Neurochem Res

DOI 10.1007/s11064-016-1919-8

ORIGINAL PAPER

Protective Effects of Chlorogenic Acid and its Metabolites

on Hydrogen Peroxide-Induced Alterations in Rat Brain Slices:
A Comparative Study with Resveratrol
Zulfiye Gul1 Celaleddin Demircan2 Deniz Bagdas3 Rifat Levent Buyukuysal1

Received: 12 November 2015 / Revised: 13 April 2016 / Accepted: 13 April 2016

Springer Science+Business Media New York 2016

Abstract The effectiveness of chlorogenic acid and its

main metabolites, caffeic and quinic acids, against oxidative stress was investigated. Resveratrol, another natural
phenolic compound, was also tested for comparison. Rat
cortical slices were incubated with 200 lM H2O2 for 1 h,
and alterations in oxidative stress parameters, such as 2, 3,
5-triphenyltetrazolium chloride (TTC) staining and the
production of both malondialdehyde (MDA) and reactive
oxygen species (ROS), were assayed in the absence or
presence of phenolic compounds. Additionally, the effectiveness of chlorogenic acid and other compounds on
H2O2-induced increases in fluorescence intensities were
also compared in slice-free incubation medium. Although
quinic acid failed, chlorogenic and caffeic acids significantly ameliorated the H2O2-induced decline in TTC
staining intensities. Although resveratrol also caused an
increase in staining intensity, its effect was not dose-dependent; the high concentrations of resveratrol tested in the
present study (10 and 100 lM) further lessened the staining
of the slices. Additionally, all phenolic compounds significantly attenuated the H2O2-induced increases in MDA
and ROS levels in cortical slices. When the IC50 values
were compared to H2O2-induced alterations, chlorogenic
acid was more potent than either its metabolites or

& Rifat Levent Buyukuysal

lrbuyuk@uludag.edu.tr
1

Department of Medical Pharmacology, Faculty of Medicine,

Uludag University, 16059 Bursa, Turkey

Department of Internal Medicine, Faculty of Medicine,

Uludag University, 16059 Bursa, Turkey

Experimental Animals Breeding and Research Center,

Faculty of Medicine, Uludag University, 16059 Bursa,
Turkey

resveratrol for all parameters studied under these experimental conditions. In slice-free experimental conditions, on
the other hand, chlorogenic and caffeic acids significantly
attenuated the fluorescence emission enhanced by H2O2
with a similar order of potency to that obtained in slicecontaining physiological medium. These results indicate
that chlorogenic acid is a more potent phenolic compound
than resveratrol and its main metabolites caffeic and quinic
acids against H2O2-induced alterations in oxidative stress
parameters in rat cortical slices.
Keywords Chlorogenic acid  Caffeic acid  Resveratrol 
Brain slices  Oxidative stress  Phenolic compounds

Introduction
Oxidative stress is an important mechanism underlying
aging and neurodegenerative diseases [13]. Because an
imbalance between intracellular reactive oxygen species
(ROS) and antioxidant defense mechanisms results in
oxidative stress and because the antioxidant defense
mechanisms in the brain are not sufficient to prevent
oxidative damage, dietary intake of a variety of antioxidants might be beneficial for protecting brain function.
Indeed, several epidemiological and clinical studies have
shown an inverse association between the consumption of
antioxidant-rich foods and the risk of the several neurodegenerative diseases, including Alzheimers and
Parkinsons diseases [4, 5]. Interest in phenolic and
polyphenolic compounds is increasing because of their
perceived beneficial effects on health. In addition to their
ability to inhibit oxidative stress, these compounds also
have antitumor [6, 7], anti-inflammatory, cardioprotective,
analgesic and antidiabetic properties [814].

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Neurochem Res

Phenolic compounds in plants, such as phenolic acids,

flavonoids or stilbenes, are generally involved in defense
against ultraviolet radiation or aggression by pathogens and
can be classified into different groups based on the number
of phenol rings, [7, 15]. Chlorogenic acid, a caffeic acid
ester linked to quinic acid, is a phenolic acid that is found
in high amounts in many types of fruit and coffee [16].
Resveratrol, a 3,5,4-trihydroxystilbene, on the other hand,
occurs naturally in edible plants and is present at lower
quantities than cinnamic acid derivatives [17, 18].
Although both chlorogenic acid and resveratrol are
among the most investigated compounds in nutritional
research, their bioavailabilities are considerably low. Due
to high and extensive metabolism in the intestine and liver,
the bioavailability of resveratrol is approximately 1 % [19,
20]. Similarly, colonic microflora plays a significant role in
the metabolism of digested chlorogenic acid; thus, most of
chlorogenic acid is broken down into caffeic and quinic
acids before absorption [21, 22]. In contrast to the high
biotransformation rate of phenolic compounds, most data
that indicate their beneficial effects have been obtained
under in vitro or in vivo conditions with their parent
compounds [21]. One possible explanation is that their
major metabolites, in addition to the parent compounds,
may also have beneficial effects in the body. Indeed, it has
been reported that the plasma concentrations of intact
parent polyphenols are often low and do not account on
their own for the increase in the antioxidant capacity of the
plasma; thus, their metabolites may also contribute to
increasing this antioxidant capacity [21]. In support of this
conclusion, caffeic acid, a main metabolite of chlorogenic
acid, also has antioxidant properties under in vitro and
in vivo conditions [23, 24]. Because no published studies to
date have compared the protective effects of chlorogenic
acid and its major metabolites in living neuronal tissue, this
study was undertaken to compare their effectiveness on
oxidative stress-induced alterations in rat brain cortical
slices. The study further compared their effectiveness to
resveratrol, another commonly investigated phenolic
compound in nutritional research.

Preparation and Incubation of Cortical Slices

Female SpragueDawley rats (3 months of age) were
obtained from the Experimental Animals Breeding and
Research Center (Bursa, Turkey). Because we previously
reported no significant differences related to sex in brain
slice studies [2527], female rats were preferred in the present study. All experimental protocols were approved by the
Local Ethics Committee for Animal Experiments, Uludag
University (Approval number: 2013-17-06), and all efforts
were made to minimize the number of animals used and their
suffering. Rats were decapitated, and their brains were
removed quickly and placed in cold physiological medium
(in mmol/L: 120 NaCl, 1.3 CaCl2, 1.2 MgSO4, 3.5 KCl, 1.2
NaH2PO4, 25 NaHCO3 and 10 glucose) that was oxygenated
with 95 % O2 and 5 % CO2. Cortical slices used in this study
were prepared from mainly frontal and parietal areas of the
rat brain. After removing of the brain, cortical tissue around
34 mm 9 810 mm in size from this area was dissected
and sliced with a McIlwain tissue chopper (Brinkmann
Instruments; Westbury, NY, USA). Slices (0.40 mm thickness) were washed with oxygenated cold physiological
medium and transferred to wells of the tissue culture incubation plate. Each well contained 2 ml of physiological
medium with an equal number of cortical slices (three slices
in each well). The incubation plate was placed in a water bath
at 37 C, and slices were incubated for 1 h to equilibrate.
During this period, the medium was changed every 15 min
with fresh medium. Following a 1 h equilibration period, the
cortical slices were incubated in either control or H2O2containing medium for another hour at 37 C. All phenolic
compounds tested in the present study were added into the
medium at the beginning of H2O2 incubation. Chlorogenic
acid and its metabolites were dissolved in distilled water.
Resveratrol, on the other hand, was dissolved in 0.1 %
dimethyl sulfoxide (DMSO). When the effects of phenolic
compounds were studied, an equal amount of distilled water
or DMSO was added into the control and H2O2 groups. At the
end of the incubation periods, the slices were used for TTC
staining or determination of tissue MDA and ROS levels.
TTC Staining

Materials and Methods

Chemicals and Drugs
Chlorogenic acid was purchased from Acros Organics
(Geel, Belgium). Caffeic acid, quinic acid, resveratrol,
2,3,5-triphenyltetrazolium
chloride
(TTC)
and
20 ,70 dichlorodihydrofluorescein diacetate (DCFH-DA)
were obtained from Sigma Chemical Co. (St. Louis, MO,
USA). Other chemicals were obtained from Merck KGaA
(Darmstadt, Germany) or Sigma Chemical Co.

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At the end of H2O2 incubation, slices were stained with

0.5 % TTC in phosphate buffer (100 mM, pH 7.4) in a
covered water bath at 37 C for 1 h. After staining, slices
were washed with cold saline and immersed in 2 ml of
ethanol-DMSO mixture (5050 %; v/v) to extract the red
formazan from the slices [28]. After a 24 h extraction in
the dark, 250 ll of the extracted liquid was transferred to
96-well plates, and absorbance values were measured at
490 nm by a plate reader (Mannheim Boehringer Photometer 4010). All absorbance values were corrected

Neurochem Res

according to tissue protein levels, and then percentage

losses in TTC staining intensities were calculated. In some
experiments, the slices were immersed in 2 ml of 4 %
formalin for 24 h in the dark to fix the red formazan. Slices
were then placed on a parafilm sheet, and photo images
were taken after drying the slices with paper towel.
MDA Assay
Tissue MDA levels were determined after 2, 4-dinitrophenylhydrazine (DNPH) derivatization as described previously [29]. Cortical slices obtained at the end of the
incubation periods were homogenized in 1 ml of 1 % sulfuric acid. A 200 ll portion of the homogenate was mixed
with 20 ll of DNPH solution (5 mM; prepared in 2 M
hydrochloric acid) and incubated for 24 h in the dark. The
mixture was then centrifuged at 10,0009g for 10 min, and
20 ll of the supernatant was injected into an HPLC system
(model PU-980 liquid chromatography pump; Jasco, Japan).
The DNPH derivative of MDA was separated on a
C18-reversed phase column (MachereyNagel GmbH,
Duren, Germany) with a flow rate of 0.7 ml/min, and the UV
detector was set at 310 nm. The mobile phase consisted of
acetonitrile-distilled water (38: 62; v/v) containing 0.2 %
(v/v) acetic acid. MDA peaks were determined according to
the retention time and confirmed by spiking with added
exogenous standard. MDA standards were prepared from
1,1,3,3 tetramethoxypropane. The MDA amounts measured
in the samples were corrected according to their tissue protein levels and expressed as pmol/mg protein.

significant increase in fluorescence intensity, and compounds with antioxidant properties can attenuate this H2O2induced increase [30]. Thus, the effectiveness of phenolic
compounds against the H2O2-induced increase in fluorescence intensity was also tested in the absence of cortical
slices in the medium. In the first series of experiments,
increasing concentrations of H2O2 were incubated in the
absence or presence of DCFH-DA (5 lM) in 2 ml of
oxygenated medium for 1 h at 37 C in the dark. In the
second series of experiments, all phenolic compounds were
incubated with H2O2 (1 mM) and DCFH-DA (5 lM) in
2 ml of oxygenated physiological medium (pH 7.5) for 1 h
at 37 C in the dark. At the end of the incubation, 200 ll of
the samples was diluted with 2 ml of distilled water, and
the fluorescence intensities were measured as indicated
above. During these studies, equal amounts of distilled
water or DMSO (0.1 %) were added into the medium as a
control for chlorogenic acid and its metabolites or for
resveratrol, respectively, and the fluorescence intensities
observed control conditions were subtracted from the
intensity values determined in the presence of phenolic
compounds.
Total Protein Assay
After homogenization of the slices in 2 ml of distilled
water, tissue protein levels were measured in 50 ll of the
homogenate [31]. All results were corrected according to
total protein levels in the slices.
Statistical Analysis

Determination of Tissue ROS Formation

Tissue ROS levels were measured with DCFH-DA (final
concentration of 5 lM). DCFH-DA was dissolved in 0.1 %
DMSO and added into the medium during H2O2 incubation
of the slices. At the end of H2O2 incubation, slices were
washed with ice-cold physiological medium and homogenized with an ultrasound homogenizer, and 200 ll of the
homogenate was used for fluorescence quantification with a
spectrofluorometer (excitation and emission wavelengths
488 and 525 nm, respectively; Jasco FP-750 spectrofluorometer). To minimize the light-induced decomposition of
DCFH-DA, all experimental procedures were performed in
the dark. Fluorescence intensities of the samples were
normalized according to their tissue protein levels.
H2O2-Induced Fluorescence in Slice-Free Incubation
Medium
We recently reported that incubating H2O2 and DCFH-DA
in slice-free oxygenated physiological medium causes a

The data were expressed as the mean SEM (standard

error of the mean) in all cases. The data were analyzed
using mix ANOVA followed by the StudentNewman
Keuls test. A p value of \0.05 was considered significant.
IC50 values with 95 % CL for all data were calculated by
an unweighted least-squares linear regression as described
by Tallarida and Murray [32].

Results
H2O2-Induced Alterations in TTC Staining, MDA
Production and ROS Production in Rat Cortical
Slices
A 1 h incubation of rat cortical slices with H2O2 caused a
dose-dependent decrease in TTC staining intensities
(Fig. 1). Tissue MDA and ROS levels, on the other hand,
increased significantly after H2O2 was added into the
medium (Fig. 1).

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Neurochem Res

Effects of Resveratrol on H2O2-Induced Alterations

in Cortical Slices: Comparison with CGA

Fig. 1 H2O2-induced alterations in TTC staining, MDA and ROS

productions in rat cortical slices. After preincubation period, cortical
slices were incubated with increasing concentrations of H2O2 for 1 h.
At the end of H2O2 incubation, slices were used for TTC staining and
determination of tissue MDA or tissue ROS levels as indicated in the
text. Data are provided as the mean SEM of 810 determinations.
*p \ 0.05, **p \ 0.01, ***p \ 0.001; significantly different from the
corresponding control value determined in the absence of H2O2

Effects of Chlorogenic Acid and its Metabolites

on H2O2-Induced Alterations in Cortical Slices
Under oxidative stress conditions, such as ischemia followed by reoxygenation, H2O2 levels actually reach hundred micromolar levels, depending on the experimental
conditions [1, 2]. Additionally, the H2O2-induced decrease
in cell viability has been observed at hundred micromolar
levels [33]. Thus, in the present study, we evaluated the
effectiveness of chlorogenic acid and other naturally
occurring compounds against 200 lM H2O2-induced
alterations in rat cortical slices.
As seen in Figs. 2a and 3, the H2O2-induced decrease in
TTC staining was ameliorated dose-dependently by
chlorogenic acid. Although even 0.1 lM chlorogenic acid
exerted a significant protective effect, staining of the slices
reached control levels at 10 lM concentration. Although
caffeic acid also protected the slices to a similar degree
(Fig. 2b), quinic acid nearly failed to protect the slices
(Fig. 2c). When calculated from the doseresponse curves,
the IC50 values of chlorogenic, caffeic and quinic acids
were 0.1 0.01, 0.22 0.01 and [100 lM, respectively
(Table 1).
As observed in the TTC staining studies, H2O2-induced
increases in tissue MDA and ROS levels were also attenuated dose-dependently by chlorogenic acid (Figs. 4a, 5a).
Although caffeic acid also protected the slices against
MDA and ROS production (Figs. 4b, 5b), its IC50 values
were significantly higher than those of chlorogenic acid
(Table 1). Although only a high concentration of quinic
acid caused a significant decrease in the tissue MDA level
(Fig. 4c), it did not alter the H2O2-induced increase in ROS
production (Fig. 5c).

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Although resveratrol also ameliorated the H2O2-induced

decrease in TTC staining intensities, its effect was not
dose-dependent. Although low concentrations of resveratrol were partially effective, increasing the concentration in
the medium caused a further decrease in the staining
intensities (Fig. 6a). Resveratrol, like chlorogenic acid,
also significantly protected against MDA and ROS production (Fig. 6b, c), but the IC50 value for each parameter
was significantly higher than that of chlorogenic acid
(Table 1).
H2O2-Induced Increase in Fluorescence Intensity
from DCFH-DA in Slice-Free Incubation Condition:
Effects of Chlorogenic Acid, its Metabolites
and Resveratrol
As we reported recently [28], a 1 h incubation of DCFHDA (5 lM) with increasing concentrations of H2O2
(501000 lM) in slice-free oxygenated physiological
medium caused significant increases in fluorescence
emission Fig. 7a). When added into the medium, chlorogenic and caffeic acids decreased the 1 mM H2O2-induced
fluorescence emission dose-dependently (Fig. 7b). The
IC50 values of chlorogenic and quinic acids were
1.51 0.02 and 5.4 0.7 lM, respectively. Although
quinic acid nearly failed, resveratrol, unexpectedly, caused
significant enhancements in the fluorescence emission
determined under this experimental condition (Fig. 7b).

Discussion
Organotypic or acute brain slice preparation is one of the
most widely used techniques in neuroscience for understanding mechanisms and identifying targets and possible
therapeutic strategies against neurological disorders, such
as stroke, Parkinsons and Alzheimers diseases [34, 35].
Although there are some limitations, such as the lack of
certain inputs and outputs that normally exist in the intact
brain, brain slices offer certain advantages over in vivo
approaches; namely, experimental conditions, such as pO2,
pH and temperature, can be controlled and maintained as
desired [36]. In contrast to cell cultures or tissue homogenates, brain slices can maintain structural integrity [35].
Additionally, because brain slices have no bloodbrain
barrier, their extracellular space is accessible to the incubation/perfusion medium and its contents [35, 37].
As an in vitro model of oxidative stress, we used H2O2
as a free radical-generating system. In the tissue, H2O2
originates from the enzymatic or spontaneous dismutation

Neurochem Res

Fig. 2 Effects of chlorogenic acid and its metabolites on the H2O2induced decrease in TTC staining intensities. Cortical slices were
incubated with 200 lM H2O2 in the presence or absence of
a chlorogenic (CGA), b caffeic (CA) and c quinic acids (QA) for
1 h. At the end of the incubation, slices were stained with 0.5 % TTC
in phosphate buffer in a covered water bath at 37 C for 1 h.
Extraction of the red formazan from the slices and measurement of

the staining intensities were performed as indicated in the text. Data

are provided as the mean SEM of 1214 determinations after
correcting the absorbance values to the tissue protein levels.
#
p \ 0.001; significantly different from the control value.
*p \ 0.05, **p \ 0.01 and ***p \ 0.001; significantly different from
the value determined with H2O2 alone

Fig. 3 Typical images of rat cortical slices stained with TTC. After
preincubation period, cortical slices were incubated in control or
200 lM H2O2-containing medium for 1 h in the absence or presence
of chlorogenic acid (CGA). At the end of the incubation, the slices

were stained with TTC and immersed in 2 ml of 4 % formalin for

24 h to fix the red formazan. Slices were then placed on a parafilm
sheet, and photo images were taken after drying the slices with paper
towel

of superoxide anions, which are by-products of a variety of

oxidases [38, 39]. Massive H2O2 production occurs during
in vivo ischemia that correlates with neuronal loss [40, 41].
The pathological consequences of H2O2 elevation are

normally prevented by endogenous antioxidants, including

the H2O2-scavenging enzymes glutathione (GSH) peroxidase and catalase [4244]. Notably, modulatory as well as
pathological consequences of H2O2 are often mediated by

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Neurochem Res

Fig. 4 Effects of chlorogenic acid and its metabolites on the H2O2induced increase in tissue MDA levels. Cortical slices were incubated
with 200 lM H2O2 in the presence or absence of a chlorogenic
(CGA), b caffeic (CA) and c quinic acids (QA) for 1 h. At the end of
the incubation, the slices were removed from the wells, and tissue
MDA levels were measured as indicated in the text. Data are provided

as the mean SEM of 810 determinations after correcting MDA

levels according to the tissue protein levels. #p \ 0.001, significantly
different from the control value. *p \ 0.05, **p \ 0.01 and
***p \ 0.001; significantly different from the value determined with
H2O2 alone

the hydroxyl radical (OH), which results from the its

catalytic decomposition on transition metals [1, 4547].
In the present study, we first aimed to determine H2O2induced alterations in cortical slices before testing the
effectiveness of chlorogenic acid and other phenolic compounds. As seen in Fig. 1, a 1 h incubation with H2O2
decreased the TTC staining intensities of the slices dosedependently. Even the lowest concentration of H2O2
(50 lM) caused a significant decrease, indicating an
impairment in mitochondrial energy metabolism of the
slices under this condition. In good agreement with these
results, H2O2 also dose-dependently increased the tissue
ROS level and MDA production, which is one of the most
frequently used indicators of lipid peroxidation (Fig. 1).
All these results clearly indicate that the cortical slice
preparation used in the present study seems to be an
appropriate model for testing the effectiveness of phenolic
compounds against oxidative stress conditions. Under
certain oxidative stress conditions, such as ischemia followed by reoxygenation, H2O2 levels can reach hundred
micromolar levels depending on the experimental conditions [48, 49]; thus, we evaluated the effectiveness of
chlorogenic acid and other naturally occurring compounds
against 200 lM H2O2 in the present study.

Because the bioavailability of chlorogenic acid is considerably low due to its significant metabolism to caffeic
and quinic acids before gastrointestinal absorption, one
possibility is that the beneficial effects of chlorogenic acid
may result from its main metabolite(s). Although caffeic
acid is also well known for its antioxidant capacity, data in
the literature that compare its activities are limited and
contradictory. The esterification of caffeic acid by a sugar
moiety decreases its antioxidant activity [50]. In support of
this observation, chlorogenic acid is a less effective
antioxidant than caffeic acid in lard and stripped corn oil
[51], in sunflower oil [52] or in fish muscle [53]. In contrast
to these observations, the binding of quinic acid to caffeic
acid causes an increase in the antioxidant activity of caffeic
acid while decreasing its H2O2 and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities [23]. Additionally, some other reports indicate that chlorogenic and
caffeic acids are equally effective antioxidants during the
autoxidation of triacylglycerols of sunflower oil [54] or
against the in vitro peroxidation of human low density
lipoproteins [55]. Most of the data comparing the effectiveness of chlorogenic acid and its metabolites have been
obtained under cell- or tissue-free in vitro conditions.
Several factors, such as hydrophilic vs lipophilic properties

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Neurochem Res

Fig. 5 Effects of chlorogenic acid and its metabolites on H2O2induced increase in tissue ROS levels. Cortical slices were incubated
with 200 lM H2O2 and 5 lM DHCF-DA in the presence or absence
of a chlorogenic (CGA), b caffeic (CA) and c quinic acids (QA) for
1 h. At the end of the incubation, the slices were removed from the
wells, and tissue ROS levels were measured as indicated in the text.

Table 1 IC50 valuesof

chlorogenic acid, its main
metabolites and resveratrol
against to H2O2-induced
alterations in rat cortical slices

Phenolic compounds

The data are provided as the mean SEM of 1012 determinations

after correcting the fluorescence intensities according the tissue
protein levels. #p \ 0.01, significantly different from the control
value. *p \ 0.05, **p \ 0.01 and ***p \ 0.001; significantly different from the value determined with H2O2 alone

Parameters measured in cortical slices

TTC staining intensity

MDA production

ROS levels

Chlorogenic acid

0.100.01

0.220.01

0.11 0.01

Caffeic acid

0.200.01

1.120.05***

5.6 0.5***

Quinic acid

[100

10.331.2***

[100

Resveratrol

1.170.38***

1.560.04***

10.05 3.1***

Data presented means SEM. IC50 value of phenolic compounds were calculated from the data given in
Figs. 2, 4, 5 and 6 by using unweighted least-squares linear regression as described by Tallarida and Murray
[30]
*** p \ 0.001 versus CGA

of phenolic compounds, may affect their diffusion inside

living cells and thus can alter their effectiveness [56].
Additionally, the activities of the enzymes involved in
either the production or scavenging of free radicals may
also be altered by phenolic compounds in living cells [24,
57]. Thus, one of the main objectives of the present study is
to compare the beneficial effects of chlorogenic acid and its
two major metabolites, caffeic and quinic acids, against
oxidative stress in a tissue-containing experimental condition. In support of this conclusion, although the in vitro

superoxide anion-scavenging activity of caffeic acid is

higher than chlorogenic acid, their protective effects
against ischemia/reperfusion determined under in vivo
conditions have been found to be almost equal [58].
In good agreement with previously reported data
obtained from different in vitro [5860] and in vivo [61,
62] experimental models, the presence of the chlorogenic
acid in the medium significantly protected the cortical
slices against H2O2-induced alterations. Although caffeic
acid also exerted similar protective effects, its potency was

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Neurochem Res
Fig. 6 Effect of resveratrol on
H2O2-induced alterations in
cortical slices: comparison with
chlorogenic acid. Cortical slices
were incubated with 200 lM
H2O2 in the presence or absence
of resveratrol for 1 h. At the end
of the incubation, the slices
were used for a TTC staining,
b determination of tissue MDA
or c tissue ROS levels as
indicated in the text. After
correcting the measured values
according to the tissue protein
levels, the results were
expressed as percentage
changes in H2O2-induced
alterations. Chlorogenic acid
(CGA)-induced changes were
re-calculated from the data
given in Figs. 2, 4 and 5. Data
are given as the mean SEM
of 810 determinations.
*p \ 0.05, **p \ 0.01,
***p \ 0.001; significantly
different than the resveratrolinduced changes

significantly less than its parent compound; when compared, the IC50 values of caffeic acid for all parameters
were higher under the present experimental conditions
(Table 1). We also observed that quinic acid, another main
metabolite of chlorogenic acid, nearly failed to change the
H2O2-induced alterations. However, this in vitro data may
not completely rule out its effectiveness because quinic
acid, which is present as a normal constituent of the diet, is
capable of converting to tryptophan and nicotinamide via
GI tract microflora, thus providing an in situ physiological
source of these essential metabolic ingredients to humans
[63].
Although resveratrol, like chlorogenic acid, also protected the slices against H2O2-induced alterations, its
potency was less than chlorogenic acid in our experimental
conditions. As discussed above, studies that compared the
antioxidant or antiradical properties of natural phenolic
compounds have been performed under cell-free conditions. However, natural antioxidants do not always behave
like antioxidants; depending on their concentrations [64
66] or environmental oxidative status [67], they can have a
pro-oxidant effect, thereby causing cellular damage. As
seen in Fig. 6a, although 0.1 and 1 lM resveratrol significantly ameliorated the TTC staining intensities, its higher
concentrations in the medium caused a further decline.
This observation may result from the deleterious effects of

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high concentrations of resveratrol, which is consistent with

a previous study that suggested a significant pro-oxidant
effect depending on its concentration [68]. Although similar pro-oxidant behaviors have also been reported with
chlorogenic and caffeic acids [11, 67], these compounds
did not exert a resveratrol-like effect on measured oxidative stress parameters under our experimental conditions.
We recently observed that incubating DCFH-DA with
increasing concentrations of H2O2 in slice-free physiological medium for 1 h at 37 C caused significant increases in
fluorescence intensities, and this method is used for testing
the efficiencies of antioxidant compounds [28]. Although
de-esterification of DCFH-DA to DCFH needs intracellular
esterases, deacetylation also occurs at physiological pH
even in the absence of esterases [69]. Additionally, compounds that are free radicals (e.g., Tempol) or can form
stable radicals (e.g., uric acid, n-propyl gallate) can cause
fluorescence emission from DHCF-DA at physiological pH
[69], which supports our observation obtained with H2O2 in
a slice-free incubation condition.
In the present study, we tested the efficiencies of phenolic compounds against 1 mM H2O2-induced fluorescence
emission at equal concentrations used in brain slice
experiments. As shown in Fig. 7, chlorogenic and caffeic
acids significantly attenuated the fluorescence emission
enhanced by H2O2 with a similar order of potency to that

Neurochem Res

Fig. 7 H2O2-induced increase in fluorescence intensity from DCFHDA in slice-free incubation condition: Effects of chlorogenic acid, its
metabolites and resveratrol. a increasing concentrations of H2O2 were
incubated in the absence or presence of DCFH-DA (5 lM) in 2 ml of
physiological medium (pH 7.4) for 1 h at 37 C. At the end of the
incubation, 200 ll of the samples was diluted with 2 ml of distilled
water, and the fluorescence intensities were measured as indicated in
the text. Data are given as the mean SEM of 49 determinations.
b DHCF-DA (5 lM) was incubated with 1 mM H2O2 in 2 ml of
physiological medium (pH 7.4) for 1 h at 37 C. Chlorogenic (CGA),
caffeic (CA), quinic (QA) acids and resveratrol were added into the
medium at the beginning of incubation, and all experimental
procedures were performed in the dark to minimize the light-induced
oxidation of DHCF-DA. At the end of the incubation, 200 ll of the
samples was diluted with 2 ml of distilled water, and the fluorescence
intensities were measured. Data are given as the mean SEM of 68
determinations. *p \ 0.05, **p \ 0.01, ***p \ 0.001; significantly
different from the value determined in the absence of phenolic
compounds

obtained in slice-containing physiological medium. Quinic

acid, as observed in a slice-containing experimental model,
nearly failed to attenuate the fluorescence emission

enhanced by H2O2. In contrast to chlorogenic acid,

resveratrol caused significant increases in fluorescence
emission under this condition. Trolox, a water-soluble
analog of vitamin E, has been shown to enhance fluorescence emission from DCFH-DA in cell-free physiological
buffer even in the absence of an oxidizing agent [69].
Because resveratrol failed to enhance the fluorescence
emission in the absence of H2O2 in the medium (data not
shown) but it further enhanced H2O2-induced emission, its
mechanism seems to be different from Trolox and probably
depends on its pro-oxidant property. All these results
obtained with chlorogenic acid and others indicate that this
slice-free experimental model seems to promise an alternative model for testing the efficiencies of compounds
against H2O2-induced ROS production. Because several
factors, such as the concentration of H2O2 or duration of
the incubation period, may alter the results obtained under
this condition, additional studies need to be performed to
determine the accuracy or precision of this method.
Although incubating DCFH-DA with H2O2 in slice-free
oxygenated physiological medium causes a significant
increase in fluorescence intensities, it remains unclear
which reactive oxygen species are responsible for the
oxidation of DCFH to highly fluorescent DCF. DCFH does
not directly react with H2O2 to form DCF; several oneelectron-oxidizing species including hydroxyl radical,
superoxide anion or hypochlorous acid (HOCl) oxidize
DCFH to DCF [70]. Because several reactive intermediates
derived from H2O2 probably cause DCFH oxidation, a
DCFH-DA probe cannot be reliably used to measure the
levels of H2O2 or other specific reactive oxygen species in
the medium, and interpretation of the specific oxygen
species should be approached with caution.
In summary, the results presented here clearly indicate
that although quinic acid failed, chlorogenic and caffeic
acids protected rat cortical slices against H2O2-induced
alterations in oxidative stress parameters. Although
resveratrol, another commonly investigated phenolic
compound, has similar beneficial effects, its potency was
less than chlorogenic and caffeic acids. Because several
factors may alter their efficiencies, the antioxidant potencies of the natural phenolic compounds obtained in cell- or
tissue-free in vitro conditions do not necessarily match
with their effectiveness obtained under in vivo or tissue
containing in vitro experimental conditions.
Acknowledgments We would like to thank Uludag University
Research Council for linguistic edition of the MS by American
Journal Experts (AJE).
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict of
interest.

123

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