A novel semisynthetic flavonoid 7-O-galloyltaxifolin upregulates heme oxygenase-1 in RAW264.7 cells via MAPK/Nrf2 pathway

Article abstract of DOI:10.1021/jm3013344

Quercetin and gallic acid are natural activators of the transcription factor Nrf2, which regulates the expression of many cytoprotective enzymes including heme oxygenase-1 (HO-1). We developed procedures for the synthesis of monogalloyl esters of quercetin and taxifolin (dihydroquercetin), namely, 3-O-galloylquercetin and 7-O-galloyltaxifolin, and examined their effect on the Nrf2 pathway in RAW264.7 cells. Unlike quercetin and free gallic acid, 3-O-galloylquercetin and natural quercetin derivatives isoquercitrin (quercetin-3-O-β-d-glucoside) and taxifolin had no effect on the expression of HO-1. In contrast, 7-O-galloyltaxifolin increased both mRNA and protein levels of HO-1 at concentrations of 25 μM and above. The induction of HO-1 by 7-O-galloyltaxifolin was primarily associated with the production of reactive oxygen species and phosphorylation of mitogen-activated protein kinases (MAPKs), including p38 MAPKs and ERKs, followed by nuclear accumulation of Nrf2 and downregulation of Keap1, a negative regulator of Nrf2. We conclude that 7-O-galloyltaxifolin upregulates HO-1 via activation of the MAPK/Nrf2 signaling pathway.

Full text of DOI:10.1021/jm3013344

Article
pubs.acs.org/jmc
A Novel Semisynthetic Flavonoid 7‑O‑Galloyltaxifolin Upregulates
Heme Oxygenase‑1 in RAW264.7 Cells via MAPK/Nrf2 Pathway
Jirí Vrba,*,† Radek Gazak,‡ Marek Kuzma,‡ Barbora Papouskova,§ Jan Vacek,† Martin Weiszenstein,†
̌
̌
́
̌
́
Vladimír Kren,‡ and Jitka Ulrichova†,∥
̌
́
†Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnev
̌
otínska
́
3,
́
Olomouc 77515, Czech Republic
‡Institute of Microbiology, Academy of Sciences of the Czech Republic, Víden
̌
ska 1083, Prague 14220, Czech Republic
́
§Regional Centre of Advanced Technologies and Materials, Department of Analytical Chemistry, Faculty of Science, Palacky
University, 17 listopadu 12, Olomouc 77146, Czech Republic
́
∥Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hnev
̌ ́
otínska 3, Olomouc
́
77515, Czech Republic
S
* Supporting Information
ABSTRACT: Quercetin and gallic acid are natural activators of
the transcription factor Nrf2, which regulates the expression of
many cytoprotective enzymes including heme oxygenase-1 (HO-
1). We developed procedures for the synthesis of monogalloyl
esters of quercetin and taxifolin (dihydroquercetin), namely, 3-O-
galloylquercetin and 7-O-galloyltaxifolin, and examined their effect
on the Nrf2 pathway in RAW264.7 cells. Unlike quercetin and free
gallic acid, 3-O-galloylquercetin and natural quercetin derivatives
isoquercitrin (quercetin-3-O-β-D-glucoside) and taxifolin had no
effect on the expression of HO-1. In contrast, 7-O-galloyltaxifolin
increased both mRNA and protein levels of HO-1 at
concentrations of 25 μM and above. The induction of HO-1 by 7-O-galloyltaxifolin was primarily associated with the
production of reactive oxygen species and phosphorylation of mitogen-activated protein kinases (MAPKs), including p38
MAPKs and ERKs, followed by nuclear accumulation of Nrf2 and downregulation of Keap1, a negative regulator of Nrf2. We
conclude that 7-O-galloyltaxifolin upregulates HO-1 via activation of the MAPK/Nrf2 signaling pathway.
heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1,
catalase, thioredoxin, and glutathione S-transferase. The activity
of Nrf2 is regulated in response to the redox imbalance caused
by oxidative or electrophilic stress.5 Under unstressed
conditions, Nrf2 activity is repressed by Kelch-like ECH-
associated protein 1 (Keap1), which forms a complex with
Nrf2, sequesters Nrf2 in the cytoplasm, and targets Nrf2 for
proteasomal degradation. In contrast, the oxidation or covalent
modification of reactive cysteine residues in Keap1 produces a
proteasome-dependent degradation of Keap1 followed by the
stabilization and nuclear accumulation of Nrf2. In the nucleus,
Nrf2 dimerizes with small Maf proteins and binds to the DNA
at the antioxidant response elements to activate transcription of
Nrf2 target genes.6,7 The nuclear accumulation of Nrf2 may
also be mediated via the phosphorylation of Nrf2 by various
protein kinase pathways such as the p38 mitogen-activated
protein kinase (p38 MAPK), extracellular signal-regulated
kinase (ERK), and c-Jun N-terminal kinase (JNK) pathways.
However, these protein kinase pathways regulate diverse
INTRODUCTION
■
Natural flavonoids are the largest group of plant-derived
polyphenol compounds. They share a common structure
consisting of two benzene rings linked through three carbons
that form an oxygen-containing heterocycle. Basic flavonoid
skeletons such as flavone and flavanone may carry various
combinations of hydroxyl and methoxyl groups. This aside,
flavonoids are predominantly found in plants as O-glycosides.1
For instance, quercetin (3,5,7,3′,4′-pentahydroxyflavone; Figure
1) may occur in the form of mono-, di-, and triglycosides
containing simple sugars, for example, glucose, galactose, and
arabinose,2 sugar derivatives, such as gallate and ferulate esters,3
and various disaccharides.2
Dietary intake of the flavonoids present in fruits, vegetables,
and beverages such as tea and wine1 is generally considered to
have beneficial health effects arising primarily from their anti-
inflammatory activity. The protective action of many flavonoids
may be ascribed, at least in part, to their ability to activate
nuclear factor erythroid 2-related factor 2 (Nrf2).4 Nrf2 is an
important cytoprotective transcription factor responsible for
both the constitutive and inducible expression of genes coding
for antioxidant and phase II detoxification enzymes such as
Received: September 17, 2012
Published: January 8, 2013
© 2013 American Chemical Society
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Figure 1. Chemical structures of tested phenolic compounds.
a
Scheme 1. Synthesis of 7-O-Galloyltaxifolin (3)
a
Reagents and conditions: (a) 3,4,5-tri-O-benzylgalloyl chloride (1.5 equiv), pyridine, 0 °C, 1 h, 23%; (b) H2−Pd/C, EtOAc, room temp, 3 h, 94%.
cellular processes, and the precise mechanisms of the
phosphorylation-dependent Nrf2 activation are not fully
understood.4,7
CHEMISTRY
■
The synthesis of 7-O-galloyltaxifolin (3) was performed by
direct galloylation of taxifolin (1) with 3,4,5-tri-O-benzylgalloyl
chloride in pyridine (Scheme 1). The chloride of the benzyl-
protected gallic acid was prepared by treating the correspond-
ing acid with oxalyl chloride solution in CH2Cl2 and a catalytic
amount of DMF immediately before the intended synthesis.
Benzyl-containing intermediate 2 was then deprotected by
catalytic hydrogenolysis with Pd on carbon, giving the final
galloyl ester 3. To achieve the galloylation of 1, we also tested
several other esterification methods, for example, the DCC/
DMAP procedure, acyl chloride/Et3N, and other methods we
had previously used for the regioselective galloylation of
silybin.12 However, these methods usually gave complex
mixtures of products containing only minute amounts of
monogalloyl esters.
The synthesis of 3-O-galloylquercetin (8) was achieved using
the strategy described in Scheme 2. The synthesis started from
naturally occurring quercetin glycoside rutin (4). Benzylation of
4 by BnBr in DMF in the presence of Cs2CO3 led, after acidic
hydrolysis of the intermediate glycoside, to 5,7,3′,4′-tetra-O-
benzylquercetin (5). Esterification of the benzyl-protected
Classic Nrf2 activators identified among natural flavonoids
are quercetin8 and epigallocatechin-3-gallate, a flavanol carrying
a galloyl ester group.9,10 It has been shown that the ability of
catechins to activate the antioxidant response element depends
on the presence of a galloyl moiety in their molecules.11
Similarly, the introduction of a galloyl moiety into the structure
of the flavonolignan silybin has been found to have
considerable influence on the biological activity of the resulting
compound.12 These findings inspired us to prepare galloyl
esters of the prototypical flavonoid quercetin and its
dihydroderivative taxifolin (dihydroquercetin; Figure 1),
which both exhibit a number of biological activities.13,14 In
this study, two monogalloyl esters, 3-O-galloylquercetin and 7-
O-galloyltaxifolin, were prepared, and their effect on the Nrf2
signaling pathway was examined in murine macrophage
RAW264.7 cells.
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a
stepwise deprotected to achieve sufficient purification. The
deprotection started by catalytic hydrogenolysis of the benzyl
groups using Pd on carbon. The crude product from the
hydrogenolysis was then extensively purified by column
chromatography to remove all side products. Pure galloyl
ester 7 bearing MOM groups protecting the galloyl moiety was
then treated with a methanolic solution of HCl, which yielded
galloyl ester 8.
Scheme 2. Synthesis of 3-O-Galloylquercetin (8)
BIOLOGICAL RESULTS
■
Cytotoxicity of Tested Compounds in RAW264.7
Cells. The effect of the tested compounds (Figure 1) on the
viability of RAW264.7 cells was examined using an MTT
reduction assay. At concentrations of 10 and 50 μM and 6 h of
exposure, the natural flavonoids quercetin, isoquercitrin, and
taxifolin caused negligible changes in cell viability (Figure 2). In
contrast, the newly synthesized galloyl esters and free gallic acid
showed moderate cytotoxicity. For instance, 6 h of cell
exposure to 50 μM 3-O-galloylquercetin, 7-O-galloyltaxifolin,
or gallic acid significantly decreased cell viability to 79%, 72%,
and 76%, respectively (Figure 2).
7-O-Galloyltaxifolin Is Converted to 7-O-Galloylquer-
cetin in RAW264.7 Cells. Prior to the expression studies, the
stability of the galloylated derivatives during the cell treatment
was examined by the HPLC/MS method. On a phenyl-based
HPLC column, 7-O-galloyltaxifolin and 3-O-galloylquercetin
gave well-resolved chromatographic peaks at 10.2 and 10.7 min,
respectively (Figure 3). Electrospray ionization/quadrupole
time-of-flight mass spectrometry (ESI-QqTOF MS) enabled
both compounds to be identified on the basis of their MS
fragmentation patterns and elemental composition determi-
nation. Using MS with negative ESI, 3-O-galloylquercetin and
7-O-galloyltaxifolin gave pseudomolecular [M − H]− peaks at
m/z 453.0446 and 455.0599, respectively. The full MS and MSE
spectra of both compounds are shown in Figure 4A,B.
When RAW264.7 cells were incubated for up to 6 h with 50
μM 3-O-galloylquercetin or 7-O-galloyltaxifolin, the uptake of
both compounds was relatively fast. After 10 min of incubation,
the intracellular concentrations of both galloyl esters reached
approximately 85% of their maximal values, which were found
after 3 h of exposure and remained almost unchanged for
additional 3 h. For instance, the yield of 3-O-galloylquercetin
was 1.75 and 1.80 nmol per 106 cells after 3 and 6 h of
incubation, respectively (data not shown). The chromatogram
of the cell extract prepared after 6 h of cell exposure to 50 μM
3-O-galloylquercetin is shown in Figure 3 (line d). After the cell
a
Reagents and conditions: (a) BnBr (6 equiv), Cs2CO3 (4.5 equiv),
DMF, 44 h at room temp, then 50 °C for 4 h; (b) EtOH/HCl (42:8,
v/v), reflux, 2 h, 23% (after two steps); (c) MOM3gallic acid, DCC/
DMAP, CH2Cl2, room temp, overnight; (d) H2−Pd/C, EtOAc, room
temp, 4 h, 35% (after steps c and d); (e) MeOH, HCl (cat.), room
temp, 3 h, 83%.
quercetin 5 by MOM-protected gallic acid using DCC/DMAP
led to an orthogonally protected galloyl ester, which was
Figure 2. Effect of tested phenolic compounds on viability of RAW264.7 cells. Cells were treated for 6 h with 0.1% DMSO (control) or with
indicated concentrations of quercetin (QUE), isoquercitrin (IQ), 3-O-galloylquercetin (3GQ), taxifolin (TAX), 7-O-galloyltaxifolin (7GT), or gallic
acid (GA). The cell viability was determined by MTT reduction assay. Data are means SD of three experiments. * p < 0.05; ** p < 0.01,
significantly decreased versus control.
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treatment, both 3-O-galloylquercetin and 7-O-galloyltaxifolin
were found to be stable with respect to their possible hydrolytic
cleavage. HPLC/MS detection of [M − H]− ions of gallic acid,
quercetin, and taxifolin at m/z 169, 301, and 303, respectively,
showed that the levels of gallic acid were undetectable and that
the intracellular amounts of quercetin or taxifolin represented
less than 4.5% of the respective parent compound. The same
quantities of hydrolytic products were also found when stock
solutions of 3-O-galloylquercetin or 7-O-galloyltaxifolin in
DMSO were analyzed prior to their addition to the cells
(data not shown). In contrast to 3-O-galloylquercetin, the
treatment of RAW264.7 cells with 7-O-galloyltaxifolin resulted
in the appearance of a specific product having a peak at 14.3
min (Figure 3, line c). The ESI-QqTOF MS analysis identified
this product as 7-O-galloylquercetin. As shown in Figure 4, the
MS and MSE spectra of 7-O-galloylquercetin (Figure 4C) were
essentially the same as those of 3-O-galloylquercetin (Figure
4A). The formation of 7-O-galloylquercetin was not observed
after the incubation of the parent compound in the culture
medium without RAW264.7 cells. These results showed that
the cells were responsible for the oxidation of 7-O-
galloyltaxifolin to 7-O-galloylquercetin, with the product
detected in cells as early as 10 min after the start of the cell
treatment. After 3 h of cell exposure to 50 μM 7-O-
galloyltaxifolin, the content of 7-O-galloyltaxifolin and 7-O-
galloylquercetin was 1.03 and 8.08 nmol per 106 cells,
respectively, and very similar ratios between the parent
compound and product were found, regardless of the
incubation time (data not shown).
Figure 3. HPLC/MS chromatograms of galloylated derivatives of
quercetin and taxifolin. Standard solutions (2 μg/mL) of 3-O-
galloylquercetin (3GQ, line a) or 7-O-galloyltaxifolin (7GT, line b)
and extracts of RAW264.7 cells treated for 6 h with 50 μM 7GT (line
c), 50 μM 3GQ (line d), or 0.1% DMSO (control) were analyzed by
HPLC/ESI-QqTOF MS. The m/z values of 453 and 455 were
monitored using selected-ion monitoring mode. 7GQ, 7-O-galloyl-
quercetin (a product of 7GT oxidation).
Exposure to 7-O-Galloyltaxifolin Increases mRNA and
Protein Levels of Heme Oxygenase-1. The study further
investigated the effect of the tested compounds on the
expression of the Nrf2 target gene Hmox1 encoding heme
oxygenase-1. As expected, quantitative real-time PCR analysis
demonstrated an induction of mouse Hmox1 gene expression in
RAW264.7 cells exposed to quercetin, which was used as a
positive control.15,16 After 6 h of cell exposure, 50 μM quercetin
significantly increased the level of Hmox1 mRNA to 4.3-fold
when normalized to Gapdh mRNA (Figure 5). A dose-
dependent increase in the expression of Hmox1 mRNA was
also found after 6 h of cell treatment with 7-O-galloyltaxifolin,
which appeared to be a more potent Hmox1 inducer than
quercetin. At concentrations of 25 and 50 μM, 7-O-
galloyltaxifolin significantly elevated Hmox1 mRNA levels to
Figure 5. Relative changes in Hmox1 gene expression by tested
phenolic compounds in RAW264.7 cells. Cells were treated for 6 h
with 0.1% DMSO (control) or with indicated concentrations of
quercetin (QUE), isoquercitrin (IQ), 3-O-galloylquercetin (3GQ),
taxifolin (TAX), 7-O-galloyltaxifolin (7GT), or gallic acid (GA). The
levels of Hmox1 mRNA were determined by quantitative real-time
PCR with results normalized to Gapdh mRNA. Data are means SD
of three experiments. * p < 0.05; ** p < 0.01; *** p < 0.001,
significantly increased versus control.
Figure 4. Full MS spectra and MSE fragmentation (insets) of (A) 3-O-
galloylquercetin, (B) 7-O-galloyltaxifolin, and (C) 7-O-galloylquerce-
tin. The MS spectra were obtained from the chromatographic peaks
shown in Figure 3. For details on MS fragmentation, see Supporting
Information.
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Figure 6. Effect of tested phenolic compounds on heme oxygenase-1 (HO-1) protein levels in RAW264.7 cells. (A) Cells were treated for 6 h with
0.1% DMSO (control) or with 50 μM quercetin (QUE), isoquercitrin (IQ), 3-O-galloylquercetin (3GQ), taxifolin (TAX), 7-O-galloyltaxifolin
(7GT), or gallic acid (GA). (B) Cells were incubated for 1−6 h in the absence or presence of 50 μM 7GT (as indicated). (C) Cells were treated for
6 h with 0.1% DMSO (control) or with 10 to 50 μM 7GT. After treatment, proteins in the whole cell lysates (20 μg/lane) were analyzed by Western
blotting and HO-1 and actin were visualized by chemiluminescent detection. Relative HO-1 band intensities normalized to actin are shown. Data are
means of three experiments.
Figure 7. Effect of 7-O-galloyltaxifolin (7GT) on nuclear and cytosolic levels of Nrf2 in RAW264.7 cells. (A) Cells were treated for 3 h with 0.1%
DMSO (control), 50 μM quercetin (QUE; positive control), or 10−50 μM 7GT. (B) Cells were incubated for 30 min to 3 h in the absence or
presence of 50 μM 7GT (as indicated). After treatment, cells were fractionated, and the proteins in nuclear and cytosolic fractions (30 μg/lane) were
analyzed by Western blotting. Nrf2 and actin were visualized by chemiluminescent detection. Relative Nrf2 band intensities normalized to actin are
shown. Data are means of three experiments.
Figure 8. Effect of 7-O-galloyltaxifolin (7GT) on Keap1 protein levels in RAW264.7 cells. (A) Cells were treated for 6 h with 0.1% DMSO (control)
or with 10−50 μM 7GT. (B) Cells were incubated for 1−6 h in the absence or presence of 50 μM 7GT (as indicated). After treatment, proteins in
the whole cell lysates (20 μg/lane) were analyzed by Western blotting, and Keap1 and actin were visualized by chemiluminescent detection. Relative
Keap1 band intensities normalized to actin are shown. Data are means of three experiments.
4.8-fold and 13.4-fold, respectively, compared with the control
(Figure 5). This aside, 6 h of exposure to 50 μM gallic acid
produced a small, statistically nonsignificant increase in the
level of Hmox1 mRNA that reached (1.5 0.3)-fold that of the
control. In contrast, the expression of Hmox1 mRNA remained
practically unaffected in cells treated with isoquercitrin, 3-O-
galloylquercetin, or taxifolin (Figure 5).
The levels of Hmox1 mRNA appeared to correlate with the
abundance of heme oxygenase-1 (HO-1) protein. In
RAW264.7 cells treated for 6 h with individual tested
compounds (50 μM), Western blot analysis showed an obvious
upregulation of HO-1 by quercetin and 7-O-galloyltaxifolin
(Figure 6A). A slight increase in the protein level of HO-1 was
also induced by gallic acid, whereas no substantial changes in
the expression of HO-1 were found after cell exposure to
isoquercitrin, 3-O-galloylquercetin, or taxifolin (Figure 6A).
The upregulation of HO-1 by 7-O-galloyltaxifolin was both
time- and dose-dependent. At a concentration of 50 μM, the
increased abundance of HO-1 was detected as early as 3 h after
the start of the incubation (Figure 6B). After 6 h of cell
treatment, 7-O-galloyltaxifolin clearly elevated the protein levels
of HO-1 at concentrations of 25 μM and above (Figure 6C).
Exposure to 7-O-Galloyltaxifolin Induces Nuclear
Accumulation of Nrf2 and Downregulates Keap1. To
characterize the mechanisms by which 7-O-galloyltaxifolin
induces Hmox1 gene expression, the nuclear and cytosolic
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Figure 9. ROS generation by 7-O-galloyltaxifolin (7GT) and effect of N-acetyl-L-cysteine (NAC) on 7GT-induced Hmox1 gene expression in
RAW264.7 cells. (A) Cells were treated for 2 h with 10−50 μM 7GT. After treatment, cells were incubated with carboxy-H2DCFDA and the
percentage of cells with increased ROS levels was evaluated by flow cytometry. Data are means SD of three experiments. * p < 0.05; ** p < 0.01,
significantly increased versus control. (B) Cells were pretreated for 30 min with 5 or 10 mM NAC and then incubated in the absence or presence of
50 μM 7GT for additional 6 h. The levels of Hmox1 mRNA were determined by quantitative real-time PCR and normalized to Gapdh mRNA.
Results are expressed as the percentage of 7GT-induced Hmox1 mRNA expression. Data are means SD of three experiments. ** p < 0.01,
significantly different from cells treated with 7GT in the absence of NAC.
Figure 10. Effect of 7-O-galloyltaxifolin (7GT) on the phosphorylation status of MAPKs in RAW264.7 cells. (A) Cells were treated for 30 min with
75 ng/mL epidermal growth factor (EGF; positive control) or incubated for 30 min to 3 h in the absence or presence of 50 μM 7GT (as indicated).
After treatment, proteins in the whole cell lysates (20 μg/lane) were analyzed by Western blotting. ERK1 phosphorylated at Thr202 and Tyr204,
ERK2 phosphorylated at Thr185 and Tyr187, and total ERK1/2 were visualized by chemiluminescent detection. (B) Cells were treated for 30 min
with 5 μM anisomycin (positive control) or 75 ng/mL EGF or incubated for 30 min to 4 h in the absence or presence of 50 μM 7GT (as indicated).
The levels of p38 MAPKs dually phosphorylated at Thr180/Tyr182 and total p38 MAPKs were analyzed in the whole cell lysates (35 μg/lane) as
mentioned above. (C) Cells were treated for 30 min with 5 μM anisomycin (positive control) or incubated for 30 min to 4 h in the absence or
presence of 50 μM 7GT (as indicated). JNKs dually phosphorylated at Thr183/Tyr185 and total JNKs were analyzed in the whole cell lysates (30
μg/lane) as mentioned above.
levels of the transcription factor Nrf2 were analyzed by Western
blotting. The treatment of RAW264.7 cells for 3 h with 50 μM
quercetin, a positive control,8 elevated the protein levels of
Nrf2 in both nuclear and cytosolic fractions (Figure 7A). After
3 h of exposure, 7-O-galloyltaxifolin was found to increase the
levels of Nrf2 in both fractions at concentrations of 25 and 50
μM (Figure 7A). A time-course experiment revealed that 50
μM 7-O-galloyltaxifolin elevated the nuclear levels of Nrf2 as
early as 1 h after the start of the incubation, while the cytosolic
Nrf2 levels slightly increased after 2 h (Figure 7B). These
results demonstrated that 7-O-galloyltaxifolin was able to
enhance the nuclear accumulation of Nrf2 as well as the total
cellular Nrf2 content. We also tested whether the upregulation
of Nrf2 by 7-O-galloyltaxifolin could be associated with the
degradation of Keap1, a negative regulator of Nrf2.7 Western
blot analysis of whole cell lysates showed that 7-O-
galloyltaxifolin dose-dependently reduced the protein levels of
Keap1 after 6 h of treatment (Figure 8A). At a concentration of
50 μM, the effect of 7-O-galloyltaxifolin was also found to be
time-dependent, while the levels of Keap1 started to decline
after 3 h of incubation (Figure 8B). Our results showed that the
nuclear accumulation of Nrf2 preceded the downregulation of
Keap1, and hence other mechanisms of 7-O-galloyltaxifolin-
induced Hmox1 gene expression needed to be taken into
account.
Antioxidant N-Acetyl-L-cysteine Suppresses 7-O-Gal-
loyltaxifolin-Induced Hmox1 Gene Expression. To
evaluate possible involvement of reactive oxygen species
(ROS) in the modulation of Hmox1 gene expression by 7-O-
galloyltaxifolin, we first examined whether cell exposure to 7-O-
galloyltaxifolin caused ROS generation. Flow cytometric
analysis using a carboxy derivative of reduced and acetylated
2′,7′-dichlorofluorescein showed that exposure of RAW264.7
cells to a potent prooxidant, tert-butyl hydroperoxide (TBHP),
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induced an increase in the cell fluorescence, indicative of
intracellular ROS production.17 Increased levels of ROS were
detected in 3−5% of untreated cells and in 25% 4% of cells
treated for 30 min with 100 μM TBHP (data not shown).
When the cells were exposed to 7-O-galloyltaxifolin, elevated
ROS levels were found after 2 h of exposure. At concentrations
of 25 and 50 μM, 7-O-galloyltaxifolin raised the cell population
with increased ROS levels to 19% and 29%, respectively (Figure
9A). We further examined whether the induction of Hmox1
gene expression by 7-O-galloyltaxifolin could be reduced by a
nonspecific ROS scavenger N-acetyl-L-cysteine (NAC).10 We
found that 30 min pretreatment of RAW264.7 cells with 5 or 10
mM NAC, neutralized with sodium bicarbonate, significantly
decreased Hmox1 mRNA levels induced by 50 μM 7-O-
galloyltaxifolin at 6 h of exposure by 90% and 94%, respectively
(Figure 9B). Moreover, we also found that 5 and 10 mM NAC
did not prevent the oxidation of 7-O-galloyltaxifolin to 7-O-
galloylquercetin (data not shown). These results suggest that
ROS are responsible for the induction of Hmox1 gene
expression by 7-O-galloyltaxifolin and that 7-O-galloylquercetin
is produced enzymatically rather than through a chemical
process.
Figure 11. Effect of MAPK inhibitors on 7-O-galloyltaxifolin (7GT)-
induced Hmox1 gene expression in RAW264.7 cells. Cells were
pretreated for 30 min with 0.1% DMSO (control), 15 μM PD98059,
15 μM SB203580, or 30 μM SP600125 and then incubated in the
absence or presence of 50 μM 7GT for additional 6 h. The levels of
Hmox1 mRNA were determined by quantitative real-time PCR and
normalized to Gapdh mRNA. Results are expressed as the percentage
of 7GT-induced Hmox1 mRNA expression. Data are means SD of
three experiments. * p < 0.05; ** p < 0.01, significantly different from
cells treated with 7GT in the absence of MAPK inhibitors.
Induction of Hmox1 Gene Expression by 7-O-
Galloyltaxifolin Involves the Activities of p38 MAPKs
and ERKs. Since ROS activate MAPK cascades,18 the study
examined the possible involvement of MAPKs in the induction
of Hmox1 mRNA expression by 7-O-galloyltaxifolin. The
activity of MAPKs is regulated by upstream activating kinases
and inactivating protein phosphatases.19 Therefore, we first
tested the effect of 7-O-galloyltaxifolin on the phosphorylation
status of MAPKs. As shown by Western blotting with phospho-
specific antibodies, epidermal growth factor (EGF) and
anisomycin were appropriate positive controls20 for stimulating
the phosphorylation of MAPKs in RAW264.7 cells. After 30
min of treatment, 75 ng/mL EGF increased the phosphor-
ylation of ERK1 but not ERK2 (Figure 10A). In contrast, 30
min of exposure to 5 μM anisomycin enhanced the
phosphorylation of p38 MAPKs (Figure 10B) and JNKs
(Figure 10C). Cell treatment for 30 min with 50 μM 7-O-
galloyltaxifolin caused a marked increase in the phosphorylation
of both ERK1 and ERK2. The levels of phosphorylated ERK1/
2 decreased somewhat with further incubation but remained
above basal levels for up to 3 h (Figure 10A). This aside, 7-O-
galloyltaxifolin was found to elevate the phosphorylation of p38
MAPKs and JNKs in a time-dependent manner. The levels of
phosphorylated p38 MAPKs were slightly elevated by 50 μM 7-
O-galloyltaxifolin after 1 h of cell exposure (Figure 10B), while
the increased phosphorylation of JNKs was detected as early as
30 min after start of the incubation (Figure 10C). Since 7-O-
galloyltaxifolin increased the phosphorylation of all three
MAPK subfamilies, we subsequently employed specific
pharmacological MAPK inhibitors PD98059, SB203580, and
SP600125 to suppress the activity of ERKs, p38 MAPKs, and
JNKs, respectively.15 Our results showed that the increase in
Hmox1 mRNA induced by 7-O-galloyltaxifolin was significantly
reduced by the inhibitors of ERKs and p38 MAPKs.
Pretreatment of RAW264.7 cells for 30 min with 15 μM
PD98059 or 15 μM SB203580 decreased the induction of
Hmox1 mRNA by 6 h of exposure to 50 μM 7-O-
galloyltaxifolin by 15% and 70%, respectively (Figure 11). In
contrast, the inhibition of JNKs by 30 μM SP600125 enhanced
the effect of 7-O-galloyltaxifolin on Hmox1 mRNA level by
90% under the same experimental conditions (Figure 11).
DISCUSSION
■
The natural flavonoids quercetin and taxifolin (dihydroquerce-
tin) exhibit a wide range of biological activities, e.g.
antioxidative, anti-inflammatory, and anticancer.13,14,21 The
molecules of both flavonoids may also serve as the backbone
for a number of potentially bioactive derivatives, such as the
glycosides produced in plants22,23 or glucuronides, sulfates, and
methyl conjugates produced by phase II xenobiotic metaboliz-
ing enzymes.24 This aside, semisynthetic 3-O-acylquercetins
with aliphatic acyl groups having 2−16 carbon atoms have been
characterized.25 In this study, we prepared galloylated
derivatives of quercetin and taxifolin and examined their effect
on the Nrf2 signaling pathway, which regulates the expression
of genes encoding many cytoprotective and stress-responsive
enzymes, including heme oxygenase-1 (HO-1).4 We synthe-
sized two galloyl esters, namely, 3-O-galloylquercetin and 7-O-
galloyltaxifolin. These compounds have not been identified in
plants to date, even though plants contain gallic acid both in the
free form and esterified to sugars, for example, in tannins, and
to certain flavonoids such as catechins and proanthocyanidins.23
Previous studies have shown that quercetin8 and gallic acid
activate the Nrf2 signaling pathway.26 Quercetin has been
consistently found to induce HO-1 expression in several cell
models.15,27 Upregulation of HO-1 has also been demonstrated
in the hearts of rats that were orally administered gallic acid.28
HO-1 is a highly inducible enzyme that catalyzes the
degradation of heme into ferrous ion, the antioxidant biliverdin,
and anti-inflammatory agent carbon monoxide. The modulation
of HO-1 activity may hence represent a potential therapeutic
target for the treatment of diseases associated with inflamma-
tion and oxidative stress, such as inflammatory bowel disease29
and atherosclerosis.30
Our study on murine macrophage RAW264.7 cells confirms
the upregulation of HO-1 expression by quercetin and to a
lesser extent by gallic acid as well. In contrast, the expression of
HO-1 was unaffected by quercetin 3-O-conjugates, including
the semisynthetic galloyl ester 3-O-galloylquercetin and natural
glycoside isoquercitrin (quercetin-3-O-glucoside). Similarly to
isoquercitrin, the other quercetin glycosides, quercitrin
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Journal of Medicinal Chemistry
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(quercetin-3-O-rhamnoside) and rutin (quercetin-3-O-rhamno-
glucoside), were also found to be unable to induce the
expression of HO-1 in RAW264.7 cells.15 In our study, the
expression of HO-1 also remained unchanged by taxifolin,
which only differs from quercetin in the absence of a 2,3-double
bond. The inactivity of taxifolin agreed with previous results
obtained from the retinal ganglion RGC-5 cell line.31 On the
other hand, we report here that galloylation of the 7-OH group
in taxifolin provided a novel HO-1 inducer, 7-O-galloyltaxifolin,
which was found to increase both the mRNA and protein levels
of HO-1 at concentrations from 25 μM upward. HPLC/MS
analyses showed that 7-O-galloyltaxifolin was readily absorbed
by RAW264.7 cells and that the galloyl ester was stable with
respect to its possible hydrolytic cleavage. In contrast, a rapid
establishment of the equilibrium between the absorbed 7-O-
galloyltaxifolin and its oxidation product 7-O-galloylquercetin
was found, with an approximate parent compound/product
ratio of 1/8. To date, several plant enzymes including flavonol
synthase, anthocyanidin synthase,32 and horseradish perox-
idase33 have been shown to catalyze the oxidation of
dihydroquercetin to quercetin. The mechanism of the
dihydroquercetin skeleton oxidation in the structure of 7-O-
galloyltaxifolin presented herein remains unclear. Nonetheless,
the presence of the galloyl moiety seems to be important,
because ungalloylated taxifolin was found not to be oxidized to
quercetin in animal cells.24,34 Since both 7-O-galloyltaxifolin
and its oxidation product 7-O-galloylquercetin were simulta-
neously present in RAW264.7 cells soon after the start of the
cell treatment, we were unable to determine which member of
the redox couple was responsible for the biological response.
Nrf2 is the primary transcription factor involved in the
induction of antioxidant response element-driven gene
expression.4 The upregulation of Nrf2 activity is associated
with the nuclear accumulation of Nrf2, arising either from the
degradation of Keap1, a negative regulator of Nrf2, or from the
phosphorylation of Nrf2 by protein kinases.7 We demonstrated
that the induction of HO-1 expression by 7-O-galloyltaxifolin
was almost completely suppressed by the antioxidant N-
acetylcysteine, suggesting involvement of ROS in mediating the
biological effect of 7-O-galloyltaxifolin. ROS are known to
activate different classes of MAPKs.18 Our experiments revealed
that cell treatment with 7-O-galloyltaxifolin induced prompt
phosphorylation of p38 MAPKs, ERK1/2, and JNKs, followed
by the nuclear accumulation of Nrf2. Moreover, we found that
the induction of HO-1 by 7-O-galloyltaxifolin required the
activities of p38 MAPKs and ERKs but not JNKs, since
pharmacological inhibitors of p38 MAPKs and ERKs decreased
7-O-galloyltaxifolin-induced mRNA levels of HO-1 by 70% and
15%, respectively. These results suggest that the introduction of
a galloyl moiety into the molecule of taxifolin brings about the
ability to induce ROS production and MAPK activation.
Although 7-O-galloyltaxifolin also downregulated Keap1, this
event occurred later than the nuclear accumulation of Nrf2. We
may therefore presume that the downregulation of Keap1 was
not the primary reason for 7-O-galloyltaxifolin-induced HO-1
expression, but the decrease in Keap1 levels could potentiate
the effect of 7-O-galloyltaxifolin during longer treatments. Our
results show that 7-O-galloyltaxifolin induces HO-1 expression
primarily through the activation of protein kinase cascades.
Similarly, it has also been demonstrated that the upregulation of
HO-1 by quercetin involves the activation of the ERK1/2 and
p38 MAPK pathways15,27 and the effect of epigallocatechin-3-
gallate on HO-1 expression has been linked with the activation
of the phosphoinositide 3-kinase/Akt and ERK1/2 pathways.10
In summary, we have shown that galloylation or glycosylation
of the 3-OH group in quercetin removes its ability to induce
HO-1 expression in RAW264.7 cells. In contrast, we have
prepared a novel HO-1 inducer by galloylation of the 7-OH
group in taxifolin. The mechanism of 7-O-galloyltaxifolin-
induced HO-1 expression involves the generation of ROS and
phosphorylation of p38 MAPKs and ERK1/2, which are
associated with activation of the Nrf2 signaling pathway. Since
7-O-galloyltaxifolin is oxidized to 7-O-galloylquercetin in
RAW264.7 cells, further research should be aimed at synthesiz-
ing 7-O-galloylquercetin and evaluating its effect on the Nrf2
pathway.
EXPERIMENTAL SECTION
Chemistry: General Methods. NMR spectra were recorded on a
■
Bruker Avance III 600 MHz spectrometer (Bruker Daltonics, Billerica,
1
MA, USA; 600.23 MHz for H, 150.93 MHz for 13C at 30 °C) in
DMSO-d6 (99.9 atom % D; Sigma-Aldrich, Steinheim, Germany).
Residual signal of solvent was used as an internal standard (δH 2.500
ppm, δC 39.60 ppm). NMR experiments: 1H NMR, 13C NMR,
gCOSY, gHSQC, and gHMBC were performed using the
1
manufacturer’s software. H NMR and 13C NMR spectra were zero
filled to 4-fold data points and multiplied by window function before
Fourier transformation. Two-parameter double-exponential Lorentz−
1
Gauss function was applied for H to improve resolution and line
broadening (1 Hz) was applied to get better 13C signal-to-noise ratio.
Chemical shifts are given in δ-scale with digital resolution justifying the
reported values to three (δH) or two (δC) decimal places.
ESI-MS spectra were measured with Micromass LC-MS Platform in
MeOH with addition of HCO2H; HRMS spectra (ESI, APCI, FAB)
were measured with a LTQ Orbitrap XL instrument (Thermo Fisher
Scientific, Waltham, MA, USA) or with a ZAB-EQ instrument (VG
Analytical, Manchester, UK).
Purity of the tested compounds was determined by HPLC, and in
all cases, the purity reached at least 95% (for respective chromato-
grams, see Supporting Information). HPLC analyses were carried out
using a Shimadzu Prominence LC analytical system consisting of LC-
20AD binary HPLC pump, SIL-20AC cooling autosampler, CTO-
10AS column oven, and SPD-20MA diode array detector (Shimadzu,
Kyoto, Japan). A Chromolith Performance RP-18e monolithic column
(100 mm × 3 mm) equipped with a guard column (5 mm × 4.6 mm)
(Merck, Darmstadt, Germany) was used with an isocratic mobile
phase CH3CN/MeOH/H2O/TFA (2:37:61:0.1, flow rate 1.8 mL/
min) at 25 °C and UV detection at 285 nm (+ scan 200−365 nm).
7-O-Galloyltaxifolin (3). Taxifolin (1; 250 mg, 0.821 mmol) and
3,4,5-tri-O-benzylgalloyl chloride (568 mg, 1.239 mmol) were
dissolved in dry pyridine (10 mL) and stirred at 0 °C for 1 h. The
reaction was stopped by diluting in ice-cold HCl (1 M, 75 mL), the
solution was extracted with EtOAc (2 × 50 mL), and the organic
layers were combined, dried (Na2SO4), evaporated, and chromato-
graphed (CHCl3/acetone/HCO2H, 95:5:1) to yield 7-O-(3′,4′,5′-tri-
O-benzylgalloyl)taxifolin (2, 138 mg, 23%) as a white amorphous solid
(ESI-MS (m/z) 749 [M + Na]+; for 1H and 13C NMR data see Tables
S1 and S2 in Supporting Information). Compound 2 (100 mg, 0.138
mmol) was dissolved in EtOAc (10 mL) and then Pd/C (100 mg, 10%
Pd) was added and stirred under H2 for 3 h (20 °C). The Pd was then
removed by filtration through Celite, which was washed with acetone,
and the solvent was evaporated. Flash chromatography (CHCl3/
MeOH/HCO2H, 9:1:0.1) yielded title compound 3 (59 mg, 94%) as a
white amorphous solid. HRMS calcd [M + Na]+ 479.0585, found
479.0583. 1H NMR (600.23 MHz, DMSO-d6, 30 °C): δ 4.771 (d, 1H,
J = 11.7, H-3), 5.172 (d, 1H, J = 11.7, H-2), 6.417 (d, 1H, J = 2.1, H-
8), 6.450 (d, 1H, J = 2.1, H-6), 6.867 (d, 1H, J = 8.1, H-5′), 6.936 (dd,
1H, J = 2.1, 8.1, H-6′), 7.101 (d, 1H, J = 2.1, H-2′), 7.214 (s, 2H, Gal
o-CH). 13C NMR (150.93 MHz, DMSO-d6, 30 °C): δ 74.14 (C-3),
85.42 (C-2), 103.31 (C-8), 104.44 (C-6), 106.01 (C-4a), 111.21 (Gal
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Journal of Medicinal Chemistry
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ortho), 116.49 (C-5′), 116.58 (C-2′), 120.35 (Gal ipso), 121.55 (C-6′),
130.01 (C-1′), 141.22 (Gal para), 146.50 (C-3′), 147.22 (Gal meta),
147.42 (C-4′), 161.05 (C-7), 164.30 (C-5), 164.30 (C-8a), 165.22
(Gal CO), 200.72 (C-4). Gal = galloyl moiety.
and the supernatants were analyzed by HPLC/MS. The chromato-
graphic separation was performed in an Agilent Zorbax Eclipse XDB-
phenyl column (150 mm × 2.1 mm i.d., 5 μm; Agilent Technologies,
CA, USA) using an Acquity UPLC system (Waters, Milford, MA,
USA) equipped with a binary solvent manager, sample manager,
column manager, and photodiode array detector. The autosampler was
conditioned at 4 °C, and the injection volume was 5 μL. A binary
gradient elution was performed at a flow rate of 0.4 mL/min; column
temperature was maintained at 35 °C. Solvent A was composed of
water with 1% (v/v) acetic acid and methanol (90:10, v/v), and
solvent B was methanol. The gradient profile was as follows: 0−14 min
10−50% B, 14−16 min 50−100% B, 16−18 min 100−10% B, 18−20
min 10% B. The Waters QqTof Premier mass spectrometer (Waters,
Manchester, U.K.) was connected to the UPLC system via an
electrospray ionization (ESI) interface. The ESI source operated in
negative ionization mode with the capillary voltage at 2.1 kV and the
sampling cone at 40 V. The source temperature and the desolvation
temperature were set to 120 and 300 °C, respectively. The cone and
desolvation gas flows were 0 and 500 L/h, respectively. Data were
acquired from 50 to 1000 Da with a 0.5 s scan time. Data acquisition
was achieved using two interleaved scan functions (MS and MSE
experiments), which enabled the simultaneous acquisition of both low
collision energy and high collision energy mass spectra from a single
experiment. The collision energy was set to 5 V for Function 1 and
20−35 V for Function 2. Acquiring data in this manner enabled the
collection of intact precursor ions as well as fragment ion information
in an unbiased manner. Postacquisition processing of the data was
performed using the program Metabolynx V4.1 (Waters, Milford, MA,
USA).
3-O-Galloylquercetin (8). 3-O-[(3″,4″,5″-Tri-O-methoxymethyl)-
galloyl]quercetin (7; 250 mg, 0.426 mmol, for the preparation see
Supporting Information) was dissolved in MeOH (15 mL),
concentrated HCl (1 mL) was added, and the resulting mixture was
stirred at room temperature for 3 h. The reaction was quenched by the
addition of a saturated solution of NaHCO3 (till neutral pH),
evaporated to dryness by coevaporation with absolute EtOH,
redissolved in acetone, filtered to remove inorganic salts, and
evaporated again. The dry residue yielded title compound 8 (160
mg, 83%) after flash chromatography (CHCl3/MeOH/HCO2H, from
95:5:1 to 90:10:1). HRMS calcd [M + Na]+ 477.0428, found
477.0427. 1H NMR (600.23 MHz, DMSO-d6, 30 °C): δ 6.197 (d, 1H,
J = 2.0, H-6), 6.435 (d, 1H, J = 2.0, H-8), 6.829 (d, 1H, J = 8.4, H-5′),
7.214 (s, 2H, Gal o-CH), 7.262 (dd, 1H, J = 2.3, 8.4, H-6′), 7.348 (d,
1H, J = 2.3, H-2′). 13C NMR (150.93 MHz, DMSO-d6, 30 °C): δ
94.32 (C-8), 99.51 (C-6), 102.83 (C-4a), 109.34 (Gal ortho), 114.87
(C-2′), 115.81 (C-5′), 116.62 (Gal ipso), 119.45 (C-1′), 120.40 (C-
6′), 129.79 (C-3), 140.73 (Gal para), 145.80 (C-3′), 145.97 (Gal
meta), 149.98 (C-4′), 155.85 (C-2), 156.81 (C-8a), 161.11 (C-5),
163.38 (Gal CO), 166.45 (C-7), 174.90 (C-4). Gal = galloyl moiety.
Biological Testing. Reagents. Quercetin, gallic acid, anisomycin,
N-acetyl-L-cysteine, PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-
benzopyran-4-one), SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfi-
nylphenyl)-5-(4-pyridyl)-1H-imidazole), SP600125 (1,9-pyrazoloan-
throne), human epidermal growth factor, and dimethyl sulfoxide
(DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Taxifolin was obtained from Amagro (Prague, Czech Republic).
Isoquercitrin (98% purity, HPLC) was prepared by the enzymatic
procedure as described previously.35 Nitrogen, argon, and helium (all
99.999%) were obtained from Linde Gas (Prague, Czech Republic).
Water for HPLC was prepared using an Ultrapur reverse osmosis-
deionizer system (Watrex, Prague, Czech Republic).
Reverse Transcription and Quantitative Real-Time PCR. After
treatment, total RNA was extracted using TRI Reagent Solution
(Applied Biosystems, Foster City, CA, USA), and the concentration of
RNA was determined by spectrophotometry at 260 nm. RNA samples
(2 μg) were reverse transcribed using a High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems) and real-time PCR was
performed in a LightCycler 480 II system (Roche Diagnostics,
Mannheim, Germany) using TaqMan Universal PCR Master Mix and
TaqMan Gene Expression Assays consisting of specific primers and
FAM dye-labeled TaqMan minor groove binder probes (Applied
Biosystems). The assay ID was Mm00516005_m1 for Hmox1 and
Mm99999915_g1 for Gapdh. Amplification conditions were 50 °C for
2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and
60 °C for 1 min. Crossing point values, equivalent to CT, were
determined automatically using the second derivative maximum
analysis. Relative changes in gene expression were calculated by the
RAW264.7 Cell Culture and Treatment. The murine macrophage
RAW264.7 cell line (No. 91062702, ECACC, Salisbury, U.K.) was
cultured at 37 °C in Dulbecco’s modified Eagle’s medium (D5796,
Sigma) supplemented with 100 U/mL penicillin, 100 μg/mL
streptomycin (Invitrogen, Carlsbad, CA, USA), and 10% fetal bovine
serum (Biochrom, Berlin, Germany) in a humidified atmosphere
containing 5% CO2. Cells were regularly subcultured before
confluence. For all experiments, RAW264.7 cells at passage 5−25
were seeded in the complete culture medium into 6-well plates at a
density of 1 × 105 cells/cm2. After 8 h of stabilization, the culture
medium was exchanged for the serum-free medium. After overnight
incubation, cells were treated in serum-free medium with the tested
compounds. Negative controls were treated with 0.1% (v/v) DMSO
only.
Cell Viability Assay. After treating RAW264.7 cells with 0.1%
DMSO (control), the tested compounds or 1.5% (v/v) Triton X-100
(positive control), the cell viability was determined using an MTT
reduction assay. In brief, cells were washed with PBS and incubated for
2 h at 37 °C in fresh serum-free medium containing 0.5 mg/mL MTT
(Sigma). After this, the medium was removed, and the intracellular
formazan produced by active mitochondria was solubilized in DMSO.
The absorbance at 540 nm was measured on a spectrophotometric
plate reader and used to calculate relative cell viability, where cells
treated with DMSO alone represented 100% viability.
HPLC/MS Analysis. After treatment, RAW264.7 cells were scraped
from the plates, collected by gentle centrifugation, washed twice with
PBS, resuspended in 0.4 mL of methanol containing 5% (v/v) acetic
acid and sonicated 10 times at 50% amplitude with a cycle set at 0.5 s
using an Ultrasonic Processor UP200s equipped with a Sonotrode
Microtip S2 sonicator probe (Hielscher, Teltow, Germany). Afterward,
the cell lysates were centrifuged for 2 min at 14 000 × g at room
temperature, and the supernatants were analyzed by HPLC/MS.
Aliquots of culture medium were diluted (1:1, v/v) in methanol
containing 5% (v/v) acetic acid, centrifuged for 2 min at 14 000 × g,
comparative CT method using the 2−ΔΔC equation with results
T
normalized to Gapdh mRNA levels.
Western Blot Analyses. To prepare total cellular extracts, cells were
washed with cold PBS, scraped from the plates, pelleted by
centrifugation for 3 min at 1500 × g and 4 °C and lysed in lysis
buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease
inhibitors Complete (Roche Diagnostics), 0.2% (m/v) sodium
dodecyl sulfate (SDS), 1% (v/v) Nonidet-P40, 1% (v/v) Triton X-
100, pH 7.4). After incubation on ice for 30 min, whole cell lysates
were centrifuged for 10 min at 16 000 × g and 4 °C, and the
supernatants were collected.
To prepare nuclear and cytosolic extracts, cells were collected from
the plates as described above, and the cell pellets were resuspended in
ice-cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.1% (v/v) Nonidet-P40, 0.5 mM dithiothreitol (DTT),
protease inhibitors Complete). The cells were allowed to swell on ice
for 10 min, centrifuged for 10 min at 12 000 × g and 4 °C, and the
supernatants were collected as the cytosolic fraction. The pellets were
vigorously resuspended in ice-cold buffer B (20 mM HEPES, pH 7.9,
420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol,
0.5 mM DTT, 0.5 mM PMSF) and incubated for 30 min on ice. The
suspensions were centrifuged for 10 min at 12 000 × g and 4 °C, and
the supernatants were collected as the nuclear fraction.
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Proteins in all samples were quantified using the Pierce BCA
Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Aliquots
containing an equal amount of protein were subjected to electro-
phoresis through 10% SDS−polyacrylamide gel, proteins were
transferred to polyvinylidene difluoride membrane by electroblotting,
and the membranes were probed with appropriate primary antibodies.
Rabbit polyclonal heme oxygenase-1 (sc-10789), rabbit polyclonal
Nrf2 (sc-722), goat polyclonal Keap1 (sc-15246), and goat polyclonal
actin (sc-1616) antibodies were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal phospho-
p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#9101), rabbit
polyclonal p44/42 MAPK (ERK1/2) (#9102), rabbit polyclonal
phospho-SAPK/JNK (Thr183/Tyr185) (#9251), rabbit polyclonal
SAPK/JNK (#9252), rabbit monoclonal phospho-p38 MAPK
(Thr180/Tyr182) (D3F9) XP (#4511), and rabbit monoclonal p38
MAPK (D13E1) XP (#8690) antibodies were obtained from Cell
Signaling Technology (Danvers, MA, USA). Primary antibodies were
visualized with rabbit anti-goat or goat anti-rabbit horseradish
peroxidase-conjugated secondary antibodies using a chemiluminescent
reaction. The relative band intensities were determined by
densitometric analysis using ImageJ software (National Institutes of
Health, Bethesda, MD, USA).
Flow Cytometric Detection of ROS. RAW264.7 cells were treated
with 7-O-galloyltaxifolin or with 100 μM tert-butyl hydroperoxide
(TBHP; positive control; Merck). After treatment, cells were
centrifuged for 3 min at 110 × g, and the medium was exchanged
for fresh serum-free medium. To detect intracellular ROS, cells were
incubated with 10 μM 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluor-
escein diacetate17 (carboxy-H2DCFDA; Invitrogen) for 15 min at 37
°C. After incubation, cells were scraped from the plate and
immediately analyzed by flow cytometry on a Cytomics FC 500
(Beckman Coulter, Fullerton, CA, USA). Cells were excited with a
488-nm argon laser line and analyzed on FL1 channel (525 nm),
counting 10 000 events per sample. The percentage of cells with
increased ROS levels was calculated using MultiCycle AV software
(Phoenix Flow Systems, San Diego, CA, USA).
of Education, Youth and Sports of the Czech Republic, and by
Grant LF_2012_010 from Palacky University.
́
ABBREVIATIONS USED
■
3GQ, 3-O-galloylquercetin; 7GT, 7-O-galloyltaxifolin; APCI,
atmospheric pressure chemical ionization; carboxy-H2DCFDA,
5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate;
EGF, epidermal growth factor; ERK, extracellular signal-
regulated kinase; ESI-QqTOF MS, electrospray ionization/
quadrupole time-of-flight mass spectrometry; GA, gallic acid;
Gapdh, glyceraldehyde-3-phosphate dehydrogenase gene;
HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid;
Hmox1, heme oxygenase-1 gene; HO-1, heme oxygenase-1;
IQ, isoquercitrin; JNK, c-Jun N-terminal kinase; Keap1, Kelch-
like ECH-associated protein 1; MAPK, mitogen-activated
protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; NAC, N-acetyl-L-cysteine; Nrf2,
nuclear factor erythroid 2-related factor 2; p38 MAPK, p38
mitogen-activated protein kinase; PD98059, 2-(2-amino-3-
methoxyphenyl)-4H-1-benzopyran-4-one; PMSF, phenylme-
thylsulfonyl fluoride; QUE, quercetin; SB203580, 4-(4-
fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-
imidazole; SP600125, 1,9-pyrazoloanthrone; TAX, taxifolin
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ASSOCIATED CONTENT
■
S
* Supporting Information
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +420 585632310. Fax: +420 585632302. E-mail:
vrbambv@seznam.cz.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr Alexander Oulton for providing linguistic
assistance. This work was supported by Grant P301/11/0767
from the Czech Science Foundation, by Grant CZ.1.05/2.1.00/
01.0030 and Grant CZ.1.05/2.1.00/03.0058 from the Ministry
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