Clarification of the role of quercetin hydroxyl groups in superoxide generation and cell apoptosis by chemical modification

Article abstract of DOI:10.1271/bbb.90253

Accumulated data have suggested that the hydroxyl groups of flavonoids are important for their bioactive function. To directly demonstrate the role of hydroxyl groups, we synthesized a derivative of quercetin, 3,7,3',4'-0- tetrabenzylquercetin (4Bn-Q) tha

Full text of DOI:10.1271/bbb.90253

Biosci. Biotechnol. Biochem., 73 (9), 2048–2053, 2009
Clarification of the Role of Quercetin Hydroxyl Groups
in Superoxide Generation and Cell Apoptosis by Chemical Modification
y
1;2;
Kozue SAKAO,1 Makoto FUJII,1;2 and De-Xing HOU
1Course of Biological Science and Technology, United Graduate School of Agricultural Sciences,
Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan
2Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University,
1-21-24 Korimoto, Kagoshima 890-0065, Japan
Received April 7, 2009; Accepted May 23, 2009; Online Publication, September 7, 2009
[doi:10.1271/bbb.90253]
Accumulated data have suggested that the hydroxyl
groups of flavonoids are important for their bioactive
function. To directly demonstrate the role of hydroxyl
groups, we synthesized a derivative of quercetin,
3,7,30,40-O-tetrabenzylquercetin (4Bn-Q) that substi-
tuted the hydroxyl groups of quercetin with benzyl
groups, and then evaluated the ability to inhibit cell
proliferation and cause apoptosis in human leukemia
(HL-60) cells. The results reveal that quercetin, but not
4Bn-Q, inhibited cell proliferation and induced apopto-
sis as characterized by DNA fragmentation, activation of
caspase-3, and PARP cleavage. Treatment with 4Bn-Q
reduced the intracellular level of quercetin-induced
superoxide, and the scavenger of superoxide, Mn (III)
tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP),
reduced the superoxide level and apoptosis induced by
quercetin. These findings directly demonstrate that the
hydroxyl groups of quercetin contributed to the gener-
ation of intracellular superoxide, consequently inhibiting
proliferation and inducing apoptosis in HL-60 cells.
it is one of the best-studied flavonoids and can inhibit
cell proliferation11,12) and induce apoptosis in a variety
of cancer cells.13–17) To directly clarify the role of the
hydroxyl groups of quercetin in its bioactive functions,
we synthesized the quercetin derivative, 3,7,30,40-
O-tetrabenzylquerctin (4Bn-Q) which substituted the
hydroxyl groups of quercetin with benzyl groups at the
3,7,30,40 positions. The bioactive function of these two
compounds was then evaluated for the ability to inhibit
cell proliferation and induce apoptosis in human
leukemia (HL-60) cells which provide a valid model
for testing antileukemic or general antitumoral com-
pounds. Our results show that quercetin significantly
inhibited cell proliferation by caspase-mediated apopto-
sis. However, 4Bn-Q lost this function. Intracellular
reactive oxygen species (ROS) data show that the
hydroxyl groups of quercetin might have contributed to
superoxide generation, consequently inhibiting prolifer-
ation and inducing apoptosis in HL-60 cells.
Materials and Methods
Key words: quercetin; chemical modification; superox-
ide; apoptosis
Synthesis of 4Bn-Q. 4Bn-Q was synthesized as previously
described.18) Briefly, a solution of quercetin (1.0 equiv.) in N,N-
dimethylformamide (DMF), K2CO3 (3.5 equiv.) and benzyl bromide
(3.5 equiv.) were mixed. After vigorously stirring at 0 ꢀC for 2 h, the
reaction mixture was warmed to room temperature over 2 h and stirred
for another 12 h. The resulting mixture was diluted with water
(400 ml), and then extracted with EtOAc. The organic layer was
washed with water and dried over MgSO4. The residue was purified by
column chromatography, using dichloromethane as the eluent, to
afford the tetrabenzylether. The chemical structure of benzylated
quercetin was determined by NMR, which was recorded on a
spectrometer (ECA-600, 600 MHz for 1H , Jeol, Japan) using [D6]
CDCl3 as the solvent, and tetramethylsilane (TMS) as the internal
standard. Chemical shifts and J values are given in Hz.
Flavonoids are polyphenolic compounds that are
widely distributed in many edible plants. Accumulated
data have revealed flavonoids to have strong anti-
oxidative effects1) and a variety of bioactivities.2–4)
Furthermore, structure-activity relationships have been
suggested by investigating the structural diversity based
on the different aglycone, glycosidation and acylation
patterns of flavonoids.5–8) In particular, the hydroxyl
groups of flavonoids have been suggested to play a
critical role in their bioactivities. For example, the
hydroxyl number of a flavonoid is important for its
radioprotective effects9) and caspase-3 activation.5) Our
previous reports have indicated that the bioactivities of
flavonoids appeared to be associated with the structure
of the B-ring.10) However, most of this speculation was
made by comparing the different number of hydroxyl
groups among flavonoids. Little evidence was obtained
from direct structural modification. In this present study,
we chose quercetin as an experimental material because
Materials and cell culture. Quercetin, protein kinase K, RNase A
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were from Sigma. The antibodies against poly (ADP) ribose
polymerase (PARP) and caspase-3 were from Cell Signaling
Technology. Fetal bovine serum (FBS) was from BioWhittaker Co.,
and Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP)
was from Calbiochem. The human leukemia (HL-60) cell line was
obtained from Cancer Cell Repository (Tohoku University, Japan)
and was cultured at 37 ꢀC with 5% CO2 in an RPMI-1640 medium
y
To whom correspondence should be addressed. Tel/Fax: +81-99-285-8649; E-mail: hou@chem.agri.kagoshima-u.ac.jp
Abbreviations: 4Bn-Q, 3,7,30,40-O-tetrabenzylquercetin; DHE, dihydroethidium; HL-60, human leukemia cell; HPF, hydroxyphenyl fluoresceꢁinꢂ;
MnTBAP, Mn (III) tetrakis (4-benzoic acid) porphyrin chloride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; O2
superoxide; OH, hydroxyl radical; ROS, reactive oxygen species
,

Quercetin-Induced Superoxide and Apoptosis
containing 10% FBS and 1% penicillin-streptomycin glutamine
(PSG).
2049
OH
OH
O
O
HO
O
Cell viability assay. The cell survival rate was measured by an
MTT assay.19) HL-60 cells were suspended at
density of
O
O
O
a
1:5 ꢃ 104 cells/well in an RPMI-1640 medium containing 10% FBS
and 1% PSG. After 24 h of preculture, the cells were treated with 25–
100 mM of quercetin or 4Bn-Q for 48 h. An MTT solution (0.5 mg/ml)
was then added to each well, and the cells incubated for another 4 h.
The resulting MTT-formazan product was dissolved by the addition of
100 ml of 0.04 N HCl-2-propanol and determined by measuring the
absorbance at 595 nm with a micro-plate reader. The cell viability is
expressed as the optical density ratio of the treated product to the
control.
OH
O
OH
O
OH
Quercetin
4Bn-Q
Fig. 1. Chemical Structures of Quercetin (left) and 3,7,30,40-O-
Tetrabenzylquercetin (4Bn-Q) (right).
100
80
DNA fragmentation assay. The DNA fragmentation assay was
carried out as described.10) HL-60 cells (1:0 ꢃ 106 cells/3 ml/dish)
were treated with 25–50 mM of quercetin or 4Bn-Q for 8 h. The cells
were harvested by centrifugation and washed in ice-cold PBS. The
resulting pellets were resuspended in a lysis buffer (50 mM Tris–HCl at
pH 8.0, 10 mM EDTA, and 0.5% SDS) plus 0.1 mg/ml of RNase A.
After 30 min of incubation at 37 ꢀC, proteinase K was added and the
mixture was incubated for another 3 h. DNA was separated by 2%
agarose gel electrophoresis and digitally imaged after staining with
ethidium bromide.
60
40
Determination of apoptosis by flow cytometry. Apoptotic DNA was
measured by flow cytometry as described previously.20) Briefly, after
being treated with each sample, HL-60 (2:0 ꢃ 106) cells were fixed
overnight in 70% ethanol at ꢁ20 ꢀC and then resuspended in PBS. The
cells were stained in 1 ml of PBS containing 10 mg/ml of RNase for
30 min in room temperature, and then 25 mg/ml of propidium iodide
was added and the mixture stained in the dark. Fluorescence emitted
from the propidium-DNA complex was detected with a flow cytometer
(CyFlow, Partec). Apoptotic nuclei were distinguished by their
hypodiploid DNA content from the diploid DNA content of normal
nuclei.
Quercetin
4Bn-Q
20
0
0
20 40 60 80 100
Concentration (µM)
Fig. 2. Influence of Quercetin and 4Bn-Q on the Viability of HL-60
Cells.
Western blot analysis. After being treated with each sample,
HL-60 (2:0 ꢃ 106) cells were lysed with a modified RIPA buffer
containing 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA,
0.1% Nonidet P-40, 1% deoxycholate, 50 mM sodium fluoride, 50 mM
sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and a
proteinase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The
lysate was sonicated three times for 10 s and then centrifuged at
14,000 g for 15 min at 4 ꢀC. Equal amounts of the lysate protein were
run on SDS–PAGEs and electrophoretically transferred to a PVDF
membrane (Amersham Pharmacia Biotech). After blotting, the
membrane was first blocked with a TBST buffer (500 mM NaCl,
20 mM Tris–HCl (pH 7.4) and 0.1% Tween 20) containing 5% non-fat
dried milk, and then incubated overnight with specific antibodies at
4 ꢀC and further incubated for 1 h with the HRP-conjugated secondary
antibody. Bound antibodies were detected by using the ECL system,
and the relative amounts of proteins associated with the specific
antibody were quantified by using Lumi Vision Imager software
(TAITEC).
HL-60 cells were placed in 96-well plates for 24 h, and then
treated with or without different concentrations of each compound
for another 48 h. The cell density was assessed colorimetrically after
staining with MTT and is expressed as the optical density ratio of the
treated to control cells at 595 nm. Cell survival is expressed as the
optical density ratio of the treated to control cells. Data are presented
as the mean ꢄ SD of three independent experiments.
Results
Synthesis of quercetin derivative, 4Bn-Q
In order to replace the hydroxyl groups of quercetin,
3,7,30,40-O-tetrabenzylquercetin was synthesized and
purified via column chromatography. The chemical
1
structure of 4Bn-Q (Fig. 1) was determined by a H-
NMR analysis as follows: 4.98 (s, 2H, OCH2Ph), 5.03
(s, 2H, OCH2Ph), 5.11 (s, 2H, OCH2Ph), 5.24 (s, 2H,
OCH2Ph), 6.42 (d, J ¼ 1:8 Hz, 1H, aromatic H), 6.45
(d, J ¼ 2:4 Hz, 1H, aromatic H), 6.94 (d, J ¼ 9:0 Hz,
1H, aromatic H), 7.22–7.45 (m, 20H, aromatic H), 7.53
(dd, J ¼ 9:0, 1.8 Hz, 1H, aromatic H), 7.70 (d, J ¼
2:4 Hz, 1H, aromatic H). Therefore, the position of the
benzyl group was assigned to the 3,7,30,40-position of
quercetin.
Intracellular ROS assay. Intracellular superoxide was measured
by using dihydroethidium (DHE) fluorescein (Molecular Probes).
DHE has been reported to be oxidized specifically by superoxide
anions21) and is one of the most frequently used fluorescence probes
for measuring intracellular superoxide. The intracellular hydroxyl
radical (ꢂOH) was measured by using hydroxyphenyl fluorescein
(HPF; Molecular Probes). HPF is a fluorescence probe to selectively
detect the hydroxyl radical and partial ONOO–, and is frequently
used to detect the intracellular hydroxyl radical.22) In brief, HL-60
cells (1:0 ꢃ 106) were treated with quercetin or 4Bn-Q for various
times and then incubated with 20 mM DHE or 10 mM HPF for 30 min
at 37 ꢀC. The cells (1:0 ꢃ 106) were analyzed at FL1 (530 nm) for
HPF and FL2 (585 nm) for DHE with a flow cytometer (CyFlow,
Partec).
Effect of quercetin and 4Bn-Q on cell proliferation
To compare the effect of quercetin and 4Bn-Q on the
proliferation of HL-60 cells, cells were treated with 25–
100 mM of each compound. After a 48-h culture, the cell
survival rate was investigated by an MTT assay. As

2050
K. SAKAO et al.
A
Quercetin
25
50
4Bn-Q
25 50
(µM)
M
C
B
Control
Quercetin
4Bn-Q
600
400
200
0
600
600
400
200
0
400
200
0
5.7%
3.9%
27.9%
0
1
2
0
1
2
0
1
2
10
10
10
10
10
10
10
10
10
PI LOG
PI LOG
DNA content
PI LOG
C
Control
Quercetin
4Bn-Q
Full-length
Cleaved
Caspase-3
PARP
Full-length
Cleaved
Fig. 3. Apoptosis Induction of HL-60 Cells by Quercetin and 4Bn-Q.
Cells were grown in the absence (control) or the presence of quercetin (25 mM) or 4Bn-Q (25 mM) for 8 h. A, DNA fragmentation. DNA was
extracted and then separated by 2.0% agarose gel electrophoresis. The gel was visualized under ultraviolet light after staining with ethidium
bromide (M, DNA marker; C, control). B, Quantification of apoptotic DNA. Harvested cells were fixed in 70% ethanol and stained with
propidium iodide, before being subjected to a flow cytometric analysis. The percentages of cells with hypodiploid DNA content represent the
fractions undergoing apoptotic DNA. C, caspase activation and PARP cleavage. Cellular lysate was applied to 15% or 10% of SDS–PAG
caspases, and PARP were detected with the corresponding specific antibodies, being visualized by a chemiluminescence ECL kit.
shown in Fig. 2, quercetin inhibited cell proliferation in
the concentration range of 25–100 mM with an IC50 (50%
inhibitory concentration) value of 58 mM, while 4Bn-Q
showed no inhibition under the same condition, losing
the ability to inhibit cell proliferation.
apoptosis, and the cleavage of poly-(ADP ribose)
polymerase (PARP), which responds to DNA strand
breaks and is used as another hallmark of apoptosis,
occured in the quercetin-treated cells (25 mM, 8 h), but
not in the 4Bn-Q-treated cells (Fig. 3C). Therefore,
quercetin might have suppressed cell proliferation by
inducing apoptosis, and 4Bn-Q lost this ability.
Effect of quercetin and 4Bn-Q on cell apoptosis
To elucidate whether quercetin and 4Bn-Q could
induce apoptosis in HL-60 cells, we characterized HL-
60 cells by several approaches. DNA fragmentation was
detected in the cells treated with 25–50 mM of quercetin,
but not in the cells treated with 25–50 mM of 4Bn-Q
(Fig. 3A). The percentage of cells with hypodiploid
DNA was further quantified as 3.9% for the control, and
27.9% and 5.7% for the treatment by quercetin or 4Bn-Q
(25 mM, 8 h), respectively (Fig. 3B). The significant
difference in apoptotic DNA was observed between
control and quercetin treatment, but not between control
and 4Bn-Q treatment. Molecular data further revealed
that the active form of caspase 3, an initiator caspase of
Effect of quercetin and 4Bn-Q on the intracellular
ROS level
Several lines of evidence have suggested that some
flavonoids induce tumor cell apoptosis, in part, by
enhancing the intracellular ROS level.23,24) To know the
mechanism underlying apoptosis induction by quercetin,
we measured the intracellular levels of various ROS in
quercetin- or 4Bn-Q-treated cells by using fluorescent
probes. As shown in Fig. 4A, quercetin markedly
increased the level of superoxide (O2ꢁꢂ), while 4Bn-Q
did not affect the level. EGCG was used as a negative
control.25) On the other hand, quercetin and 4Ben-Q did

Quercetin-Induced Superoxide and Apoptosis
2051
B
A
.
-.
Hydroxyl radical ( OH)
Superoxide (O2
)
300
250
200
150
100
50
Quercetin
300
250
200
150
100
50
Quercetin
EGCG
4Bn-Q
Control
Control
4Bn-Q
EGCG
0
0
100
101
102
100
101
102
FL1-LOG (HPF)
FL2-LOG (DHE)
Fig. 4. Influence of Quercetin and 4Bn-Q on Intracellular ROS Levels.
HL-60 cells were grown in the absence (control) or presence of quercetin (25 mM) or 4Bn-Q (25 mM) for 30 min. The harvested cells were then
respectively incubated with 20 mM of DHE or 10 mM of HPF for another 30 min and analyzed by flow cytometry as described in the Materials and
Methods section.
not markedly affect the level of the hydroxyl radical
(ꢂOH) (Fig. 4B). As a positive control, EGCG markedly
increased the intracellular level of ꢂOH.23) We also
performed a time-course assay from 15 min to 120 min
to measure the levels of these ROS, and obtained similar
results to that with the treatment for 30 min (data not
shown). These data suggest that the presence of
hydroxyl groups might be essential for quercetin to
generate superoxide, because 4Bn-Q, which substituted
the hydroxyl groups of quercetin by benzalytion, lost
this ability. It is also interesting that ROS generated by
quercetin and EGCG were different, although both
compounds could induce apoptosis in HL-60 cells,
suggesting they may have a different mechanism for
inducing apoptosis.
tion of the hydroxyl groups of quercetin has been done
by acylation26) and methylation.27) It has been reported
that the acylated derivative of quercetin was hydrolyzed
in cultured cells,28) and that the methylated-derivative of
quercetin was easily metabolized by glycosylation.29) To
substitute the hydroxyl group of quercetin by a stable
group, we synthesized a benzylated derivative of
quercetin (4Q-B). Our results reveal that quercetin
inhibited cell proliferation and induced apoptosis, but
that 4Bn-Q had lost this ability. These data indicate that
the bioactivity of quercetin appears to be associated with
the presence of the hydroxyl groups. The question
remains as to that how the hydroxyl groups of quercetin
affect cell proliferation or apoptosis induction. It has
been reported that flavonoids such as apigenin, querce-
tin, myricetin and kaempferol were able to induce
apoptosis in HL-60 cells by elevating the intracellular
ROS level, although it is unclear which ROS were
essential for apoptosis induction by quercetin.5) Super-
oxide and hydroxyl radicals are major intracellular ROS.
To demonstrate whether these ROS would be involved
in quercetin-induced apoptosis, we examined the intra-
cellular levels of superoxide and hydroxyl radicals with
specific fluorescent probes. The data revealed that
treatment with quercetin increased the level of super-
oxide, and the 4Bn-Q had no such effect. On the other
hand, there was no significant change in the levels of the
hydroxyl radicals between the two compounds. Thus,
the hydroxyl groups of quercetin might contribute to
the generation of superoxide, rather than the hydroxyl
radical.
MnTBA, a scavenger of superoxide, reduces intra-
cellular superoxide and cell apoptosis
To further demonstrate whether the increased super-
oxide level by quercetin was associated with cell
proliferation inhibition and apoptosis induction, we used
the scavenger of superoxide, MnTBAP, to scavenge
quercetin-induced intracellular superoxide. The cells
were treated with quercetin in the presence or absence of
MnTBAP, and the intracellular superoxide level and cell
apoptosis were then determined by flow cytometry. As
shown in Fig. 5A, pretreatment with MnTBAP reduced
the level of quercetin-induced superoxide, although the
superoxide was not completely reduced to the control
level. Moreover, the percentage of apoptotic DNA in the
cells pretreated with MnTBAP pretreatment was 13.9%,
lower than that in the cells treated by quercetin alone
(21.5%) (Fig. 5B).
To further clarify whether the increased superoxide
level by quercetin was linked to cell proliferation
inhibition and apoptosis induction, a scavenger of
superoxide (MnTBAP) was used. The results revealed
that MnTBAP reduced quercetin-induced superoxide,
although the superoxide was not completely reduced to
the control level. Moreover, the percentage of apoptotic
DNA (13.9%) in the cells pretreated with MnTBAP
was lower than that in the quercetin-treated cells
(21.5%) (Fig. 5B). Our data demonstrate, at least in
part, that superoxide contributed to quercetin-induced
apoptosis.
Discussion
Accumulated data have suggested that the hydroxyl
groups of flavonoids play critical roles in their bioactive
functions. To directly demonstrate the role of hydroxyl
groups, we synthesized quercetin derivative 4Bn-Q,
which substituted the hydroxyl groups with benzyl
groups, and then evaluated its ability to suppress cell
proliferation in HL-60 cells, this being a valid model for
testing antileukemic or general antitumoral compounds
and for clarifying the molecular mechanism. Substitu-
Finally, the hydroxyl group at position 5 could not be
benzylated in the present study. This may have been due

2052
K. SAKAO et al.
A
-.
Superoxide (O2
)
300
250
200
150
100
50
Quercetin
+ MnTBAP
Quercetin
Control
MnTBAP
0
10
0
1
2
10
10
FL2-LOG (DHE)
B
Control
Quercetin
Quercetin + MnTBAP
MnTBAP
348
232
116
0
348
232
116
0
348
232
116
0
348
232
116
0
4.7 %
13.9 %
21.5 %
5.5 %
0
1
2
0
1
2
0
1
2
0
1
2
10
10
10
10
10
10
10
10
10
10
10
10
PI LOG
PI LOG
PI LOG
PI LOG
DNA content
Fig. 5. Effect of MnTBAP on Quercetin-Induced Superoxide and Apoptosis.
A, Intracellular superoxide production in HL-60 cells. Cells were pretreated with MnTBAP (100 mM) for 1 h, and then treated with quercetin
(25 mM) for another 30 min. The harvested cells were incubated with 20 mM of DHE for 30 min and analyzed by flow cytometry as described in
the Materials and Methods section. B, Quantification of apoptotic DNA. HL-60 cells were pretreated with MnTBAP (100 mM) for 1 h and then
treated with quercetin for 8 h. The harvested cells were fixed in 70% ethanol and stained with propidium iodide, before being subjected to flow
cytometric analysis. The percentages of cells with hypodiploid DNA contents represent the fractions undergoing apoptotic DNA.
to its poor reactivity, because the hydrogen bond
between the hydroxyl group at position 5 and the keto
group at position 4 was quite stable under the present
synthetic conditions. On the other hand, we also
attempted to benzylate the hydroxyl groups at only
positions 30 and 40 to clarify the role of the B-ring
hydroxyl groups in intracellular ROS generation. How-
ever, the hydroxyl groups at positions 3 and 7 revealed
reactivity as high as that at positions 30 and 40 under the
present synthetic conditions. We therefore obtained a
tetrabenzylquercetin (4Bn-Q) which is a benzylated
derivative of quercetin at 3,7,30,40 positions. Based on
our data and other reports, we can conclude that these
hydroxyl groups, at least, contributed to generation of
intracellular ROS. To clarify the role of the hydroxyl
groups at individual positions, we intend to obtain the
different derivatives of quercetin by modifying the
synthetic conditions.
Acknowledgments
This work was partly supported by funding from
the Frontier Science Research Center of Kagoshima
University to D-X. Hou.
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