Sei sulla pagina 1di 11

Neurochem Res DOI 10.1007/s11064-016-1919-8

Neurochem Res DOI 10.1007/s11064-016-1919-8 ORIGINAL PAPER Protective Effects of Chlorogenic Acid and its Metabolites on

ORIGINAL PAPER

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 Gul 1 Celaleddin Demircan 2 Deniz Bagdas 3 Rifat Levent Buyukuysal 1

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 oxida- tive stress was investigated. Resveratrol, another natural phenolic compound, was also tested for comparison. Rat cortical slices were incubated with 200 lM H 2 O 2 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 effec- tiveness of chlorogenic acid and other compounds on H 2 O 2 -induced increases in fluorescence intensities were also compared in slice-free incubation medium. Although quinic acid failed, chlorogenic and caffeic acids signifi- cantly ameliorated the H 2 O 2 -induced decline in TTC staining intensities. Although resveratrol also caused an increase in staining intensity, its effect was not dose-de- pendent; the high concentrations of resveratrol tested in the present study (10 and 100 l M) further lessened the staining of the slices. Additionally, all phenolic compounds sig- nificantly attenuated the H 2 O 2 -induced increases in MDA and ROS levels in cortical slices. When the IC 50 values were compared to H 2 O 2 -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

2 Department of Internal Medicine, Faculty of Medicine, Uludag University, 16059 Bursa, Turkey

3 Experimental Animals Breeding and Research Center, Faculty of Medicine, Uludag University, 16059 Bursa, Turkey

Faculty of Medicine, Uludag University, 16059 Bursa, Turkey resveratrol for all parameters studied under these experi-

resveratrol for all parameters studied under these experi- mental conditions. In slice-free experimental conditions, on the other hand, chlorogenic and caffeic acids significantly attenuated the fluorescence emission enhanced by H 2 O 2 with a similar order of potency to that obtained in slice- containing 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 H 2 O 2 -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 [ 1 3 ]. 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 antioxi- dants 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 neu- rodegenerative diseases, including Alzheimer’s and Parkinson’s 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 [ 8 14 ].

123

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.

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 2 0 ,7 0 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.

123

Preparation and Incubation of Cortical Slices

Female Sprague–Dawley 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 [ 25 27 ], female rats were preferred in the pre- sent 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 CaCl 2 , 1.2 MgSO 4 , 3.5 KCl, 1.2 NaH 2 PO 4 , 25 NaHCO 3 and 10 glucose) that was oxygenated with 95 % O 2 and 5 % CO 2 . 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 3–4 mm 9 8–10 mm in size from this area was dissected and sliced with a McIlwain tissue chopper (Brinkmann Instruments; Westbury, NY, USA). Slices (0.40 mm thick- ness) were washed with oxygenated cold physiological medium and transferred to wells of the tissue culture incu- bation 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 H 2 O 2 - containing medium for another hour at 37 C. All phenolic compounds tested in the present study were added into the medium at the beginning of H 2 O 2 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 H 2 O 2 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

At the end of H 2 O 2 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 (50–50 %; v/v) to extract the red formazan from the slices [ 28 ]. After a 24 h extraction in the dark, 250 l l of the extracted liquid was transferred to 96-well plates, and absorbance values were measured at 490 nm by a plate reader (Mannheim Boehringer Pho- tometer 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-dinitro- phenylhydrazine (DNPH) derivatization as described pre- viously [ 29 ]. Cortical slices obtained at the end of the incubation periods were homogenized in 1 ml of 1 % sul- furic acid. A 200 l l portion of the homogenate was mixed with 20 l l 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 10,000 9 g for 10 min, and 20 l l 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 C 18 -reversed phase column (Macherey–Nagel 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 pro- tein levels and expressed as pmol/mg protein.

Determination of Tissue ROS Formation

significant increase in fluorescence intensity, and com- pounds with antioxidant properties can attenuate this H 2 O 2 - induced increase [30 ]. Thus, the effectiveness of phenolic compounds against the H 2 O 2 -induced increase in fluores- cence intensity was also tested in the absence of cortical slices in the medium. In the first series of experiments, increasing concentrations of H 2 O 2 were incubated in the absence or presence of DCFH-DA (5 l M) 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 H 2 O 2 (1 mM) and DCFH-DA (5 l M) 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 l l 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 l l of the homogenate [ 31 ]. All results were corrected according to total protein levels in the slices.

Statistical Analysis

Tissue ROS levels were measured with DCFH-DA (final concentration of 5 l M). DCFH-DA was dissolved in 0.1 %

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 Student–Newman–

DMSO and added into the medium during H 2 O 2 incubation

Keuls test. A p value of \0.05 was considered significant.

of the slices. At the end of H 2 O 2 incubation, slices were

IC

50 values with 95 % CL for all data were calculated by

washed with ice-cold physiological medium and homoge-

an

unweighted least-squares linear regression as described

nized with an ultrasound homogenizer, and 200 l l of the

by

Tallarida and Murray [32 ].

homogenate was used for fluorescence quantification with a spectrofluorometer (excitation and emission wavelengths 488 and 525 nm, respectively; Jasco FP-750 spectrofluo-

Results

rometer). 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.

H 2 O 2 -Induced Alterations in TTC Staining, MDA Production and ROS Production in Rat Cortical Slices

H 2 O 2 -Induced Fluorescence in Slice-Free Incubation Medium

We recently reported that incubating H 2 O 2 and DCFH-DA in slice-free oxygenated physiological medium causes a

A 1 h incubation of rat cortical slices with H 2 O 2 caused a

dose-dependent decrease in TTC staining intensities (Fig. 1 ). Tissue MDA and ROS levels, on the other hand, increased significantly after H 2 O 2 was added into the medium (Fig. 1 ).

123

Neurochem Res

Neurochem Res Fig. 1 H 2 O 2 -induced alterations in TTC staining, MDA and ROS

Fig. 1 H 2 O 2 -induced alterations in TTC staining, MDA and ROS productions in rat cortical slices. After preincubation period, cortical slices were incubated with increasing concentrations of H 2 O 2 for 1 h. At the end of H 2 O 2 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 8–10 determinations. * p \ 0.05, **p \ 0.01, ***p \ 0.001; significantly different from the corresponding control value determined in the absence of H 2 O 2

Effects of Chlorogenic Acid and its Metabolites on H 2 O 2 -Induced Alterations in Cortical Slices

Under oxidative stress conditions, such as ischemia fol- lowed by reoxygenation, H 2 O 2 levels actually reach hun- dred micromolar levels, depending on the experimental conditions [ 1 , 2 ]. Additionally, the H 2 O 2 -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 l M H 2 O 2 -induced alterations in rat cortical slices. As seen in Figs. 2 a and 3 , the H 2 O 2 -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. 2 b), quinic acid nearly failed to protect the slices (Fig. 2 c). When calculated from the dose–response curves, the IC 50 values of chlorogenic, caffeic and quinic acids were 0.1 ± 0.01, 0.22 ± 0.01 and [100 l M, respectively (Table 1 ). As observed in the TTC staining studies, H 2 O 2 -induced increases in tissue MDA and ROS levels were also atten- uated dose-dependently by chlorogenic acid (Figs. 4 a, 5 a). Although caffeic acid also protected the slices against MDA and ROS production (Figs. 4 b, 5 b), its IC 50 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. 4 c), it did not alter the H 2 O 2 -induced increase in ROS production (Fig. 5 c).

123

Effects of Resveratrol on H 2 O 2 -Induced Alterations in Cortical Slices: Comparison with CGA

Although resveratrol also ameliorated the H 2 O 2 -induced decrease in TTC staining intensities, its effect was not dose-dependent. Although low concentrations of resvera- trol were partially effective, increasing the concentration in the medium caused a further decrease in the staining intensities (Fig. 6 a). Resveratrol, like chlorogenic acid, also significantly protected against MDA and ROS pro- duction (Fig. 6 b, c), but the IC 50 value for each parameter was significantly higher than that of chlorogenic acid (Table 1 ).

H 2 O 2 -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 DCFH- DA (5 l M) with increasing concentrations of H 2 O 2 (50–1000 l M) in slice-free oxygenated physiological medium caused significant increases in fluorescence emission Fig. 7 a). When added into the medium, chloro- genic and caffeic acids decreased the 1 mM H 2 O 2 -induced fluorescence emission dose-dependently (Fig. 7 b). The IC 50 values of chlorogenic and quinic acids were 1.51 ± 0.02 and 5.4 ± 0.7 l M, respectively. Although quinic acid nearly failed, resveratrol, unexpectedly, caused significant enhancements in the fluorescence emission determined under this experimental condition (Fig. 7 b).

Discussion

Organotypic or acute brain slice preparation is one of the most widely used techniques in neuroscience for under- standing mechanisms and identifying targets and possible therapeutic strategies against neurological disorders, such as stroke, Parkinson’s and Alzheimer’s 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 pO 2 , pH and temperature, can be controlled and maintained as desired [ 36 ]. In contrast to cell cultures or tissue homo- genates, brain slices can maintain structural integrity [35 ]. Additionally, because brain slices have no blood–brain barrier, their extracellular space is accessible to the incu- bation/perfusion medium and its contents [ 35 , 37 ]. As an in vitro model of oxidative stress, we used H 2 O 2 as a free radical-generating system. In the tissue, H 2 O 2 originates from the enzymatic or spontaneous dismutation

Neurochem Res

Neurochem Res Fig. 2 Effects of chlorogenic acid and its metabolites on the H 2 O

Fig. 2 Effects of chlorogenic acid and its metabolites on the H 2 O 2 - induced decrease in TTC staining intensities. Cortical slices were incubated with 200 l M H 2 O 2 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 12–14 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 H 2 O 2 alone

from the value determined with H 2 O 2 alone Fig. 3 Typical images of rat

Fig. 3 Typical images of rat cortical slices stained with TTC. After preincubation period, cortical slices were incubated in control or 200 l M H 2 O 2 -containing medium for 1 h in the absence or presence of chlorogenic acid (CGA). At the end of the incubation, the slices

of superoxide anions, which are by-products of a variety of oxidases [ 38 , 39 ]. Massive H 2 O 2 production occurs during in vivo ischemia that correlates with neuronal loss [40 , 41 ]. The pathological consequences of H 2 O 2 elevation are

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

normally prevented by endogenous antioxidants, including the H 2 O 2 -scavenging enzymes glutathione (GSH) peroxi- dase and catalase [ 42 44 ]. Notably, modulatory as well as pathological consequences of H 2 O 2 are often mediated by

123

Neurochem Res

Neurochem Res Fig. 4 Effects of chlorogenic acid and its metabolites on the H 2 O

Fig. 4 Effects of chlorogenic acid and its metabolites on the H 2 O 2 - induced increase in tissue MDA levels. Cortical slices were incubated with 200 l M H 2 O 2 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

the hydroxyl radical ( OH), which results from the its catalytic decomposition on transition metals [ 1 , 45 47 ]. In the present study, we first aimed to determine H 2 O 2 - induced alterations in cortical slices before testing the effectiveness of chlorogenic acid and other phenolic com- pounds. As seen in Fig. 1 , a 1 h incubation with H 2 O 2 decreased the TTC staining intensities of the slices dose- dependently. Even the lowest concentration of H 2 O 2 (50 l M) caused a significant decrease, indicating an impairment in mitochondrial energy metabolism of the slices under this condition. In good agreement with these results, H 2 O 2 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 fol- lowed by reoxygenation, H 2 O 2 levels can reach hundred micromolar levels depending on the experimental condi- tions [ 48 , 49 ]; thus, we evaluated the effectiveness of chlorogenic acid and other naturally occurring compounds against 200 l M H 2 O 2 in the present study.

123

as the mean ± SEM of 8–10 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 H 2 O 2 alone

Because the bioavailability of chlorogenic acid is con- siderably 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 H 2 O 2 and 1,1-diphenyl-2-picryl- hydrazyl (DPPH) radical scavenging activities [ 23 ]. Addi- tionally, 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 effec- tiveness 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

Neurochem Res

Neurochem Res Fig. 5 Effects of chlorogenic acid and its metabolites on H 2 O 2

Fig. 5 Effects of chlorogenic acid and its metabolites on H 2 O 2 - induced increase in tissue ROS levels. Cortical slices were incubated with 200 l M H 2 O 2 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.

The data are provided as the mean ± SEM of 10–12 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 differ- ent from the value determined with H 2 O 2 alone

Table 1 IC 50 valuesof chlorogenic acid, its main metabolites and resveratrol against to H 2 O 2 -induced alterations in rat cortical slices

Phenolic compounds

Parameters measured in cortical slices

 

TTC staining intensity

MDA production

ROS levels

Chlorogenic acid

0.10±

0.01

0.22

±0.01

0.11 ± 0.01

Caffeic acid

0.20±

0.01

1.12

±0.05

***

5.6 ± 0.5***

Quinic acid

[100

10.33± 1.2

***

[100

Resveratrol

1.17± 0.38***

1.56 ±0.04 ***

10.05 ± 3.1 ***

Data presented means ± SEM. IC 50 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 condi- tion. 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 [58 60 ] and in vivo [ 61 , 62 ] experimental models, the presence of the chlorogenic acid in the medium significantly protected the cortical slices against H 2 O 2 -induced alterations. Although caffeic acid also exerted similar protective effects, its potency was

123

Neurochem Res

Fig. 6 Effect of resveratrol on H 2 O 2 -induced alterations in cortical slices: comparison with chlorogenic acid. Cortical slices were incubated with 200 l M H 2 O 2 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 H 2 O 2 -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 8–10 determinations. * p \ 0.05, ** p \ 0.01, ***p \ 0.001; significantly different than the resveratrol- induced changes

different than the resveratrol- induced changes significantly less than its parent compound; when com-

significantly less than its parent compound; when com- pared, the IC 50 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 H 2 O 2 -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 pro- tected the slices against H 2 O 2 -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 condi- tions. 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. 6 a, although 0.1 and 1 l M resveratrol signif- icantly ameliorated the TTC staining intensities, its higher concentrations in the medium caused a further decline. This observation may result from the deleterious effects of

123

high concentrations of resveratrol, which is consistent with

a previous study that suggested a significant pro-oxidant

effect depending on its concentration [ 68 ]. Although sim- ilar 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 oxida- tive stress parameters under our experimental conditions. We recently observed that incubating DCFH-DA with increasing concentrations of H 2 O 2 in slice-free physiolog-

ical 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, com- pounds 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 H 2 O 2 in

a slice-free incubation condition. In the present study, we tested the efficiencies of phe- nolic compounds against 1 mM H 2 O 2 -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 H 2 O 2 with a similar order of potency to that

Neurochem Res

Neurochem Res Fig. 7 H 2 O 2 -induced increase in fluorescence intensity from DCFH- DA

Fig. 7 H 2 O 2 -induced increase in fluorescence intensity from DCFH- DA in slice-free incubation condition: Effects of chlorogenic acid, its metabolites and resveratrol. a increasing concentrations of H 2 O 2 were incubated in the absence or presence of DCFH-DA (5 l M) 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 4–9 determinations. b DHCF-DA (5 l M) was incubated with 1 mM H 2 O 2 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 6–8 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 H 2 O 2 . 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 fluores- cence 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 H 2 O 2 in the medium (data not shown) but it further enhanced H 2 O 2 -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 alter- native model for testing the efficiencies of compounds against H 2 O 2 -induced ROS production. Because several factors, such as the concentration of H 2 O 2 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 H 2 O 2 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 H 2 O 2 to form DCF; several one- electron-oxidizing species including hydroxyl radical, superoxide anion or hypochlorous acid (HOCl) oxidize DCFH to DCF [ 70 ]. Because several reactive intermediates derived from H 2 O 2 probably cause DCFH oxidation, a DCFH-DA probe cannot be reliably used to measure the levels of H 2 O 2 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 H 2 O 2 -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 poten- cies 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

Neurochem Res

References

1.

Halliwell B (1992) Reactive oxygen species and the central nervous system. J Neurochem 59:1609–1623

2.

Halliwell B (2006) Oxidative stress and neurodegeneration:

Where are we now? J Neurochem 97:1634–1658. doi:10.1111/j.

3.

Ischiropoulos H, Beckman JS (2003) Oxidative stress and nitra- tion in neurodegeneration: cause, effect, or association? J Clin Invest 111:163–169. doi:10.1172/JCI200317638

4.

Thomas B (2009) Parkinson’s disease: from molecular pathways in disease to therapeutic approaches. Antioxid Redox Signal 11:2077–2082. doi: 10.1089/ars.2009.2697

5.

Zhu X, Lee HG, Perry G, Smith MA (2007) Alzheimer disease, the two-hit hypothesis: an update. Biochim Biophys Acta Mol Basis Dis 1772:494–502. doi: 10.1016/j.bbadis.2006.10.014

6.

Cai Y, Luo Q, Sun M, Corke H (2004) Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci 74:2157–2184. doi:10.1016/j.

7.

Balasundram N, Sundram K, Samman S (2006) Phenolic com- pounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem 99:191–203. doi: 10.1016/j.foodchem.2005.07.042

8.

Bagdas D, Etoz BC, Gul Z et al (2014) Chlorogenic acid enhances abdominal skin flap survival based on epigastric artery in nondiabetic and diabetic rats. Ann Plast Surg. doi:10.1097/

9.

Bagdas D, Ozboluk HY, Cinkilic N, Gurun MS (2014) Antinoci- ceptive effect of chlorogenic acid in rats with painful diabetic neuropathy. J Med Food 17:730–732. doi:10.1089/jmf.2013.2966

10.

Bagdas D, Cam Etoz B, Inan Ozturkoglu S et al (2014) Effects of systemic chlorogenic acid on random-pattern dorsal skin flap survival in diabetic rats. Biol Pharm Bull 37:361–370

11.

Bagdas D, Etoz BC, Gul Z et al (2015) In vivo systemic chlorogenic acid therapy under diabetic conditions: wound healing effects and cytotoxicity/genotoxicity profile. Food Chem Toxicol 81:54–61. doi:10.1016/j.fct.2015.04.001

12.

Bagdas D, Gul NY, Topal A et al (2014) Pharmacologic overview of systemic chlorogenic acid therapy on experimental wound healing. Naunyn Schmiedebergs Arch Pharmacol 387:1101–1116.

13.

Kanno Y, Watanabe R, Zempo H et al (2013) Chlorogenic acid attenuates ventricular remodeling after myocardial infarction in mice. Int Heart J 54:176–180

14.

Li Y, Shen D, Tang X et al (2014) Chlorogenic acid prevents isoproterenol-induced hypertrophy in neonatal rat myocytes. Toxicol Lett 226:257–263. doi: 10.1016/j.toxlet.2014.02.016

15.

Rispail N, Morris PM, Webb KJ (2005) Phenolic compounds:

extraction and analysis. In: Ma´ rquez AJ, Stougaard J, Udvardi MK, Parniske M, Spaink HP, Saalbach G, Webb KJ, Chiurazzi M, Ma´ r- quez AJ (eds) Lotus japonicus handbook. Springer, Netherlands, pp 349–354

16.

Clifford MN (1999) Chlorogenic acids and other cinnamates— nature, occurrence and dietary burden. J Sci Food Agric 79:362–372. doi: 10.1002/(SICI)1097-0010(19990301)79:3\362:

17.

Sharma P (2011) Cinnamic acid derivatives: a new chapter of various pharmacological activities. J Chem Pharm Res 3:403–423

18.

Avanesyan AA, Pashkov AN, Simonyan NA et al (2009) Anti- radical activity of cinnamic acid derivatives. Pharm Chem J 43:249–250. doi: 10.1007/s11094-009-0285-0

19.

Wenzel E, Somoza V (2005) Metabolism and bioavailability of trans-resveratrol. Mol Nutr Food Res 49:472–481. doi:10.1002/

123

20. Yu C, Geun Shin Y, Chow A et al (2002) Human, rat, and mouse metabolism of resveratrol. Pharm Res 19:1907–1914. doi:10.

21. Azuma K, Ippoushi K, Nakayama M et al (2000) Absorption of chlorogenic acid and caffeic acid in rats after oral administration.

J Agric Food Chem 48:5496–5500. doi:10.1021/jf000483q

22. Gonthier M-P, Verny M-A, Besson C et al (2003) Chlorogenic acid bioavailability largely depends on its metabolism by the gut microflora in rats. J Nutr 133:1853–1859

23. Sroka Z, Cisowski W (2003) Hydrogen peroxide scavenging, antioxidant and anti-radical activity of some phenolic acids. Food Chem Toxicol 41:753–758

24. Wang S-H, Chen C-S, Huang S-H et al (2009) Hydrophilic ester- bearing chlorogenic acid binds to a novel domain to inhibit xanthine oxidase. Planta Med 75:1237–1240. doi:10.1055/s-0029-1185521

25. Gu¨ rsoy M, Bu¨ yu¨ kuysal RL (2008) Resveratrol protects rat striatal slices against anoxia-induced dopamine release. Neurochem Res 33:1838–1844. doi:10.1007/s11064-008-9645-5

26. Gu¨ rsoyM, Bu¨ yu¨ kuysal RL (2010)Mechanism of S100b release from rat cortical slices determined under basal and stimulated conditions. Neurochem Res 35:429–436. doi:10.1007/s11064-009-0075-9

27. Bu¨ yu¨ kuysal RL, Mete B (1999) Anoxia-induced dopamine release from rat striatal slices: involvement of reverse transport mechanism. J Neurochem 72:1507–1515

28. Preston E, Webster J (2000) Spectrophotometric measurement of experimental brain injury. J Neurosci Methods 94:187–192

29. Pilz J, Meineke I, Gleiter CH (2000) Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative.

J Chromatogr B Biomed Sci Appl 742:315–325

30. Demircan C, Gul Z, Buyukuysal RL (2014) High glutamate attenuates S100B and LDH outputs from rat cortical slices

enhanced by either oxygen-glucose deprivation or menadione. Neurochem Res 39:1232–1244. doi: 10.1007/s11064-014-1301-7

31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem

193:265–275

32. Tallarida RJ, Murray RB (1987) Manual of pharmacologic cal- culations with computer programs, 2nd edn. Springer, New York

33. Feeney CJ, Frantseva MV, Carlen PL et al (2008) Vulnerability

of glial cells to hydrogen peroxide in cultured hippocampal slices. Brain Res 1198:1–15. doi: 10.1016/j.brainres.2007.12.049

34. Cho S, Wood A, Bowlby MR (2007) Brain slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics. Curr Neuropharmacol 5:19–33. doi:10.2174/

35. De Simoni A, Yu LMY (2006) Preparation of organotypic hip- pocampal slice cultures: interface method. Nat Protoc 1:1439–1445.

36. Schurr A, Rigor BM (eds) (1990) Cerebral ischemia and resus- citation. CRC Press Phillis JW, Boca Raton

37. Lein PJ, Barnhart CD, Pessah IN (2011) Acute hippocampal slice preparation and hippocampal slice cultures. Methods Mol Biol 758:115–134. doi: 10.1007/978-1-61779-170-3_8

38. Cross AR, Jones OT (1991) Enzymic mechanisms of superoxide production. Biochim Biophys Acta 1057:281–298

39. Clapp PA, Davies MJ, French MS, Gilbert BC (1994) The bac- tericidal action of peroxides; an E.P.R. spin-trapping study. Free Radic Res 21:147–167

40. Hyslop PA, Hinshaw DB, Scraufstatter IU et al (1995) Hydrogen peroxide as a potent bacteriostatic antibiotic: implications for host defense. Free Radic Biol Med 19:31–37

41. Zhang J, Piantadosi CA (1992) Mitochondrial oxidative stress after carbon monoxide hypoxia in the rat brain. J Clin Invest 90:1193–1199. doi:10.1172/JCI115980

Neurochem Res

42. Cohen G (1994) Enzymatic/nonenzymatic sources of oxyradicals and regulation of antioxidant defenses. Ann N Y Acad Sci

738:8–14

43. Desagher S, Glowinski J, Premont J (1996) Astrocytes protect neu- rons from hydrogen peroxide toxicity. J Neurosci 16:2553–2562

44. Sokolova T, Gutterer JM, Hirrlinger J et al (2001) Catalase in astroglia-rich primary cultures from rat brain: immunocyto- chemical localization and inactivation during the disposal of hydrogen peroxide. Neurosci Lett 297:129–132

45. Avshalumov MV, Chen BT, Rice ME (2000) Mechanisms underlying H(2)O(2)-mediated inhibition of synaptic transmis- sion in rat hippocampal slices. Brain Res 882:86–94

46. Pellmar TC, Neel KL, Lee KH (1989) Free radicals mediate peroxidative damage in guinea pig hippocampus in vitro. J Neu- rosci Res 24:437–444. doi: 10.1002/jnr.490240314

47. Sah R, Schwartz-Bloom RD (1999) Optical imaging reveals elevated intracellular chloride in hippocampal pyramidal neurons after oxidative stress. J Neurosci 19:9209–9217

48. Halliwell B, Gutteridge JM (1990) Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 186:1–85

49. Halliwell B (1994) Free radicals, antioxidants, and human dis- ease: curiosity, cause, or consequence? Lancet (London, Eng- land) 344:721–724

50. Cuvelier M-E, Richard H, Berset C (1992) Comparison of the antioxidative activity of some acid-phenols: strucgure-activity relationship. Biosci Biotechnol Biochem 56:324–325. doi:10.

51. Chen JH, Ho C-T (1997) Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. J Agric Food Chem 45:2374–2378. doi:10.1021/jf970055t

ˇ

52. Milic´ BL, Djilas SM, C anadanovic´ -Brunet JM (1998) Antiox- idative activity of phenolic compounds on the metal-ion break-

down of lipid peroxidation system. Food Chem 61:443–447.

53. Medina I, Gallardo JM, Gonzalez MJ et al (2007) Effect of molecular structure of phenolic families as hydroxycinnamic acids and catechins on their antioxidant effectiveness in minced fish muscle. J Agric Food Chem 55:3889–3895. doi:10.1021/

54. Marinova EM, Toneva A, Yanishlieva N (2009) Comparison of the antioxidative properties of caffeic and chlorogenic acids. Food Chem 114:1498–1502. doi: 10.1016/j.foodchem.2008.11.

045

55. Cheng J-C, Dai F, Zhou B et al (2007) Antioxidant activity of hydroxycinnamic acid derivatives in human low density lipoprotein: mechanism and structure–activity relationship. Food Chem 104:132–139. doi:10.1016/j.foodchem.2006.11.012

56. Chu Y-F, Brown PH, Lyle BJ et al (2009) Roasted coffees high in lipophilic antioxidants and chlorogenic acid lactones are more neuroprotective than green coffees. J Agric Food Chem 57:9801–9808. doi:10.1021/jf902095z

57. Quincozes-Santos A, Bobermin LD, Latini A et al (2013) Resveratrol protects C6 astrocyte cell line against hydrogen

peroxide-induced oxidative stress through heme oxygenase 1. PLoS One 8:e64372. doi: 10.1371/journal.pone.0064372

58. Sato Y, Itagaki S, Kurokawa T et al (2011) In vitro and in vivo

antioxidant properties of chlorogenic acid and caffeic acid. Int J Pharm 403:136–138. doi:10.1016/j.ijpharm.2010.09.035

59. Kim J, Lee S, Shim J et al (2012) Caffeinated coffee, decaf- feinated coffee, and the phenolic phytochemical chlorogenic acid up-regulate NQO1 expression and prevent H 2 O 2 -induced apop- tosis in primary cortical neurons. Neurochem Int 60:466–474.

60. Oboh G, Agunloye OM, Akinyemi AJ et al (2012) Comparative study on the inhibitory effect of caffeic and chlorogenic acids on key enzymes linked to alzheimer’s disease and some pro-oxidant induced oxidative stress in rats’ brain-in vitro. Neurochem Res 38:413–419. doi: 10.1007/s11064-012-0935-6

61. Kwon S-H, Lee H-K, Kim J-A et al (2010) Neuroprotective effects of chlorogenic acid on scopolamine-induced amnesia via anti-acetylcholinesterase and anti-oxidative activities in mice. Eur J Pharmacol 649:210–217. doi:10.1016/j.ejphar.2010.09.001

62. Heitman E, Ingram DK (2014) Cognitive and neuroprotective effects of chlorogenic acid. Nutr Neurosci doi:10.1179/

63. Pero RW, Lund H, Leanderson T (2009) Antioxidant metabolism induced by quinic acid. Increased urinary excretion of tryptophan and nicotinamide. Phytother Res 23:335–346. doi: 10.1002/ptr.

64. Erdem MG, Cinkilic N, Vatan O et al (2012) Genotoxic and anti- genotoxic effects of vanillic acid against mitomycin C-induced genomic damage in human lymphocytes in vitro. Asian Pac J Cancer Prev 13:4993–4998

65. Galati G, Sabzevari O, Wilson JX, O’Brien PJ (2002) Prooxidant activity and cellular effects of the phenoxyl radicals of dietary flavonoids and other polyphenolics. Toxicology 177:91–104

66. Pasciu V, Posadino AM, Cossu A et al (2010) Akt downregula- tion by flavin oxidase-induced ROS generation mediates dose- dependent endothelial cell damage elicited by natural antioxi- dants. Toxicol Sci 114:101–112. doi: 10.1093/toxsci/kfp301

67. Carru C, Pasciu V, Sotgia S et al (2011) The oxidative state of LDL is the major determinant of anti/prooxidant effect of coffee on cu catalysed peroxidation. Open Biochem J 5:1–8. doi:10.

68. Gadacha W, Ben-Attia M, Bonnefont-Rousselot D et al (2009) Resveratrol opposite effects on rat tissue lipoperoxidation: pro- oxidant during day-time and antioxidant at night. Redox Rep 14:154–158. doi: 10.1179/135100009X466131

69. Kalinich JF, Ramakrishnan N, McClain DE (1997) The antioxi- dant Trolox enhances the oxidation of 2 0 ,7 0 -dichlorofluorescin to 2 0 ,7 0 -dichlorofluorescein. Free Radic Res 26:37–47

70. Kalyaraman B, Darley-Usmar V, Davies KJA, Dennery PA, Forman HJ, Grisham MB, Mann GE, Moore K, Roberts LJ, Ischiropoulos H et al (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limita- tions. Free Radic Biol Med 52:1–6

123