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Int J Food Sci Nutr, Early Online: 1–7

! 2014 Informa UK Ltd. DOI: 10.3109/09637486.2014.931362

RESEARCH ARTICLE

Flavonols and flavan-3-ols as modulators of xanthine oxidase and

manganese superoxide dismutase activity
Danila Di Majo1, Maurizio La Guardia2, Gaetano Leto1, Marilena Crescimanno1, Carla Flandina1, and
Marco Giammanco1
1
Unit of Physiology and Pharmacology, Department DIGISPO, University of Palermo, Italy and 2Division of Cell Biology, Department STEBICEF,
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University of Palermo, Italy

Abstract Keywords
Experiments were performed to assess the dose-dependent effects of quercetin, kaempferol, Antioxidant, dose response, functional food,
(+) catechin, and () epicatechin on superoxide radical production through the modulation of inhibitor, pro-oxidant, structure-activity
manganese superoxide dismutase and xanthine oxidase activities. The experiments were relationship
carried out at flavanoid concentrations ranging from 1 mM to 100 mM. This investigation
highlighted that flavonols induced opposite effects on superoxide radical production at History
different doses, i.e. pro-oxidant at the highest concentration (100 mM) and anti-oxidant at the
lowest concentration (1 mM). Similar behaviors were observed for xanthine oxidase with flavan- Received 15 May 2014
3ols. The diastereoisomer (the catechin) acted as a stronger radical scavenger than the Accepted 22 May 2014
Published online 27 June 2014
For personal use only.

epicatechin. However, flavan-3ols were less pro-oxidant than flavonols: in fact, the addition
of the superoxide dismutase enzyme was able to cancel the flavan-3ols’ pro-oxidant effect.
This study also shows that the absence of the 4-carbonyl group conjugated with the 2–3
double bonds in the heterocyclic ring cancelled the pro-oxidant effect of flavan-3ols.
The opposite dose-dependent effects of flavonols suggest that they may be used as either a
pro-oxidant or antioxidant.

The antioxidant properties of food are differently influenced

Introduction by the presence of a specific polyphenolic molecule (Di Majo
et al., 2005, 2008).
Flavonoids are a subclass of polyphenols that are devoid of
Among the enzymes involved in ROS formation and modu-
calories. However, growing evidence indicates that the introduc-
lation, manganese superoxide dismutase (Mn-SOD) is of pivotal
tion of these substances into the human diet may have physio-
importance in an oxygen-rich atmosphere, as demonstrated by
logical benefits. Therefore, according to the definition of
several in vivo studies in various models (Duttaroy et al., 2003;
‘‘nutraceutical’’ (Dillard & German, 2000), the use of polyphe-
Wicks et al., 2009). Altered Mn-SOD expression or activity has
nols as ‘‘nutraceuticals’’ in food preparations (Sajilata et al.,
been observed in neurodegenerative diseases, such as Alzheimer’s
2008) or as dietary-ingested compounds has been suggested.
disease (Esposito et al., 2006) and cancer (Dhar & St Clair, 2012;
In particular, clinical observations have reported the thera-
Oberley, 2005).
peutic benefits of these molecules in some pathological conditions
Many other studies report that flavonoids appear to contribute
such as myocardial ischemia, liver disease, and atherosclerosis
to the regulation of the expression and/or activity of enzymes such
(Siasos et al., 2013; Williamson & Manach, 2005). Furthermore,
as cyclooxygenase, lipoxygenase, and nitric oxide synthase
experimental findings suggest that these molecules are endowed
(Santangelo et al., 2007). However, studies regarding the possible
with chemopreventive and antitumor properties (Del Rio et al.,
dose-dependent effect of these molecules on the activity of these
2013; Gonzàlez-Vallinas et al., 2013; Huang et al., 2010). The
antioxidant enzymes are currently lacking, even though clinical
therapeutic effects of flavonoids may be explained in part by their
evidence suggests that chemoprevention via phenolic phytochem-
antioxidant activity due to their ability to (i) inhibit specific
icals may be an inexpensive, readily applicable, acceptable, and
enzymes involved in reactive oxygen and nitrogen species
effective approach to the clinical management of cancer progres-
(ROS/RNS) formation, (ii) act as scavenger of ROS/RNS,
sion (Luk et al., 2007). Although the consumption of flavonoid/
(iii) chelate trace elements, and/or (iv) up-regulate antioxidant
phenolics is considered safe, their use for therapeutic or
genes (Gong & Chen, 2003; Joven et al., 2014).
chemopreventive purposes has been reported to occasionally
induce side effects such as liver failure, dermatitis, anemia, male
reproductive disorders, and, in cases of an estrogen-like chemical
structure of the compound, breast cancer (Galati & O’Brien,
Correspondence: Danila Di Majo, Unit of Physiology and Pharmacology, 2004). As these effects were found to be dose-dependent, this
Department DIGISPO, University of Palermo, Italy. E-mail: danila. phenomenon must be taken into consideration when considering
dimajo@unipa.it any long-term preventive or therapeutic use of these molecules
 2 D. Di Majo et al. Int J Food Sci Nutr, Early Online: 1–7

(Fresco et al., 2006). Therefore, more information about the health
benefits and the possible risks of dietary supplements or herbal
medicines are needed to better define their pharmacological
profile and their clinical usefulness. On the basis of these
findings, we have undertaken some investigations to evaluate the
effects of two flavonols (kaempferol and quercetin) and two
flavan-3ols (catechin and epicatechin) on superoxide radical
production through the concentration-dependent modulation of
Mn-SOD and XO to better define the potential nutraceutical
interest of these compounds. Furthermore, some studies have
shown that the chemical structure of flavonoid compounds may
affect the antioxidant activity of food and that there is a clear
relationship between a planar flavonoid structure and antioxidant
activity (Di Majo et al., 2005). In particular, these studies
highlight that the total number of hydroxyl and methoxyl groups
and their position on the ring may influence the extent
and the mechanism of antioxidant and pro-oxidant activities
(Finotti & Di Majo, 2003). We have therefore undertaken
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administration of these molecules (Kroon et al., 2004,
Manach et al., 2004). The current available evidence indicates
that although quercetin is not found in the plasma after
oral administration, it exerts biologically demonstrable systemic
effects, whereas its metabolites show weak activity in vitro and
often appear to be completely inactive (Perez-Vizcaino
et al., 2012). According to Perez-Vizcaino, glucuronidated
derivatives transport quercetin and deliver it to tissues as the
free aglycone, which is the final effector. A similar phenomenon
may be observed with kaempferol (O’Leary et al., 2001) and
flavanols (Serra et al., 2011; Ottaviani et al., 2011), suggesting
that the effects described above may also be extended to other
flavonoids.
Materials and methods
Sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3),

experiments with molecules of two classes of flavonoids,
i.e. flavonols and flavan-3ols, to specifically clarify the influence
of the carboxyl group at the C4 position of the heterocyclic
ring conjugated with the C2-C3 double bond on enzymatic
activities (Figure 1). As data for the influence of the stereochem-
ical configuration of flavanols on their biological activities are
scant, parallel studies were performed to assess whether both
flavan-3-ol diastereoisomers have the same effect on the XO and
Mn-SOD enzymes.
These studies were performed using the aglycone forms
but not on the methylated, glucurono-, and sulfo-conjugated
metabolites. According to some authors, the studies on quercetin
For personal use only.
ethylene diaminetetraacetic acid (EDTA), XTT (2,3-bis[2-meth-
oxy-4-nitro-sulfo-phenyl]-2H-tetrazolium-5-carboxanilide), xan-
thine (X), xanthine oxidase (EC 1.1.3.22) from buttermilk
(0.6 units/mg protein), Mn-SOD from bovine erythrocytes (EC
1.15.1.1), quercetin, kaempferol, and (+) catechin and ()
epicatechin were purchased from Sigma-Aldrich (St Louis,
MO). All the stock solutions were prepared with water purified
using a Milli-Q system (Millipore, Billerica, MA). XTT and X
were dissolved in a 50 mM sodium carbonate buffer (pH 9.8) at
room temperature and at 45  C, respectively. To allow complete
superoxide radical consumption, Mn-SOD was used at a concen-
tration of 0.3 mM. Stock solutions of flavonoids were prepared in

aglycone and other flavonoids are questionable because only ethanol at 1 mM concentration and then diluted just before the
metabolites were determined in the plasma after the oral assay in a range of concentrations between 1 mM and 100 mM.

Figure 1. Flavonoid backbone and chemical Flavonols

structure of flavonols and flavan-3ol OH
sub-groups.
OH OH

HO HO O
O

O O
OH OH
Kaempferol
Quercetin

3'
2' 4'
8 1
9 O 1' B
7 2 5'
A C 6'
6
10 3
5 4

Flavan-3ols

OH OH

OH OH

HO HO
O R O R

S OH R OH

OH OH
(−) epicatechin
(+) catechin
 DOI: 10.3109/09637486.2014.931362 Flavonols and flavan-3-ols as modulators of XO and MnSOD 3

SOD activity assay samples. The statistical analysis was performed using the
XLSTAT 2012 statistics package for Excel. p Values 0.05
The effects of different compounds on XO and Mn-SOD activities
were considered statistically significant.
were determined by a spectrophotometric assay using the X/XO/
XTT system, as previously reported (Farines et al., 2004).
Briefly, at the same time, three quartz cuvettes (a, b, and c)
Results
were prepared. In the first cuvette (a), each tested compound and
XO without SOD were added to the reagents to evaluate the Effect of flavonols on XO and Mn-SOD
capacity of the compounds to scavenge superoxide radicals
Table 1 summarizes the dose-dependent pro-oxidant or anti-
directly or to inhibit xanthine oxidase. In the second (b), XO and
oxidant effects of kaempferol and quercetin on the XO enzyme.
SOD without the sample (flavonoids) were added to the mixture
These compounds at concentrations between 25 and 100 mM
of reagents to analyze the capacity of SOD to abolish the radical
resulted in a significant increase in superoxide anion levels when
production of the X/XO-XTT system. In the last cuvette (c), SOD
compared with the controls. Consequently, they revealed a pro-
was added to the reagents of the first cuvette to evaluate the
oxidant action. This phenomenon is most likely the consequence
combined action of the compounds on XO and SOD. The reagents
of an intrinsic pro-oxidant activity of flavonols. These results are
were distributed to all three quartz cuvettes (final volume 3 mL)
in good agreement with the data reported by Lòpez-Lòpez et al.
by adding 50 mM sodium carbonate buffer (pH 9.8), 67 mL of
(2004). In fact, according to these authors, quercetin undergoes
NaEDTA (3 mM), 67 mL of XTT (0.8 mM), 67 mL of xanthine
auto-oxidation and generates superoxide radicals in a dose-
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(3 mM), and 67 mL of xanthine oxidase, with 400 mL of SOD only

dependent manner.
added to the c and b cuvettes.
A marked production of radicals was also observed at
Ethanol was added to the SOD-free (control) cuvette. The
concentrations of quercetin higher than 25 mM. The maximal
reaction was initiated by the addition of XO solution at a
production of superoxide radicals occurred at 100 mM (Table 1),
concentration of 60 mUmL1. The change in absorbance at
and kaempferol induced effects similar as quercetin. However,
470 nm was monitored for 30 minutes with a spectrophotometer
kaempferol at concentrations between 25 mM and 75 mM induced
Beckman 640 set at 25  C.
a more consistent production of radicals compared to quercetin.
The modulation of relative SOD activity (RA) due to the tested
Table 1 shows that kaempferol produced the same amount of
compounds was expressed as:
  superoxide radicals (79%) at concentrations of 50 and 100 mM.
ðc  bÞ When Mn-SOD was added at the same concentration of 0.3 mM,
RA ¼ 1  100 the amount of superoxide radicals was reduced to 20% (Table 2)
ða  bÞ
with 50 mM kaempferol.
For personal use only.

where a is the absorbance without SOD in the presence of the
compound tested, b is the absorbance of SOD in the system (X/
XO-XTT) w/o the tested compound, c is the absorbance of SOD
on the system (X/XO-XTT) in presence of the tested compound.
If RA 0, the absorbance of the solution containing SOD in
the presence of the compound (c) is lower than that determined in
the solution without SOD and in the presence of tested compound
(a). This means that the compound has no effect on SOD activity.
In fact, the enzyme is completely or partially able to neutralise the
superoxide radicals produced by the X/XO/compound system.
Conversely, RA values 0 indicates that they act as potent pro-
oxidants or as inhibitors of the SOD enzyme.
However, when kaempferol was used at the concentration of
100 mM, the addition of Mn-SOD was not able to cancel the
production of radicals (Table 2), and the system (X/XO/XTT/
SOD) with the compound showed a higher production of
Table 1. Dose-dependent effects of quercetin, kaempferol, (+) catechin,
and () epicatechin on xanthine oxidase. The values are expressed as the
mean ± standard deviation of least three independent determinations.
RA is the relative activity of compounds on the enzyme. When RA 40,
the compound tested increased superoxide radical production, in contrast
to RA 50.
Activity on the XO (% RA)

Xanthine oxidase activity assay Molecules Concentrations (mM) RA 0 " [O

2 ] RA 0 # [O
2 ]

The assay uses the X/XO/XTT system but without SOD. Kaempferol 100 79.46 ± 1.56
75 69.56 ± 0.95
The reagents and their concentrations were the same as used in
50 76.90 ± 3.17
the superoxide dismutase assay. 25 43.03 ± 4.20
The relative activity of the compounds, expressed in percent- 10 8.47 ± 0.02
age, was calculated with the following equation: 1 18.75 ± 4.44
  Quercetin 100 74.00 ± 0.46
ðb  aÞ 75 61.76 ± 2.63
RA ¼ 100
a 50 25.85 ± 6.05
25 22.61 ± 3.38
where (a) is the absorbance of the X/XO-XTT system without the 10 9.06 ± 2.35
compounds (control) and (b) is the absorbance of the solution 1 37.08 ± 3.17
containing flavonoids. Catechin 100 69.32 ± 3.67
When RA 40, the absorbance of the solution containing the 75 22.01 ± 2.36
50 26.04 ± 1.99
compounds is higher than the control. When RA 50, the 25 81.67 ± 6.37
absorbance of formazan is lower in the presence of the compound 10 108.65 ± 6.74
than in the control. 1 36.67 ± 5.72
Epicatechin 100 75.67 ± 3.07
75 65.29 ± 1.31
Statistical analysis 50 44.77 ± 1.29
Data are reported as the mean value ± standard deviation (SD). 25 55.19 ± 3.20
10 40.52 ± 1.60
The significance of differences between the parameters con-
1 55.07 ± 3.39
sidered in this study was assessed by Student’s t-test for unpaired
 4 D. Di Majo et al. Int J Food Sci Nutr, Early Online: 1–7

Table 2. Dose-dependent effects of quercetin, kaempferol, (+) catechin,

and () epicatechin on both enzymes. The values are expressed as the
mean ± standard deviation of least three independent determinations.
RA is the relative activity of compounds on the enzyme. When RA 40,
the modulating action of the compound tested on both enzymes reduced
superoxide radical production, in contrast to RA 50.

Relative activity on both

the enzymes (% RA)
Molecules Concentrations (mM) RA 0 # [O
2 ] RA 0 " [O
2 ]

Kaempferol 100 32.41 ± 3.46

75 2.18 ± 0.15
50 20.26 ± 8.27
25 36.06 ± 3.04 Figure 3. Dose-dependent effects of flavonols (+) catechin ( ) and ()
10 46.29 ± 8.63 epicatechin ( ) on superoxide anion production, as evaluated by the SOD
1 97.99 ± 3.30 assay. Data are expressed as the mean ± standard deviation of last three
Quercetin 100 9.97 ± 3.11 independent determinations.
75 8.51 ± 3.17
50 26.90 ± 10.87
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25 44.92 ± 3.06 superoxide radicals. According to Pauff & Hille, (2009), the
10 53.31 ± 0.12 antioxidant effects may be due either to the direct reaction with
1 95.10 ± 3.55 superoxide radicals (radical scavenger effect) or to the inhibition
Catechin 100 37.85 ± 3.96 of XO enzyme activity. Our results demonstrate that quercetin had
75 28.61 ± 4.79
50 51.98 ± 7.66
a greater inhibitory effect on XO than kaempferol (Table 1).
25 31.26 ± 5.73 These findings are consistent with those of Cos et al. (1998),
10 32.18 ± 3.44 which suggests that quercetin might be classified into category C
1 93.52 ± 7.7 (xanthine oxidase inhibitors with an additional superoxide-
Epicatechin 100 25.16 ± 6.63 scavenging activity), whereas kaempferol falls into category B,
75 22.69 ± 1.37 namely, xanthine oxidase inhibitors without any additional
50 57.80 ± 3.07
superoxide-scavenging activity. Consistent with the results of
25 39.11 ± 3.19
10 52.14 ± 6.47 Lin et al. (2002), the inhibiting effect of flavonols can be in
For personal use only.

1 105.54 ± 5.91
part explained by the high capacity of these molecules to interact
with the molybdopterin moiety present in the active site of the
XO enzyme. In addition, this study showed that when SOD was
added to the system (Table 2), no significant difference between
the two molecules was observed at the lowest concentrations
(p40.05).

Modulator effects of flavan-3ols on XO and Mn-SOD

Figure 2. Dose-dependent effects of flavonols quercetin ( ) and
kaempferol ( ) on superoxide anion production, as evaluated by the
SOD assay. Data are expressed as the mean ± standard deviation of last
three independent determinations.
superoxide radicals than the control (system without the
compound).
This effect may be explained by an inhibition of SOD activity
that is not the consequence of a saturation effect of the enzyme.
The dose-dependent effects of flavonols on superoxide radical
production via the modulation of XO and Mn-SOD are shown in
Figure 2. The differences observed between the two compounds
further confirm the findings by Cao et al. (1997), indicating that
the number of OH groups in the aromatic ring and their positions
are of pivotal importance in determining the anti/pro-oxidant
action of polyphenols. Quercetin, which carries two electron-
donating groups on the B-ring, showed a pro-oxidant activity
lower than that of kaempferol, which has only one OH group.
At concentrations lower than 25 mM, quercetin and kaempferol
acted as antioxidants. In fact, these molecules were able to reduce
Regarding flavan-3ols, it is important to note that catechin and
epicatechin are stereoisomers, each of which exists in two
enantiomeric forms. The four distinct stereochemical configur-
ations of these flavanols are () epicatechin, (+) epicatechin, ()
catechin, and (+) catechin. It has been reported that the
stereochemical configuration of flavan-3ols deeply influences
their uptake and metabolism in humans (Ottaviani et al., 2011)
and that the plasma levels of () epicatechin and (+) catechin are
higher than those of the other two stereoisomers.
Therefore, we focused our attention on the () epicatechin and
(+) catechin diastereoisomers. Figure 3 shows the dose-dependent
effects of (+) catechin and () epicatechin on superoxide anion
production, as evaluated by the SOD assay. The results in Table 1
show the effects of flavan-3-ols on XO activity. Similarly to
flavonols, these compounds increased the production of super-
oxide radicals at the highest concentrations (50 mM), an effect
that is likely due to an intrinsic pro-oxidant activity. Catechin was
found to be less pro-oxidant than kaempferol. In fact, the latter
molecule increased radical production at 25 mM. Conversely, at
the same concentration, catechin reduced the radical production
by the system (Table 1). These results are in agreement with those
of Marozien_e (2012), reporting that flavonoids exhibit pro-
oxidant cytotoxicity in mammalian cells due to the formation of
free radicals and that this effect increases as the redox potential of
the phenoxyl radical/phenol couple decreases. These data indicate
that flavonols induce a stronger cytotoxic effect on mammalian
cells than flavan-3ols. These results may be in part explained as
follows: due to auto-oxidation, flavonols may produce a more
 DOI: 10.3109/09637486.2014.931362 Flavonols and flavan-3-ols as modulators of XO and MnSOD 5

Kaempferol
O-semiquinone radical intermediate (K.−)
OH
O−
.

HO O HO O
+ O2 O2.− + 2H+ +
OH O
OH O O
OH
O

HO O

C
O−
OH O
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O− O
C

OH
OH

O2 −e− O
For personal use only.

O O

O2.− +
O
OH
OH

O-Quinone

Figure 4. Auto-oxidation of kaempferol and quinone/quinomethide tautomerisation of kaempferol oxidate.

stable radical that can be delocalized on the C and A rings
(Figure 4); instead, the semiquinone radical of catechins remains
localized on the B ring. Hence, the semiquinone radical of flavan-
3-ols is less stabilized by resonance, and, as a consequence, the
auto-oxidation reaction is less promoted (Figure 5a). According to
Mochizuki et al. (2002), catechins undergo multi-step auto-
oxidation at concentrations higher than 40 mM (Figure 5a), with
the formation of a semiquinone radical on the B ring, as
confirmed by ESR analysis. This radical may continue to react
until it transforms into a quinone. The superoxide radical
generated by the first reaction (Figure 5a) can react with other
molecules of catechin (Figure 5b). This reaction is fostered by the
strong oxidant activity of the superoxide radical (Ered ¼ 0.89 V)
versus O2 (Ered ¼ 0.16 V). For this reason, O 2 itself partici-
pates into the auto-oxidation of catechins.
At low concentrations (525 mM), catechin behaved as a radical
scavenger of superoxide anion. At these concentrations, it is
possible that the auto-oxidation of catechin (Figure 5a) was not
complete but may be stopped at the level of semiquinone radical
formation because the generated superoxide radical reacted
with more molecules of catechin (Figure 5b), thus producing
hydrogen peroxide and other semiquinone radicals (faster reac-
tion). Among flavan-3-ols, catechins showed a radical scavenger
activity that was stronger than epicatechins (Table 1). In fact, this
effect was observed in the entire range of 25 mM to 1 mM.
Furthermore, epicatechin behaved as a radical scavenger only at
a lower concentration (1 mM). The addition of SOD was able
to inhibit the pro-oxidant activity of flavan-3-ols in a dose-
dependent manner (Table 2). At low concentrations, the effect
of SOD was stronger because the radical-scavenging reaction
(Figure 5b) might be accelerated by the disproportion of
superoxide radical (Figure 5c).
Conclusions
The results presented here show that marked differences can be
observed between flavonoids with different structural character-
istics with regard to their ability to inhibit or stimulate superoxide
radical production through a modulation of XO and Mn-SOD.
The study of the structure-activity relationship on these com-
pounds showed that flavonols (quercetin and kaempferol) differ
from flavan-3ols (catechin and epicatechin) in their pro-oxidant
effect. The absence of the carbonyl group at C-4 and of the double
bond between C-2 and C-3 of the heterocyclic ring resulted in a
dose-dependent loss of the pro-oxidant effect. This may be due to
the saturation of the double bond, which destroys the conjugation
and coplanarity of the flavan-3ols structure. These data further
demonstrate the opposite dose-dependent effects of flavonols on
XO and Mn-SOD. At concentrations 75 mM, these molecules
showed a high pro-oxidant activity, and the addition of Mn-SOD
 6 D. Di Majo et al.
Figure 5. (a) Auto-oxidation reaction of (a)
catechin at higher concentrations; (b) free HO
radical-scavenging behavior of catechin at O O−
low concentrations; (c) the dismutation of
superoxide free radical by the enzyme OH
superoxide dismutase. OH
(b)
HO
O
OH
OH
(c)
SOD
2O2.− + 2H+
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was not able to abolish this effect. However, they acted as
antioxidants at concentrations lower than 25 mM. These opposite
dose-dependent effects we observed suggest exercising caution in
the use of flavonols for clinical purposes. Unlike flavonols,
flavan-3-ols showed antioxidant activity at all the tested
concentrations.
Our study demonstrates that two diastereoisomers exhibit
different properties on XO but that no difference was observed
following the addition of Mn-SOD. This study also shows that the
a-hydroxyl on the C-3 of heterocyclic ring (catechin) is important
For personal use only.
for the radical-scavenging activity of flavan-3ols.
Therefore, our results suggest that flavan-3ols, at low concen-
trations, may act as radical scavengers and therefore show strong
antioxidant effects, whereas they may undergo auto-oxidation at
higher concentrations, showing a weaker antioxidant potential.
Declaration of interest
The authors declare that they have no conflicts of interest.
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