exist predominantly as their conjugated metabolites in the
blood and tissues after phase II metabolism.39 The conjugated
metabolites may have different biological properties in tissues
and cells than the corresponding parent polyphenols. Increased
Another explanation that cannot be ignored was that the
number and position of the hydroxyl groups serve as the
critical structural dominants for the antioxidant activities of
polyphenols. It has been reported that the compounds with
2″,5″-dihydroxyl substituents on the phenyl ring at position 4
2186
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
afforded the best antioxidant values in the oxygen radical
absorbance capacity assay.46 In future studies, the positions of
the hydroxyl groups replaced by methoxy groups in M1 and
M2 should be investigated by NMR, and the appreciable
differences could contribute detailed insights to the antioxidant
mechanisms.
Xuebo Liu − College of Food Science and Engineering,
Northwest A&F University, Yangling 712100, China;
orcid.org/0000-0001-6370-2868
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c07521
Nrf2 is commonly recognized as a redox-sensitive tran-
scription factor that functions as a key regulator of the
expression of several antioxidant enzymes via its interaction
with ARE and protects cells against cytotoxicity caused by
oxidative stress.47 Previous research indicated that the
protective effects of CA against oxidative stress could be partly
explained by its role in regulating the Nrf2 antioxidative
defense system.48 M1 and M2 treatments have been found to
be more active than CA in increasing the nuclear accumulation
of Nrf2 proteins and HO-1 and NQO1 expression in H2O2-
induced HepG2 cells. This is consistent with our hypothesis
that the methylated metabolites of CA protected the HepG2
cells against oxidative stress through Nrf2 activation. More-
over, we found significant changes in the phosphorylation
levels of p38, JNK, and p56 by M1 and M2 treatment, which
could partly explain how the MAPK and NF-κB pathways are
also involved in the protective effect of the methylated
metabolites of CA.
Overall, the current study found that M1 and M2 were more
effective than CA in alleviating H2O2-induced oxidative stress
by activating the Nrf2 signaling pathway in HepG2 cells. The
methoxy groups of M2 were hypothesized to be responsible for
the antioxidant functions. Thus, the methylated metabolites of
CA could be potential candidates that contribute to the
biological efficacies of CA. Future studies should focus on
whether the methylated metabolites of CA have the ability to
cross the BBB and exhibit neuroprotective effects in animal
models of neurodegenerative diseases.
Author Contributions
Conception and design of the research: X.C., S.D., W.B., and
X.L.; performed experiments: X.C., R.G., S.D., W.B., and B.Z.;
analyzed data: X.C.; interpretation of the results of experi-
ments: X.C., and Y.D.; prepared figures: X.C., S.D., and G.Y.;
drafted the manuscript: X.C., S.D., Y.D., F.L., and B.Z. All the
authors read and approved the final manuscript.
Funding
This work was supported by the National Natural Science
Foundation of China (no. 31671859), Science and Technology
project of Shenzhen, China (JCYJ20180306172311983), and
China Postdoctoral Science Foundation (2017 T100775).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Life Science Research Core Services (LSRCS) at
NWAFU for the ESI-HRMS data and the Teaching and
Research Core Facility at the College of Life Science, NWAFU,
for their support in this work.
ABBREVIATIONS
■
AD, Alzheimer’s disease; AMPK, adenosine 5′-monophosphate
(AMP)-activated protein kinase; BBB, blood brain barrier;
COMT, catechol-O-methyltransferase; HO-1, heme oxygenase
1; LPS, lipopolysaccharide; MAPK, mitogen-activated protein
kinase; NF-κB, nuclear factor-κB; Nrf2, nuclear factor-
erythroid 2-related factor 2; NQO-1, NAD(P)H: quinone
oxidoreductase-1; SAM, S-adenosyl-L-methionine
AUTHOR INFORMATION
■
Corresponding Author
REFERENCES
Yutang Wang − College of Food Science and Engineering,
Northwest A&F University, Yangling 712100, China;
orcid.org/0000-0002-1097-4365; Phone: +86-029-
87092325; Email: wyt991023@nwsuaf.edu.cn; Fax: +86-
029-87091917
■
(1) Abbasi, B. H.; Tian, C.-L.; Murch, S. J.; Saxena, P. K.; Liu, C.-Z.
Light-enhanced caffeic acid derivatives biosynthesis in hairy root
cultures of Echinacea purpurea. Plant Cell Rep. 2007, 26, 1367−1372.
(2) Majewski, M.; Lis, B.; Juskiewicz, J.; Ognik, K.; Borkowska-
Sztachanska, M.; Jedrejek, D.; Stochmal, A.; Olas, B. Phenolic
fractions from dandelion leaves and petals as modulators of the
antioxidant status and lipid profile in an in vivo study. Antioxidants
2020, 9, 131.
Authors
Xiaowen Chang − College of Food Science and Engineering,
Northwest A&F University, Yangling 712100, China
Shan Dong − Shenzhen Academy of Metrology and Quality
Inspection, National Nutrition Food Testing Center
(Guangdong), Shenzhen 518000, China
Wenliang Bai − Shenzhen Academy of Metrology and Quality
Inspection, National Nutrition Food Testing Center
(Guangdong), Shenzhen 518000, China
́
́
(3) Llorach, R.; Tomas-Barberan, F. A.; Ferreres, F. Lettuce and
chicory byproducts as a source of antioxidant phenolic extracts. J.
Agric. Food Chem. 2004, 52, 5109−5116.
(4) Lee, J.; Scagel, C. F. Chicoric acid found in basil (Ocimum
basilicum L.) leaves. Food Chem. 2009, 115, 650−656.
(5) Lu, Q.-B.; Wan, M.-Y.; Wang, P.-Y.; Zhang, C.-X.; Xu, D.-Y.;
Liao, X.; Sun, H.-J. Chicoric acid prevents PDGF-BB-induced VSMC
dedifferentiation, proliferation and migration by suppressing ROS/
NF-KB/mTOR/P70S6K signaling cascade. Redox Biol. 2018, 14,
656−668.
Yan Di − College of Food Science and Engineering, Northwest
A&F University, Yangling 712100, China
Ruijuan Gu − College of Food Science and Engineering,
Northwest A&F University, Yangling 712100, China
Fuguo Liu − College of Food Science and Engineering,
Northwest A&F University, Yangling 712100, China;
Department of Chemistry, The Chinese University of Hong
Kong, Shatin, New Territories 999077, Hong Kong, China;
orcid.org/0000-0002-1645-0976
(6) Lin, Z.; Neamati, N.; Zhao, H.; Kiryu, Y.; Turpin, J. A.;
Aberham, C.; Strebel, K.; Kohn, K.; Witvrouw, M.; Pannecouque, C.;
Debyser, Z.; De Clercq, E.; Rice, W. G.; Pommier, Y.; Burke, T. R.
Chicoric acid analogues as HIV-1 integrase inhibitors. J. Med. Chem.
1999, 42, 1401−1414.
(7) Xiao, H.; Wang, J.; Yuan, L.; Xiao, C.; Wang, Y.; Liu, X. Chicoric
acid induces apoptosis in 3T3-L1 preadipocytes through ROS-
mediated PI3K/Akt and MAPK signaling pathways. J. Agric. Food
Chem. 2013, 61, 1509−1520.
Beita Zhao − College of Food Science and Engineering,
Northwest A&F University, Yangling 712100, China
2187
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
(8) Zhu, X.; Huang, F.; Xiang, X.; Fan, M.; Chen, T. Evaluation of
the potential of chicoric acid as a natural food antioxidant. Exp. Ther.
Med. 2018, 16, 3651−3657.
(26) Wang, P.; Peng, X.; Wei, Z.-F.; Wei, F.-Y.; Wang, W.; Ma, W.-
D.; Yao, L.-P.; Fu, Y.-J.; Zu, Y.-G. Geraniin exerts cytoprotective effect
against cellular oxidative stress by upregulation of Nrf2-mediated
antioxidant enzyme expression via PI3K/AKT and ERK1/2 pathway.
Biochim. Biophys. Acta 2015, 1850, 1751−1761.
(27) Tan, X.; Li, L.; Wang, J.; Zhao, B.; Pan, J.; Wang, L.; Liu, X.;
Liu, X.; Liu, Z. Resveratrol prevents acrylamide-induced mitochon-
drial dysfunction and inflammatory responses via targeting circadian
regulator Bmal1 and Cry1 in hepatocytes. J. Agric. Food Chem. 2019,
67, 8510−8519.
(28) Cai, Y.; Martens, G. A.; Hinke, S. A.; Heimberg, H.; Pipeleers,
D.; Van de Casteele, M. Increased oxygen radical formation and
mitochondrial dysfunction mediate beta cell apoptosis under
conditions of AMP-activated protein kinase stimulation. Free Radical
Biol. Med. 2007, 42, 64−78.
(29) Ghavipour, M.; Sotoudeh, G.; Ghorbani, M. Tomato juice
consumption improves blood antioxidative biomarkers in overweight
and obese females. Clin. Nutr. 2015, 34, 805−809.
(30) Kalinina, E. V.; Chernov, N. N.; Novichkova, M. D. Role of
glutathione, glutathione transferase, and glutaredoxin in regulation of
redox-dependent processes. Biochemistry 2014, 79, 1562−1583.
(31) Chen, Z.-H.; Na, H.-K.; Hurh, Y.-J.; Surh, Y.-J. 4-
Hydroxyestradiol induces oxidative stress and apoptosis in human
mammary epithelial cells: possible protection by NF-KB and ERK/
MAPK. Toxicol. Appl. Pharmacol. 2005, 208, 46−56.
(32) Hybertson, B. M.; Gao, B.; Bose, S. K.; McCord, J. M.
Oxidative stress in health and disease: the therapeutic potential of
Nrf2 activation. Mol. Aspects Med. 2011, 32, 234−246.
(33) Li, L.; Popko, J. L.; Zhang, X.-H.; Osakabe, K.; Tsai, C.-J.; Joshi,
C. P.; Chiang, V. L. A novel multifunctional O-methyltransferase
implicated in a dual methylation pathway associated with lignin
biosynthesis in loblolly pine. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
5461−5466.
(9) Manach, C.; Scalbert, A.; Morand, C.; Rémésy, C.; Jiménez, L.
Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 2004,
79, 727−747.
(10) Mena, P.; Bresciani, L.; Brindani, N.; Ludwig, I. A.; Pereira-
Caro, G.; Angelino, D.; Llorach, R.; Calani, L.; Brighenti, F.; Clifford,
M. N.; Gill, C. I. R.; Crozier, A.; Curti, C.; Del Rio, D. Phenyl-
gamma-valerolactones and phenylvaleric acids, the main colonic
metabolites of flavan-3-ols: synthesis, analysis, bioavailability, and
bioactivity. Nat. Prod. Rep. 2019, 36, 714−752.
(11) Diao, Z.; Li, J.; Liu, Q.; Wang, Y. In-vivo metabolite profiling of
chicoric acid in rat plasma, urine and feces after oral administration
using liquid chromatography quadrupole time of flight mass
spectrometry. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci.
2018, 1081−1082, 8−14.
(12) Walle, T. Methoxylated flavones, a superior cancer chemo-
preventive flavonoid subclass? Semin. Cancer Biol. 2007, 17, 354−362.
(13) Xu, H.; Li, Y.; Che, X.; Tian, H.; Fan, H.; Liu, K. Metabolism of
salvianolic acid A and antioxidant activities of its methylated
metabolites. Drug Metab. Dispos. 2014, 42, 274−281.
(14) Chao, J.; Li, H.; Cheng, K.-W.; Yu, M.-S.; Chang, R. C.-C.;
Wang, M. Protective effects of pinostilbene, a resveratrol methylated
derivative, against 6-hydroxydopamine-induced neurotoxicity in SH-
SY5Y cells. J. Nutr. Biochem. 2010, 21, 482−489.
(15) Rahimifard, M.; Maqbool, F.; Moeini-Nodeh, S.; Niaz, K.;
Abdollahi, M.; Braidy, N.; Nabavi, S. M.; Nabavi, S. F. Targeting the
TLR4 signaling pathway by polyphenols: a novel therapeutic strategy
for neuroinflammation. Ageing Res. Rev. 2017, 36, 11−19.
(16) Seeram, N. P.; Zhang, Y. J.; Sartipipour, M.; Henning, S. M.;
Lee, R. P.; Harris, D. M.; Moro, A.; Heber, D. Pharmacokinetics and
tissue disposition of urolithin A, an ellagitannin-derived metabolite, in
mice. FASEB J. 2007, 21, A1081.
(17) Yuan, T.; Ma, H.; Liu, W.; Niesen, D. B.; Shah, N.; Crews, R.;
Rose, K. N.; Vattem, D. A.; Seeram, N. P. Pomegranate’s
neuroprotective effects against Alzheimer’s disease are mediated by
urolithins, its ellagitannin-gut microbial derived metabolites. ACS
Chem. Neurosci. 2016, 7, 26−33.
(18) Forrester, S. J.; Kikuchi, D. S.; Hernandes, M. S.; Xu, Q.;
Griendling, K. K. Reactive oxygen species in metabolic and
inflammatory signaling. Circ. Res. 2018, 122, 877−902.
(19) Subramaniam, S. R.; Chesselet, M.-F. Mitochondrial
dysfunction and oxidative stress in Parkinson’s disease. Prog Neurobiol.
2013, 106-107, 17−32.
(20) Bhattacharyya, A.; Chattopadhyay, R.; Mitra, S.; Crowe, S. E.
Oxidative stress: an essential factor in the pathogenesis of gastro-
intestinal mucosal diseases. Physiol. Rev. 2014, 94, 329−354.
(21) Jiang, T.; Sun, Q.; Chen, S. Oxidative stress: A major
pathogenesis and potential therapeutic target of antioxidative agents
in Parkinson’s disease and Alzheimer’s disease. Prog Neurobiol. 2016,
147, 1−19.
(34) Lu, H.; Meng, X.; Yang, C. S. Enzymology of methylation of tea
catechins and inhibition of catechol-O-methyltransferase by (-)-epi-
gallocatechin gallate. Drug Metab. Dispos. 2003, 31, 572−579.
(35) Okushio, K.; Suzuki, M.; Matsumoto, N.; Nanjo, F.; Hara, Y.
Methylation of tea catechins by rat liver homogenates. Biosci.,
Biotechnol., Biochem. 1999, 63, 430−432.
̇
(36) Khan, H.; Sureda, A.; Belwal, T.; Çetinkaya, S.; Suntar, I.;
̈
Tejada, S.; Devkota, H. P.; Ullah, H.; Aschner, M. Polyphenols in the
treatment of autoimmune diseases. Autoimmun. Rev. 2019, 18, 647−
657.
(37) Gessner, D. K.; Ringseis, R.; Eder, K. Potential of plant
polyphenols to combat oxidative stress and inflammatory processes in
farm animals. J. Anim. Physiol. Anim. Nutr. 2017, 101, 605−628.
(38) Baur, J. A.; Sinclair, D. A. Therapeutic potential of resveratrol:
the in vivo evidence. Nat. Rev. Drug Discovery 2006, 5, 493−506.
(39) DuPont, M. S.; Bennett, R. N.; Mellon, F. A.; Williamson, G.
Polyphenols from alcoholic apple cider are absorbed, metabolized and
excreted by humans. J. Nutr. 2002, 132, 172−175.
(40) Chen, T.-Y.; Kritchevsky, J.; Hargett, K.; Feller, K.; Klobusnik,
R.; Song, B. J.; Cooper, B.; Jouni, Z.; Ferruzzi, M. G.; Janle, E. M.
Plasma bioavailability and regional brain distribution of polyphenols
from apple/grape seed and bilberry extracts in a young swine model.
Mol. Nutr. Food Res. 2015, 59, 2432−2447.
(22) Liu, Q.; Fang, J.; Chen, P.; Die, Y.; Wang, J.; Liu, Z.; Liu, X.
Chicoric acid improves neuron survival against inflammation by
promoting mitochondrial function and energy metabolism. Food
Funct. 2019, 10, 6157−6169.
(23) Liu, Q.; Chen, Y.; Shen, C.; Xiao, Y.; Wang, Y.; Liu, Z.; Liu, X.
Chicoric acid supplementation prevents systemic inflammation-
induced memory impairment and amyloidogenesis via inhibition of
NF-KB. FASEB J. 2017, 31, 1494−1507.
(24) Weinert, C. H.; Wiese, S.; Rawel, H. M.; Esatbeyoglu, T.;
Winterhalter, P.; Homann, T.; Kulling, S. E. Methylation of catechins
and procyanidins by rat and human catechol-O-methyltransferase:
metabolite profiling and molecular modeling studies. Drug Metab.
Dispos. 2012, 40, 353−359.
(25) Liu, Y.; Cheng, Y.; Li, J.; Wang, Y.; Liu, Y. Epoxy stearic acid,
an oxidative product derived from oleic acid, induces cytotoxicity,
oxidative stress, and apoptosis in HepG2 cells. J. Agric. Food Chem.
2018, 66, 5237−5246.
(41) Penalver, P.; Zodio, S.; Lucas, R.; de-Paz, M. V.; Morales, J. C.
̃
Neuroprotective and anti-inflammatory effects of pterostilbene
metabolites in human neuroblastoma SH-SY5Y and RAW 264.7
macrophage cells. J. Agric. Food Chem. 2020, 68, 1609−1620.
(42) Wang, J.; Ferruzzi, M. G.; Ho, L.; Blount, J.; Janle, E. M.; Gong,
B.; Pan, Y.; Gowda, G. A. N.; Raftery, D.; Arrieta-Cruz, I.; Sharma, V.;
Cooper, B.; Lobo, J.; Simon, J. E.; Zhang, C.; Cheng, A.; Qian, X.;
Ono, K.; Teplow, D. B.; Pavlides, C.; Dixon, R. A.; Pasinetti, G. M.
Brain-targeted proanthocyanidin metabolites for Alzheimer’s disease
treatment. J. Neurosci. 2012, 32, 5144−5150.
(43) Liu, Q.; Wang, Y.; Xiao, C.; Wu, W.; Liu, X. Metabolism of
chicoric acid by rat liver microsomes and bioactivity comparisons of
chicoric acid and its metabolites. Food Funct. 2015, 6, 1928−1935.
2188
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
(44) Wang, Y.; Xie, G.; Liu, Q.; Duan, X.; Liu, Z.; Liu, X.
Pharmacokinetics, tissue distribution, and plasma protein binding
study of chicoric acid by HPLC-MS/MS. J. Chromatogr. B: Anal.
Technol. Biomed. Life Sci. 2016, 1031, 139−145.
(45) Zhou, F.; Shen, T.; Duan, T.; Xu, Y.; Khor, S. C.; Li, J.; Ge, J.;
Zheng, Y.; Hsu, S.; De Stefano, J.; Yang, J.; Xu, L. H.; Zhu, X. Q.
Antioxidant effects of lipophilic tea polyphenols on diethylnitros-
amine/phenobarbital-induced hepatocarcinogenesis in rats. In Vivo
2014, 28, 495−503.
́
(46) Bonache, M. A.; Moreno-Fernandez, S.; Miguel, M.; Sabater-
́
Munoz, B.; Gonzalez-Muniz, R. Small library of triazolyl polyphenols
̃ ̃
correlating antioxidant activity and stability with number and position
of hydroxyl groups. ACS Comb. Sci. 2018, 20, 694−699.
(47) Ma, Q. Role of Nrf2 in oxidative stress and toxicity. Annu. Rev.
Pharmacol. Toxicol. 2013, 53, 401−426.
(48) Wang, Y.; Diao, Z.; Li, J.; Ren, B.; Zhu, D.; Liu, Q.; Liu, Z.; Liu,
X. Chicoric acid supplementation ameliorates cognitive impairment
induced by oxidative stress via promotion of antioxidant defense
system. RSC Adv. 2017, 7, 36149−36162.
2189
https://dx.doi.org/10.1021/acs.jafc.0c07521
J. Agric. Food Chem. 2021, 69, 2179−2189