Food and Chemical Toxicology 62 (2013) 448–455

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Appraisal of in vitro and in vivo antioxidant activity potential of cornelian

cherry leaves
Engin Celep a, Ahmet Aydın b, Hasan Kırmızıbekmez a, Erdem Yesilada a,⇑
a
Yeditepe University, Faculty of Pharmacy, Department of Pharmacognosy, Atasehir, 34755 Istanbul, Turkey
b
Yeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Atasehir, 34755 Istanbul, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: The antioxidant activity of the 80% methanolic extract of Cornus mas L. leaves (CMM) was evaluated by
Received 3 May 2013 various methods both in vitro and in vivo. In vitro screening tests indicated that CMM had high antioxi-
Accepted 2 September 2013 dant activity in terms of free radical scavenging and metal reducing activity. In vivo antioxidant activity
Available online 11 September 2013
studies in normal healthy rats demonstrated that the total antioxidant capacity of liver homogenates
were increased, although no changes were observed in the activities of antioxidant enzymes superoxide
Keywords: dismutase, catalase and glutathione peroxidase or in the level of lipid peroxidation. Studies on CCl4-trea-
Cornus mas L.
ted rats also showed that CMM restored the activities of antioxidant enzymes, lowered the level of lipid
In vitro antioxidant activity
In vivo antioxidant activity
peroxidation and elevated the total antioxidant capacities of both the total blood and liver homogenates
Antihepatotoxic activity of the animals. Further activity-guided fractionation studies led to the isolation of gallic acid, a
Gallic acid well-known antioxidant, as one of the active components.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction et al., 2009) were reported. Interestingly, in spite of this ethnobo-

tanical background, the phytochemical composition of the corne-
Cornus mas L., known as ‘‘cornelian cherry’’ in English and lian cherry leaves was largely unexplored. Recently, ellagic acid
‘‘kızılcık’’ in Turkish, is one of the two species belonging to the and gallic acid were reported to be the main components of C.
family Cornaceae in the Turkish flora. It is native to Central and mas leaves (Leskovac et al., 2007), while rutin (Savikin et al.,
Southeastern Europe and Southwest Asia. It is a deciduous shrub 2009) and ursolic acid were the other ingredients (Jager et al.,
or small tree with a height of 3–5 m and red drupe fruits. Its fruits 2009).
are consumed freshly or as marmalade and jam (Celep et al., 2012; Oxidative stress is attributed to the etiology of several diseases
Popović et al., 2012). They are traditionally used against diarrhea, such as diabetes, cancer and cardiovascular disorders. Therefore, it
common colds, diabetes and to lose weight (Baytop, 1999; Pieroni is important to increase the body’s antioxidant potential for fight-
et al., 2003). ing against oxidative stress. For this purpose, it has been widely
The infusion prepared from leaves of C. mas are traditionally advised that individuals increase their intake of dietary antioxi-
used in the treatment of hyperglycemia, hypertension and in dants. Regular consumption of fruits and vegetables rich in natural
wound healing, and consumed as tea not only in Turkey but also antioxidants is associated with reducing the risk of various
among Turkish immigrants living in Germany (Pieroni et al., diseases (Ames et al., 1993). Since the cornelian cherry leaves are
2005; Yesßilada et al., 1999). Although there are a number of studies reported to be used against diabetes in ethnobotanical literature,
reporting the in vitro antioxidant activity of its fruits (Ersoy et al., and the relationship between diabetes and oxidative stress was
2011; Pantelidis et al., 2007; Popović et al., 2012), studies on the already revealed (Aydin et al., 2001a), the present study aimed at
antioxidant potential of the C. mas leaves are scarce. Previously, detailed investigation of both in vitro and in vivo antioxidant
the methanol extract of cornelian cherry leaves was shown to potentials of C. mas leaves. Firstly, total phenolic, flavonoid and
possess a high in vitro antioxidant activity against DPPH radical proanthocyanidin contents were determined in the leaf extract,
as well as inhibited lipid peroxidation (Savikin et al., 2009). On since they are well-known for their antioxidant potentials. Besides,
the other hand, a significant radioprotective potential of the leaf eight different in vitro methods for the purpose of a detailed inves-
extract against radiation-induced tissue damages (Leskovac et al., tigation of the antioxidant activity of CMM were employed. Since
2007) and cytotoxic activity against cancer cell lines (Savikin the antioxidant molecules exert their activities with different
mechanisms, these methods were chosen in order to reveal the
antioxidant activity profile of the test materials thoroughly. In
⇑ Corresponding author. Tel.: +90 216 578 0000x3021; fax: +90 216 578 0068. addition to in vitro screening tests, in vivo activity tests were
E-mail address: yesilada@yeditepe.edu.tr (E. Yesilada).

0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2013.09.001
 E. Celep et al. / Food and Chemical Toxicology 62 (2013) 448–455 449

employed on both normal healthy and hepatotoxin-treated 2.7. Determination of total proanthocyanidin content
animals. For this purpose, the activities of primary antioxidant en-
The total proanthocyanidin content of samples was measured using the method
zymes, superoxide dismutase (SOD), catalase (CAT) and glutathi- described in our previous study (Celep et al., 2012). Briefly, 2.5 mL of 1% vanillin in
one peroxidase (GSH-Px), as well as malondialdehyde levels as a methanol and 2.5 mL of 9 M HCl in methanol were added to properly diluted ex-
sign of lipid peroxidation and the total antioxidant capacities on tract. After incubation at 30 °C for 20 min, the absorbance was measured at
both total blood samples and liver homogenates of the treated 500 nm, and the results were expressed as mg epigallocatechingallate equivalents
(EGCG-E) per g dry extract.
animals were measured. Finally, in vitro antioxidant activity
guided-fractionation studies were carried out in order to isolate
the antioxidant compound(s) from the active extract. 2.8. In vitro antioxidant activity studies

2.8.1. DPPH radical-scavenging activity

DPPH radical-scavenging activity of samples was performed as described previ-
2. Materials and methods
ously (Celep et al., 2012). One thousand microliters of properly diluted samples
were separately added to 5000 lL of 0.1 mM DPPH solution prepared in methanol
2.1. Chemicals
just before use. The mixture was vortexed and incubated in the dark for 50 min
at room temperature. The absorbance was measured at 517 nm. Water was used
All chemicals, enzymes and standards used in the experiments were purchased
in the control group instead of samples, and the same procedure was applied. Butyl-
from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals were of analytical
ated hydroxy toluene was used as the reference substance. DPPH scavenging activ-
grade. Doubly distilled deionized water (Milli-Q Millipore 18.2 MX/cm 25 °C) was
ity of the control group, the reference substance and the samples were calculated as
used in all experiments.
follows:

DPPH radical scavenging activity ð%Þ ¼ ½ðAbscontrol  Abssample Þ=Abscontrol   100

2.2. Plant materials
where Abscontrol is the absorbance value of the control group, and Abssample is the
The leaves of C. mas were collected from the wilderness in Susurluk, Balıkesir absorbance of the samples.
province of Turkey in June 2010. The plant material was authenticated by Prof.
Dr. Erdem Yesilada, one of the authors before any process. A voucher specimen
was deposited in the Herbarium of Faculty of Pharmacy, Yeditepe University (YEF 2.8.2. Superoxide radical-scavenging activity
10005). The leaves were dried under shade and later powdered. The superoxide radical-scavenging activity of samples was measured by a
method previously described with following modifications (Aydin et al., 2001a).
Briefly, 50 lL of properly diluted extract were mixed with 1700 lL of substrate
2.3. Extraction and fractionation solution containing 0.05 mmol/L xanthine sodium and 0.025 mmol/L 2-(4-iodo-
phenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) in a buffer solution
Two hundred and eighty grams of powdered leaves were macerated 24 h with containing 50 mmol/L CAPS and 0.94 mmol/L EDTA (pH 10.2). Then 250 lL of xan-
2200 mL of 80% MeOH. Then, the macerates were extracted twice at 45 °C using the thine oxidase (80 U/L) was added to the mixture and absorbance increase was mon-
same solvent for 4 h with continuous stirring, then filtered and lyophilized (the itored at 505 nm for 3 min. Fifty microliters of distilled water or various reference
yield was 55.44 g, 19.86%). concentrations in place of sample were used as blank or standard determinations.
Then, 45 g of the 80% methanolic extract (CMM) was dissolved in 100 mL of Gallic acid was used as reference substance.
MeOH 90% and extracted with n-hexane (4  100 mL) in a separatory funnel. Com-
bined hexane extract was evaporated under reduced pressure to yield ‘‘Hexane Fr.’’ 2.8.3. Ferric reducing antioxidant power (FRAP)
(1.8%). MeOH was removed from the remaining extract, diluted with H2O to Antioxidants are used as reductants in a redox-linked colorimetric method in
100 mL, and further fractioned by successive solvent extractions with chloroform this assay (Celep et al., 2012). Ten microliters of properly diluted samples and
(4  100 mL), ethyl acetate (4  100 mL) and n-butanol saturated with H2O 30 lL of Milli Q water were mixed with 260 lL of FRAP reagent in a microplate.
(4  100 mL). Each organic layer was evaporated to dryness under reduced pressure The mixture was incubated at 37 °C for 30 min. After the incubation period, the
to yield ‘‘CHCl3 Fr.’’ (11.2%), ‘‘EtOAc Fr.’’ (5.58%), ‘‘n-BuOH Fr.’’ (22.07%), respectively, absorbance was read at 593 nm using a 96-well microplate reader. Water was used
while the aqueous phase was lyophilized, ‘‘R-H2O Fr.’’ (59.36%) (Fig. 1). for the control group instead of sample. A standard curve was prepared using differ-
ent concentrations of FeSO47H2O (0.25–2 mM). Butylated hydroxy toluene was
used as the reference substance. The results were expressed as mM FeSO4 per g
2.4. Isolation of the active constituent material.

Two and a half grams of EtOAc Fr. was subjected to column chromatographic
separation on polyamide with MeOH (0 ? 100%) as mobile phase, and they were 2.8.4. Cupric reducing antioxidant capacity (CUPRAC)
grouped based on their TLC profile into 10 different fractions. The cupric ion reducing capacities of the samples were determined according to
Fr. 11–12 (78.8 mg) was separated by Sephadex LH-20 (12 g) column chroma- the method of Apak et al. (2004) with slight modifications. One milliliter of each of
tography using MeOH as eluent, yielding compound 1 (10.2 mg). The chemical 10 mM CuSO4, 7.5 mM neocuproine, and 1 M ammonium acetate buffer (pH 7.0)
structure of compound 1 was identified as gallic acid by comparison of its spectro- solutions were mixed in a test tube. Later, 0.5 mL of sample solution was added,
scopic data (1H and 13C NMR) with those in the literature (Chanwitheesuk et al., and the final volume was completed to 4.1 mL with Milli Q water. Following the
2007). 1 h incubation at room temperature, the absorbance was recorded at 450 nm.
Ascorbic acid was used as the standard substance, and the results were given as
mg ascorbic acid equivalent (AAE) per g material.
2.5. Determination of total phenolic content
2.8.5. Ferrous ion-chelating activity
The method described previously was used for the measurement of total pheno-
Ferrous ion-chelating activities of the samples were measured by the method
lic content (Celep et al., 2012). A blue molybdenum–tungsten complex is produced
described previously (Celep et al., 2012). One milliliter of each sample was added
in the presence of phenolics in this assay.
to 50 lL of 2 mM FeCl24H2O and 3.7 mL of distilled water. After the addition of
According to this method, 20 lL of properly diluted samples were mixed with
5 mM ferrozine, the mixture was incubated for 10 min at room temperature. Later,
Folin–Ciocalteu reagent and Na2CO3 (20%). After a 30 min incubation period at
the absorbance was read at 562 nm. The activity was calculated by using the same
45 °C, the absorbance was read at 765 nm. All experiments were performed tripli-
formula which was used for the DPPH radical scavenging activity. EDTA was used as
cate and results were expressed as mg gallic acid equivalents (GAE) per dried ex-
reference compound.
tract (g).

2.8.6. Total antioxidant capacity

2.6. Determination of total flavonoid content The total antioxidant capacities of samples were evaluated by using the phosp-
homolybdenum method with slight modifications (Celep et al., 2012). Three hun-
Five hundred milliliters of properly diluted samples were mixed with AlCl3 dred microliters of samples were mixed with 3 mL of reagent solution consisting
(10%) and 1 M sodium acetate. Following the incubation period at room tempera- of 28 mM sodium phosphate monobasic, 4 mM ammonium molybdate and 0.6 M
ture for 30 min, the absorbance was read at 415 nm. Total flavonoid contents of sulfuric acid. The mixture was incubated at 95 °C for 90 min, and later the absor-
the samples were expressed as mg quercetin equivalents (QE) per g dried extract bance was read at 695 nm. The total antioxidant capacities were expressed as mg
(Celep et al., 2012). ascorbic acid equivalent per g dry extract.
450 E. Celep et al. / Food and Chemical Toxicology 62 (2013) 448–455

CM
Leaves
(280 g)

45 °C, 4 h (x2) 80% MeOH (2.2 L)

CM-MeOH
(55.61 g, 19.86%)

+ MeOH : H2O (90:10)

n-Hexane (4 x 100 mL)

MeOH : H2O evaporated

Residue CM-n-Hexane
+ H2O (100 mL) (0.81 g, 1.8%)

CH2Cl2 (4 x 100 mL)

CM-CH2Cl2 Aqueous Layer

(5.04 g, 11.2%)

EtOAc (4 x 100 mL)

Aqueous Layer CM-EtOAc

(2.51 g, 5.58%)
n-BuOH (4 x 100 mL)

CM-R-H2O CM-n-BuOH
(26.71 g, 59.36%) ( 9.93 g, 22.07%)

Fig. 1. The extraction process.

2.8.7. Bleaching assay of b-carotene in a linoleic acid system temperature and humidity were controlled with 12 h light and dark cycle, and they
This assay was performed according to a method described before (Celep et al., had free access to food and water. The experimental protocol was approved by the
2012). Briefly, 20 lL of linoleic acid and 200 lL of Tween 20 were pipetted to a Ethical Committee of Yeditepe University Experimental Medicine Research Institute
100 mL-round bottom flask. 1000 lL of b-carotene solution in chloroform (Decision number: 2012/246), and the use of animals was in compliance with US
(0.2 mg/mL) was added. After chloroform was removed in a rotatory evaporator, National Institute of Health Guide for Care and Use of Laboratory Animals.
50 mL of distilled water was added to the oily residue with shaking vigorously to
form an emulsion. 5000 lL aliquots of the emulsion were pipetted into test tubes
containing 200 lL sample or distilled water or reference and left for incubation at
2.9.2. Experimental protocol on normal rats
50 °C for 1 h. The absorbance was read a 470 nm. BHT was used as positive control.
Rats were divided into 3 groups with 6 animals in each group. Control group re-
The antioxidant activity was expressed as inhibition percentage relative to control,
ceived distilled water containing 0.5% carboxymethylcellulose (CMC) p.o. for
using the formula previously described (Celep et al., 2012).
21 days. Reference group received 50 mg/kg bodyweight silymarin p.o., and the test
group received 80% methanolic leaf extract of C. mas (500 mg/kg bodyweight) for
2.8.8. Trolox equivalent antioxidant capacity (TEAC)
21 days. The animals were decapitated on day 22. Their blood samples were col-
5000 lL of 7 mM 2,20-azinobis-(3-ethyl-benzothiazoline-6-sulfonate) (ABTS)
lected and their livers were dissected.
was reacted with 88 lL of 140 mM potassium persulfate solution (final concentra-
tion equals to 2.45 mM) overnight in the dark to generate ABTS+ radical cation.
Later, ABTS+ solution was diluted with ethanol to an absorbance of 0.7 ± 0.05 at
734 nm. 20 lL of properly diluted samples was mixed with 2000 lL of diluted 2.9.3. Experimental protocol on CCl4-treated rats
ABTS+ solution. The absorbance was measured 6 min after the initial mixing at This experimental protocol was accomplished according to the method de-
734 nm. TEAC was expressed as lM Trolox equivalent per g dry extract (Celep scribed by Huang and colleagues with slight modifications (Huang et al., 2010). Rats
et al., 2012). in this protocol were divided into 6 groups. Control group received distilled water
containing 0.5% CMC p.o. for 5 days and olive oil (1 mL/kg bodyweight, s.c.). CCl4-
2.9. In vivo antioxidant studies treated group received 0.5% CMC for 5 days, and a 1:1 mixture of CCl4 and olive
oil (2 mL/kg bodyweight, s.c.) on days 2 and 3. Test group animals were adminis-
2.9.1. Test animals tered 80% methanolic extract of C. mas leaves at a dose of 100, 200 and 500 mg/
Male Sprague–Dawley rats, weighing 200–250 g, were used in the course of kg bodyweight for 5 days. Additionally, they received a dose of 1:1 CCl4 and olive
study. The animals were provided by the Yeditepe University Experimental Re- oil (2 mL/kg bodyweight, s.c.) on days 2 and 3, 30 min after the administration of
search Center (YUDETAM). They were kept in Plexiglas cages in a room of which the extracts. Reference group received 50 mg/kg bodyweight silymarin p.o. for
 E. Celep et al. / Food and Chemical Toxicology 62 (2013) 448–455 451

5 days, and also received 1:1 CCl4 and olive oil (2 mL/kg bodyweight, s.c.) on days 2 2.10. Statistics
and 3, 30 min after the administration of silymarin. Animals were decapitated on
day 6, blood was collected and their livers dissected for further analysis. The experiments were performed in triplicate. The results were expressed as
mean ± standard deviation. Statistical comparisons were made using one way anal-
ysis of variance (ANOVA) followed by Student–Newman–Keuls post hoc test for
multiple comparisons. Besides, Pearson correlation coefficients were calculated.
2.9.4. Preparation of total blood samples
Statistically significant difference was defined as p < 0.05.
Total blood samples were prepared according to the method described previ-
ously (Aydin et al., 2001a). After the decapitation of animals, fresh blood was col-
lected into the tubes containing EDTA. Each blood sample in the tubes was 3. Results and discussion
turned upside and down without shaking and lysed with cold distilled water
(1:4), stored in refrigerator at 4 °C for 15 min and the cell debris was removed by
centrifugation (2000g for 10 min at 4 °C). 3.1. Total phenol/total flavonoid/total proanthocyanidin contents of
CMM

2.9.5. Preparation of liver homogenates Phenolic compounds are known to have the ideal chemical
After the decapitation process, liver of each rat was removed and frozen imme- properties for antioxidant activity since they act both as hydrogen
diately on dry ice. The frozen tissues were stored at –80 °C until further use. 10% (w/
and electron donors and have the ability of chelating metal ions
v) homogenates were prepared by mincing and homogenizing 1 g of liver in 9 mL
ice cold KCl solution (1.15%) with a glass homogenizer. The homogenate was cen-
(Leopoldini et al., 2011). Therefore, a substantial portion of antiox-
trifuged at 4400g and 4 °C for 15 min. The resultant supernatant was used for the idant properties of plant extracts have been attributed to their phe-
determination of enzyme activities and lipid peroxidation. nolic content. Then, total phenolic, total flavonoid and total
anthocyanidin contents of CMM were determined and results are
shown in Table 1. Total phenolic content was found to be
2.9.6. Determination of protein content in total blood samples or liver homogenates 342.6 ± 10.7 mg GAE/g, which was much higher (56.9 mg GAE/g)
The protein content of the samples was determined according to the Lowry
than reported previously by Savikin et al. (2009). This difference
method (Lowry et al., 1951).
might possibly be due to the difference in extraction process, since
they employed methanol as vehicle and Soxhlet apparatus for
2.9.7. Determination of the activities of antioxidant enzymes extraction. While, total flavonoid and total proanthocyanidin con-
The superoxide dismutase (SOD) activity was measured according to previously tents of the extract were estimated to be 72.83 ± 3.1 mg QE/g and
described method by Aydin et al. (2001a,b). SOD activity was expressed as U/mg 298 ± 11.8 mg EGCG-E/g of dry extract, respectively.
protein.
In order to measure the catalase (CAT) activity the reaction mixture was com-
posed of 50 mM phosphate buffer pH 7.0, 10 mM H2O2 and total blood sample or 3.2. In vitro antioxidant activity of CMM
liver homogenate. The reduction rate of H2O2 was followed for 240 nm for 30 s at
room temperature (Aebi, 1984). CAT activity was expressed as U/mg protein. DPPH, superoxide radical scavenging and TEAC tests were per-
For determination of the glutathione peroxidase (GSH-Px) activity, total blood formed in order to determine the general free radical scavenging
was diluted 15 times, and tissue homogenate was diluted 17 times before the assay.
990 lL of the reaction mixture was pipetted into a 1 mL cuvette. Then, 10 lL total
abilities, since free radicals are one of the main causes of oxidative
blood or tissue homogenate is added and the mixture was incubated for 5 min at stress (Kehrer, 1993). CMM exerted high DPPH radical scavenging
room temperature. The reaction was initiated with the addition of 10 lL of tert-bu- activity (EC50: 165 ± 10.2 lg/mL), which was very near to that of
tyl hydroperoxide and the decrease in NADPH absorbance was followed at 340 nm BHT, a reference compound (EC50: 133 ± 6.4 lg/mL). However,
for 3 min. The difference in absorbance per minute is calculated. GSH-Px activity
the superoxide radical scavenging activity of the same extract
was expressed as U/mg protein.
was much lower than the reference compound gallic acid (Table
2). Since TEAC assay is accomplished by generating ABTS radical,
2.9.8. Determination of lipid peroxidation levels it gives information about the free radical scavenging capacity of
Since malondialdehyde (MDA) is an end-product and a reliable sign of lipid per- the samples. The result in Table 2 demonstrated that the extract
oxidation, the MDA levels both in total blood samples and liver homogenates were had the activity of 869 ± 20.52 ± lM Trolox equivalent/g of dry ex-
measured. The principle of the assay depends on the reaction of MDA with thiobar- tract, pointing out a good radical scavenging activity.
bituric acid (TBA) and forming a pink colored MDA–TBA complex.
The MDA levels were determined in total blood samples according to the meth-
Fe3+ reduction is an important aspect regarding electron dona-
od described by Aydin et al. (2001a). Two hundred and fifty microliters of each of tion which is one of the major mechanisms of antioxidant activity.
total blood samples, phosphate buffer and trichloroacetic acid were mixed in FRAP test is frequently used to measure the antioxidant capacity.
1.5 mL-Eppendorf tubes. The mixtures were vortexed and kept on ice bags in refrig- As shown in Table 2, CMM showed better ferric ion reducing activ-
erator (4 °C) for 2 h. At the end of the period, the tubes were centrifuged at
ity than the reference compound, BHT (3.44 ± 0.009 vs.
4400 rpm and 4 °C for 10 min. 500 lL of the supernatant was mixed with 38 lL
of EDTA and 125 lL of TBA. The mixtures were vortexed and kept on boiling water 3.02 ± 0.07), which is expressed as mM FeSO4 equivalent/g of dry
bath for 15 min. The absorbance was recorded at 532 nm. 1,1,3,3-tetramethoxypro- extract.
pane was used as standard for plotting the calibration curve. The results were given Since Cu2+ also takes part in the formation of free radicals just
as nmol/mg protein. like Fe3+, the reduction of cupric ion indicates another mechanism
The MDA levels in the liver homogenates were determined according to the
method described by Jamall and Smith (1985). Two hundred microliters of sample
reflecting the antioxidant potential (Aydin et al., 2001b). CUPRAC
or standard substance, 200 lL of sodium dodecyl sulfate, 1500 lL of acetic acid test, which is a relatively new method on metal ion reducing com-
solution and 1500 lL of thiobarbituric acid solution were mixed. The volume was pared to FRAP, is used as an antioxidant capacity index particularly
completed to 4000 lL with Milli Q water. The tubes were kept at 95 °C for 1 h. At for dietary polyphenolics. The result is shown in Table 2 as
the end of this period, they were cooled under tap water. Later, 2000 lL of the mix-
214.45 ± 2.15 mg ascorbic acid mg/g of dry extract.
ture was added to 2000 lL of trichloroacetic acid. They were centrifuged at 1000g
for 10 min. The absorbance of the supernatant was read at 532 nm. The same cali- Interestingly, the extract did not show any metal ion chelating
bration curve that was used for total blood MDA assay was also used here. The re- activity at the concentrations of 1.2 and 5 mg/mL (EDTA, reference
sults were expressed as nmol/mg protein. compound, had an EC50 value of 9.1 ± 0.4 lg/mL). However, as
mentioned above, the extract had good reducing activity on both
ferric and cupric ions. These data demonstrated that an extract
2.9.9. Determination of total antioxidant capacity of total blood samples and tissue with a good rate of metal reducing activity does not necessarily
homogenates
The same method described in Section 2.8.8 was used here with the only excep-
show good metal chelating activity.
tion of using total blood samples or tissue homogenates instead of plant extracts. Total antioxidant capacity test (TOAC) is based on the reduction
The results were expressed as lM Trolox equivalent per mg protein. of Mo6+ to Mo5+. Consequently, it gives general information about
452 E. Celep et al. / Food and Chemical Toxicology 62 (2013) 448–455

the whole reduction capacity of the samples. The result is given in investigated the antioxidant capacity of apricot in Wistar rats after
Table 2, as 320.17 ± 10.10 mg AAE/g of dry extract. 5-months of oral administration. They reported that no changes in
b-carotene bleaching assay is based on the discoloration of b- the activities of SOD, CAT or GSH-Px were observed in the liver
carotene owing to its reaction with linoleic acid-generated free homogenates of normal rats.
radicals in an emulsion system. In the presence of an antioxidant Lipid peroxidation is also one of the most important mecha-
compound, this degradation process is prevented. It also reflects nisms contributing to oxidative stress. Hence, the measurement
the ability to inhibit the lipid peroxidation in vitro (Celep et al., of lipid peroxidation is an important indicator in the assessment
2012). The extract exerted a high rate of activity (93 ± 2.4%), which of antioxidant potential. We calculated the amount of MDA, which
was very near to that of BHT (96 ± 2.6%). is one of the most important by-products of lipid peroxidation.
According to the results given in Table 3, no significant changes
3.3. In vivo antioxidant activity of CMM were observed in the MDA levels of the liver tissue or the blood
of normal rats administered either with extract or silymarin
Although in vitro antioxidant assays are widely employed for (p > 0.05). Pradeep and colleagues stated that 30 days of silymarin
the assessment of antioxidant activity potential of plants, the re- administration did not cause any alterations in the MDA levels of
sults may sometimes be conflicting with the results obtained from normal rats, which is in favor our results (Pradeep et al., 2007). An-
in vivo. The main reason behind this aspect lies on the bioavailabil- other important point about lipid peroxidation that should be
ity of active compounds, as well as their metabolism in the body. highlighted is the correlation between b-carotene bleaching assay
As mentioned earlier, most of the health benefits of fruits and and MDA levels. b-carotene bleaching assay is, as previously men-
vegetables have been attributed to their polyphenolic and flavo- tioned, used as a screening tool for the inhibition of lipid peroxida-
noid contents. However, their whole mechanism of antioxidant tion in vitro. However, we observed that cornelian cherry leaf
activity remains unclear in spite of indefinite numbers of extract showed good activity in b-carotene bleaching assay, it did
researches conducted in this area. First, the absorption of polyphe- not induce any decrease in the levels of MDA in normal rats.
nols in organism is rather low, especially that of anthocyanins. In order to measure the total antioxidant capacity in both total
Another factor is the short half-life of polyphenols in plasma, blood samples and liver homogenates, TEAC assay was used.
which are usually in the range of a couple of hours. In addition According to results given in Table 3, the extract induced an in-
of poor absorption, polyphenols and especially flavonoids are crease of 20.51% (p < 0.01), while the reference drug silymarin also
extensively metabolized in liver and intestine when ingested. provided an increase of 25.38% (p < 0.001) in total antioxidant
Flavonoids are excellent substrates and inducers of phase II capacity of total blood samples, when compared to control group.
enzymes. These factors may cause limitations in the capability of In addition to these results, the total antioxidant capacity of the li-
dietary polyphenols in performing antioxidant activity in vivo. ver homogenates of the animals treated with CMM increased as
Therefore, in order to ascertain the role of dietary antioxidants 29.19% (p < 0.001) and that of silymarin as 31.55% (p < 0.001).
fully, in vivo tests are incredibly necessary, though they are so The increased antioxidant capacity in total blood and liver tissues
few (Lotito and Frei, 2006). following the administration of CMM could indicate a direct
In order to find out any possible effects of CMM on normal met- absorption of several antioxidant phytochemicals (Robles-Sánchez
abolic conditions, normal rats were used. The treatment was per- et al., 2011).
formed orally for 21 successive days in order to observe the sub- After accomplishing in vitro screening tests and in vivo antioxi-
acute effects and the oral bioavailability of active components. dant activity studies on healthy rats with normal metabolic condi-
For the purpose of evaluating in vivo antioxidant potential, we tions, the antioxidant potential was studied under elevated
measured the activities of SOD, CAT and GSH-Px since they are oxidative stress. Carbon tetrachloride is a highly toxic chemical
the first line of defense against reactive oxygen species in the agent which is extensively used to evaluate the hepatoprotective
organism by converting the active oxygen molecules into non-toxic potential of drugs and plant extracts. Its hepatotoxic effects are a
compounds (Ames et al., 1993). They are also referred as primary result of its biotransformation process. CCl4 is primarily
antioxidant enzymes (Aydin et al., 2001b). SOD dismutates super- accumulated in hepatic parenchyma cells and metabolized to

oxide radicals into hydrogen peroxide and molecular oxygen. CAT trichloromethyl ðCCl3 Þ radical by cytochrome P450-dependent

further detoxifies hydrogen peroxide into water. GSH-Px also par- monooxygenases in liver. Then CCl3 radical reacts rapidly with
ticipates in the detoxification of hydrogen peroxide. Hence, these oxygen to form the highly reactive trichloromethyl peroxyl radical
enzymes act mutually and compose the enzymatic antioxidant ðCCl3 O2 Þ. This radical binds to cellular macromolecules and causes
capacity against ROS. peroxidative damage in lipid membranes of the adipose tissue. It
After 21-day treatment, no statistically significant changes were also facilitates the removal of hydrogen atoms from unsaturated
observed (p > 0.05) in the activities of these enzymes of both total lipids. In addition, it causes auto-oxidation of the polyenic fatty
blood and liver homogenates, comparing to the control group. Also, acids found in the membrane phospholipids, leading to the lipid
no changes were observed in the positive control group (silymarin) peroxidation. Consequently, by-products like MDA are generated
(Table 3). This consequence was somehow expected since the con- in extremely high amount, resulting in the loss of cell membrane
sumption of the studied extract did not induce the synthesis of integrity and tissue damage (Recknagel et al., 1989).
these enzymes in normal rats. Otherwise, this would be an inter- In order to evaluate the dose-depended response of CMM, it was
vention to the homeostasis of the body. These results are in accor- administered at increasing doses of 100, 200 and 500 mg/kg body
dance with the study conducted by Ozturk et al. (2009). They weight, p.o. The tests indicated that CCl4 administration caused a

Table 1
Total phenolic, flavonoid and proanthocyanidin contents of the 80% MeOH extract of C. mas leaves (CMM).a

Total phenolic contentb Total flavonoid contentc Total proanthocyanidin contentd

CMM 342.6 ± 10.7 72.83 ± 3.1 298 ± 11.8
a
Results were expressed as the mean of triplicates ± standard deviation (SD).
b
Total phenolic content was expressed as mg gallic acid equivalents (GAE) per g dried extract ± SD.
c
Total flavonoid content was expressed as mg quercetin equivalents (QE) per g dried extract ± SD.
d
Total proanthocyanidin content was expressed as mg epigallocathecin gallate equivalents (EGCG-E) per g dried extract ± SD.
 E. Celep et al. / Food and Chemical Toxicology 62 (2013) 448–455 453

Table 2
In vitro antioxidant activities of the 80% MeOH extract of C. mas leaves (CMM).a

Samples DPPH radical scavenging Superoxide radical scavenging FRAPd CUPRACe b-Carotene bleaching TOACg TEACh
activityb activityc assayf
CMM 165 ± 10.2+ 1.59 ± 0.27+ 3.44 ± 0.09+ 214.45 ± 2.15a 93 ± 2.4+ 320.17 ± 10.10+ 869 ± 20.52+
BHTA 133 ± 6.4# 3.02 ± 0.07# 96 ± 2.6#
Gallic 0.18 ± 0.01#
acid
+,#
Values with different letters within a column were significantly different (p < 0.05).
a
Results were expressed as the averages of triplicates ± standard deviation (SD).
b
EC50, expressed in lg/mL.
c
EC50, expressed in mg/mL.
d
Ferric reducing antioxidant power was expressed as mM FeSO4 equivalents per g extract.
e
Copper reducing antioxidant capacity was expressed as mg ascorbic acid equivalents per g extract.
f
The results of b-carotene bleaching assay was given as % in 1 mg/mL extract or reference compound.
g
Total antioxidant capacity was expressed as mg ascorbic acid equivalents per g extract.
h
Trolox equivalent antioxidant capacity was expressed as lM Trolox equivalent in per g extract.
A
Butylated hydroxy toluene (as reference).

vast amount of increase in the MDA levels both in total blood peroxidation. Nevertheless, Verma et al. (2009) suggested an oppo-
(p < 0.001) and liver homogenates (p < 0.001), as expected (Table site comment and stated that the declined activity of these en-
4). Although MDA levels tend to decrease in doses of 100 and zymes causes lipid peroxidation. Another point of view is that
200 mg/kg, the differences were not statistically significant the decrease in the antioxidant activity may be due to the inhibi-
(p > 0.05). While significant decreases of 26.23% (p < 0.05) and tion of protein biosynthesis or oxidative modifications of these
34.80% (p < 0.01) were seen in the doses of 500 mg/kg for CMM proteins. Free radicals generated might readily give reaction with
and 50 mg/kg for silymarin group, respectively. In terms of liver amino acid residues of proteins leading to severe modifications
MDA levels, both 200 and 500 mg/kg administration caused statis- in the structure of these proteins and consequently to inactivation
tically significant decrease (p < 0.01). The activity of 500 mg/kg of these enzymes. Although there are some conflicting results
CMM in liver tissue was higher than that of the total blood (Table about the mechanism of decrease in the enzyme activity, it is obvi-
4). We also found that silymarin administration caused a decrease ously related to elevated free radical production. The results given
of 41.92% (p < 0.001), which was very close to that reported by Chen in Table 4 indicated that administration of CMM provided the res-
et al. (2011) at the same dose level and period of treatment (45.45%). toration of these enzymes. Administration in doses of 100 mg/kg
It was stated that restoration in the levels of lipid peroxidation could (p < 0.05), 200 (p < 0.05) and 500 mg/kg (p < 0.01) resulted in sig-
be related to the ability of samples to scavenge ROS, thus preventing nificant increases in the activity of SOD in total blood samples. In
further damage to membrane lipids (Pradeep et al., 2007). terms of liver SOD, all of the groups, except the 100 mg/kg
As discussed in detail above, SOD, CAT and GSH-Px enzymes are treated-group, again showed statistically significant activities. It
the primary antioxidant enzymes, and referred as the first line of should be reminded that CMM also showed strong activity against
defense against oxidative stress. As given in Table 4, the activities superoxide radical according to our in vitro test results.
of all three enzymes were dramatically decreased both in total In 500 mg/kg-treated group of CMM, GSH-Px activities of both
blood and liver tissues after CCl4 administration. These data are total blood and liver tissues were significantly elevated (p < 0.01).
in accordance with a vast number of studies that used CCl4 as an The increase in the total blood GSH-Px levels (20.37%) was very
oxidative stress inducer. However, there are some conflicting close to that of liver tissues (19.51%). The increase in the activity
explanations in literature regarding the decrease in the activities of GSH-Px enzyme of this group was near to the activity in silym-
of these enzymes. For example Ozturk et al. (2009), Pradeep arin-treated one. Although there were numerical increases of
et al. (2007) annotated the decreased enzyme activities with the enzyme levels in 100 mg/kg and 200 mg/kg-treated groups, these
elevated free radical generation during the metabolism of CCl4; differences were statistically not significant (p > 0.05).
therefore the depletion of enzymes was a consequence of their The same results are valid for the CAT activities. There was a
excessive utilization in scavenging these radicals. In addition to significant increase in the enzyme levels in both 500 mg/kg
these comments, Hsouna et al. (2011) associated the reduced CMM-treated and the silymarin groups. The differences were
activity of these enzymes with the enhancement in the lipid statistically significant.

Table 3
In vivo effects of the 80% MeOH extract of Cornus mas leaves on total blood and liver SOD, CAT, GSH-Px activities, MDA levels and TEAC in normal rats.

Group Total blood Liver

SODa CATb GSH-Pxc MDAd TEACe SODa CATb GSH-Pxc MDAd TEACe
I 120.9 ± 11.1f 434.13 ± 24.21 76.2 ± 9.4 3.53 ± 0.34 30.38 ± 2.45 151.5 ± 17.9 854.15 ± 42.5 81.5 ± 5.3 3.89 ± 0.2 39.02 ± 3.75
II 126.5 ± 9.84 430.81 ± 30.8 78.27 ± 8.15 3.32 ± 0.33 36.61 ± 2.15 155 ± 21.1 837 ± 14.3 84.31 ± 4.73 3.60 ± 0.4 50.41 ± 3.1
(20.51%)** (29.19%)***
III 125.9 ± 13.76 440.03 ± 36.2 79.2 ± 7.5 3.39 ± 0.36 38.09 ± 3.21 158.4 ± 17.2 829.15 ± 26.3 84.8 ± 5.10 3.48 ± 0.28 51.33 ± 6.58
(25.38%)*** (31.55%)***

CMM: methanolic extract of Cornus mas, group I: control, group II: CMM 500 (mg/kg), group III: Silymarin 50 (mg/kg).
a
Superoxide dismutase activities were expressed as U/mg protein.
b
Catalase activities were expressed as U/mg protein.
c
Glutathione peroxidase activities were expressed as U/mg protein.
d
Malondialdehyde levels were expressed as nmol/mg protein.
e
Trolox equivalent antioxidant capacities were expressed as lM Trolox equivalent/mg protein.
f
Results were expressed as mean ± SD, n = 6 in each group.
**
p < 0.01 (compared to control group).
***
p < 0.001 (compared to control group).
454 E. Celep et al. / Food and Chemical Toxicology 62 (2013) 448–455

Table 4
In vivo effects of the 80% MeOH extract of Cornus mas leaves on total blood and liver SOD, CAT, GSH-Px activities, MDA levels and TEAC in CCl4-treated rats.

Group Total blood Liver

SODa CATb GSH-Pxc MDAd TEACe SODa CATb GSH-Pxc MDAd TEACe
f
I 139.6 ± 13.6 630.5 ± 44 89.61 ± 4.2 3.71 ± 0.37 34.16 ± 3.72 175.7 ± 16.7 737.4 ± 76.2 88.61 ± 5.73 4.26 ± 0.7 48.32 ± 2.3
II 90.13 ± 6.2++ 517 ± 34.2++ 67.54 ± 4.9+++ 9.34 ± 2.03+++ 19.79 ± 1.77+++ 127.6 ± 12.6++ 319 ± 28.2+++ 70.71 ± 4.12+++ 13.24 ± 3.7+++ 29.59 ± 3.1+++
III 97.11 ± 9.4 530 ± 53.9 70.31 ± 6.4 8.46 ± 1.10 22.19 ± 1.36 136.6 ± 6.7 370.5 ± 54.6 77.58 ± 5.55 10.98 ± 1.8 31.08 ± 0.9
(7.74%)*,g
IV 99.53 ± 3.21 564 ± 47.4 74.40 ± 7.1 7.44 ± 1.02 23.94 ± 1.00 158.6 ± 23.3 427.1 ± 35.5 78.9 ± 7.74 9.03 ± 1.6 33.67 ± 2.2
(10.42%)* (17.33%)** (19.51%)* (25.3%)* (31.8%)** (13.79%)*
V 102.3 ± 3.87 589 ± 35.9 81.30 ± 7.3 6.89 ± 1.46 25.34 ± 1.43 159.1 ± 19 587.5 ± 86.7 82.91 ± 6.07 8.04 ± 2.5 39.17 ± 3.6
(13.5%)** (14.01%)* (20.37%)* (26.23%)* (28.48%)** (19.8%)* (37%)*** (14.71%)* (38.46%)** (32.04%)***
VI 107.34 ± 5.6 608 ± 48.7 84.83 ± 11 6.08 ± 0.82 28.83 ± 2.42 169.7 ± 20 625.2 ± 21.5 86.5 ± 6.79 7.69 ± 0.75 44.91 ± 3.9
(19.09%)*** (21.56%)** (25.6%)** (34.8%)** (31.35%)*** (24.81%)** (49%)*** (18.2%)** (41.92)*** (51.77)***

CMM: methanolic extract of Cornus mas, group I: control, group II: CCl4, group III: CMM 100 (mg/kg) + CCl4, group IV: CMM 200 (mg/kg) + CCl4, group V: CMM 500 (mg/
kg) + CCl4, group VI: Silymarin 50 (mg/kg) + CCl4.
a
Superoxide dismutase activities were expressed as U/mg protein.
b
Catalase activities were expressed as U/mg protein.
c
Glutathione peroxidase activities were expressed as U/mg protein.
d
Malondialdehyde levels were expressed as nmol/mg protein.
e
Trolox equivalent antioxidant capacities were expressed as lM Trolox equivalent/mg protein.
f
Results were expressed as mean ± SD, n = 6 in each group.
g
The percentage activity was calculated as: (the amount in test group  the amount in CCl4 group)/the amount in CCl4 group  100.
++
p < 0.01 (compared to the control group).
+++
p < 0.001 (compared to the control group).
*
p < 0.05 (compared to CCl4 group).
**
p < 0.01 (compared to CCl4 group).
***
p < 0.001 (compared to CCl4 group).

We also investigated the TEAC of both the total blood and the blood. The increase in 500 mg/kg group (28.49%) was very close
liver tissues. As previously pointed out, it is important to appraise to that of silymarin group (31.55%). Similar results were obtained
the total antioxidant capacity of biological molecules, since the for the liver TEAC measurements; 200 and 500 mg/kg of CMM
non-enzymatic antioxidant defenses also play a major role against and silymarin significantly increased the total antioxidant capac-
oxidative stress. The TEAC results of total blood, given in Table 4, ity. Pradeep et al. (2007) found that silymarin treatment restored
revealed that CCl4-administration severely decreased the levels of the depleted levels of non-enzymatic antioxidants caused by
TEAC in both total blood and liver tissues (p < 0.001). The reason diethylnitrosamine. Fraschini et al. (2002) reported that glutathi-
for this decline was most probably due to the increased levels of one levels are elevated during silymarin treatment, and GSH
free radical generation. Feillet-Coudray et al. (1999) studied the li- homeostasis in the system was maintained. These data exerted
pid peroxidation and antioxidant status in experimental diabetes. that antioxidant molecules act not only in enzymatic defenses
They reported that streptozotocin administration decreased the but also in non-enzymatic defense parameters. The reason for
levels of plasma antioxidant capacities in rats significantly. Accord- elevated TEAC values following the extract treatment was most
ing to these data, we can possibly assume that TEAC levels of the probably due to the same reason. These results indicated that
biological fluids severely decreased after treated with oxidative in vivo antioxidant activity of 500 mg/kg of CMM was close to that
stress inducing agents. of silymarin. Therefore, it may be postulated that CMM might be
The results of TEAC experiments demonstrated that administra- practiced as an substitute for silymarin. Although the efficient dose
tion of 200 mg/kg (p < 0.01), 500 mg/kg (p < 0.01) of the extract of CMM was 10 times higher (500 mg vs. 50 mg), it should be bear-
and 50 mg/kg of silymarin (p < 0.001) increased the TEAC of total ing in mind that CMM was a crude extract, while silymarin is a
purified fraction composed of flavonolignans. On the other hand,
the leaves of cornelian cherry are used against diabetic complaints
Table 5
in Turkish folk medicine, indicating that they are probably safe for
Antioxidant activities of the fractions from the MeOH extract of C. mas leaves and the human use. However, further studies are warranted to obtain
isolated compound (1). clearer results for toxicity profile.
DPPHA FRAPB
Following the in vivo activity experiments, activity guided frac-
a
tionation of CMM was conducted. In vitro assays were used for this
CM – n-Hexane 466 ± 15.2 1.03 ± 0.07a
CM – CHCl3 287 ± 10.4b 2.80 ± 0.15b
purpose in order to avoid sacrificing more animals. DPPH radical
CM – EtOAc 141 ± 6.5c 4.08 ± 0.18c scavenging activity and ferric reducing antioxidant activity were
CM – n-BuOH 372 ± 19.8d 2.17 ± 0.14d chosen because these tests cover the most important mechanisms
CM – R-H2O 518 ± 17.3e 0.67 ± 0.07e of antioxidant activity: i.e., free radical scavenging and metal ion
Gallic acid (1) 96 ± 11.3f 4.47 ± 0.21f
reducing. The results indicated that ethyl acetate fraction had the
BHTC 133 ± 6.4g 3.02 ± 0.10g
highest activity on both tests as shown in Table 5. Further chro-
Results were expressed as the mean of triplicates ± standard deviation (SD) (n: 3). matographic studies led to the isolation of gallic acid, a well known
a–g
Values with different letters within a column were significantly different
antioxidant molecule. The activity studies demonstrated that gallic
(p < 0.05).
A
DPPH radical scavenging activity was expressed as EC50 in lg/mL.
acid had higher activity than ethyl acetate fraction. Lin et al. (2011)
B
FRAP activity was expressed as mM FeSO4 equivalents per g dry material (the also isolated the same molecule from the 70% acetone extract of
concentration of the material is 1 mg/mL). Cornus officinalis fruits, while it is being reported for the first time
C
Butylated hydroxytoluene (as reference compound). from C. mas leaves.
 E. Celep et al. / Food and Chemical Toxicology 62 (2013) 448–455 455

4. Conclusion Fraschini, F., Demartini, G., Esposti, D., 2002. Pharmacology of silymarin. Clin. Drug
Invest. 22, 51–65.
Hsouna, A. Ben., Saoudi, M., Trigui, M., Jamoussi, K., Boudawara, T., Jaoua, S., Feki,
The results of this study has clearly exhibited that CMM pos- A.El., 2011. Characterization of bioactive compounds and ameliorative effects of
sesses significant antioxidant activity against various antioxidant Ceratonia siliqua leaf extract against CCl4 induced hepatic oxidative damage and
renal failure in rats. Food Chem. Toxicol. 49, 3183–3191.
systems in vitro. In vivo studies on normal animals also confirmed
Huang, B., Ban, X., He, J., Zeng, H., Zhang, P., Wang, Y., 2010. Hepatoprotective and
that CMM increased the total antioxidant capacity in normal rats, antioxidant effects of the methanolic extract from Halenia elliptica. J.
whereas they did not induce any changes in the activities of anti- Ethnopharmacol. 131, 276–281.
Jager, S., Trojan, H., Kopp, T., Laszczyk, M.N., Scheffler, A., 2009. Pentacyclic
oxidant enzymes. Studies on CCl4-treated animals indicated that
triterpene distribution in various plants. Molecules 14, 2016–2031.
CMM restored the lowered activities of antioxidant enzymes, Jamall, I.S., Smith, J.C., 1985. Effects of cadmium on glutathione peroxidase,
reduced the level of lipid peroxidation, and increased the deterio- superoxide dismutase, and lipid peroxidation in the rat heart: a possible
rated total antioxidant capacity of these animals. Through activity- mechanism of cadmium cardiotoxicity. Toxicol. Appl. Pharmacol. 80, 33–42.
Kehrer, J.P., 1993. Free radicals as mediators of tissue injury and disease. Crit. Rev.
guided fractionation procedures from the CMM, gallic acid, a well- Toxicol. 23, 21–48.
known antioxidant compound, was isolated from the ethyl acetate Leopoldini, M., Russo, N., Toscano, M., 2011. The molecular basis of working
fraction, which demonstrated the highest activities in in vitro tests. mechanism of natural polyphenolic antioxidants. Food Chem. 125, 288–306.
Leskovac, A., Joksic, G., Jankovic, T., Savikin, K., Menkovic, N., 2007. Radioprotective
Gallic acid was also reported as one of the main ingredients of C. properties of the phytochemically characterized extracts of Crataegus
mas leaves (Leskovac et al., 2007). All of these results demonstrated monogyna, Cornus mas and Gentianella austriaca on human lymphocytes
that the consumption of C. mas leaves, which is rich in antioxidant in vitro. Planta Med. 73, 1169–1175.
Lin, M., Liu, H., Huang, W., Huang, C., Wu, T., Hsu, F., 2011. Evaluation of the
polyphenolics, might increase the total antioxidant capacity of the potential hypoglycemic and beta-cell protective constituents isolated from
body. These results evidenced the use of C. mas leaves in Turkish Corni Fructus to tackle insulin-dependent diabetes mellitus. J. Agric. Food
folk medicine against cardiovascular disorders and Chem. 59, 7743–7751.
Lotito, S.B., Frei, B., 2006. Consumption of flavonoid-rich foods and increased plasma
hyperglycaemia.
antioxidant capacity in humans: cause, consequence, or epiphenomenon? Free
Rad. Biol. Med. 41, 1727–1746.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement
Conflict of Interest with the folin phenol reagent. J. Biol. Chem. 193, 265–275.
Ozturk, F., Gul, M., Ates, B., Ozturk, I.C., Cetin, A., Vardi, N., Otlu, A., 2009. Protective
effect of apricot (Prunus armeniaca L.) on hepatic steatosis and damage induced
The authors declare no conflict of interest.
by carbon tetrachloride in Wistar rats. Brit. J. Nutr. 102, 1767–1775.
Pantelidis, G., Vasilakakis, M., Manganaris, G., Diamantidis, G., 2007. Antioxidant
capacity, phenol, anthocyanin and ascorbic acid contents in raspberries,
References blackberries, red currants, gooseberries and cornelian cherries. Food Chem.
102, 777–783.
Aebi, H., 1984. Catalase in vitro. Meth. Enzymol. 105, 121–126. Pieroni, A., Giusti, M.E., Münz, H., Lenzarini, C., Turkovic, G.T., Turkovic, A., 2003.
Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the Ethnobotanical knowledge of the Istro-Romanians of Zejane in Croatia.
degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90, 7915–7922. Fitoterapia 74, 710–719.
Apak, R., Güçlü, K., Özyürek, M., Karademir, S.E., 2004. Novel total antioxidant Pieroni, A., Muenz, H., Akbulut, M., Basßer, K.H.C., Durmusßkahya, C., 2005. Traditional
capacity index for dietary polyphenols and vitamins C and E, using their cupric phytotherapy and trans-cultural pharmacy among Turkish migrants living in
ion reducing capability in the presence of neocuproine: CUPRAC method. J. Cologne, Germany. J. Ethnopharmacol. 102, 69–88.
Agric. Food Chem. 52, 7970–7981. Popović, B.M., Štajner, D., Slavko, K., Sandra, B., 2012. Antioxidant capacity of
Aydin, A., Orhan, H., Sayal, A., Ozata, M., Sahin, G., Isßimer, A., 2001a. Oxidative stress cornelian cherry (Cornus mas L.) – comparison between permanganate reducing
and nitric oxide related parameters in type II diabetes mellitus: effects of antioxidant capacity and other antioxidant methods. Food Chem. 134, 734–741.
glycemic control. Clin. Biochem. 34, 65–70. Pradeep, K., Mohan, C.V.R., Gobianand, K., Karthikeyan, S., 2007. Silymarin
Aydin, A., Sayal, A., Isßımer, A., 2001. Serbest Radikaller ve Antioksidan Savunma modulates the oxidant–antioxidant imbalance during diethylnitrosamine
Sistemi, Gülhane Askeri Tıp Akademisi Basımevi, Ankara. induced oxidative stress in rats. Eur. J. Pharmacol. 560, 110–116.
Baytop, T., 1999. Türkiye’de Bitkilerle Tedavi, second ed. Nobel Tıp Kitabevleri, Recknagel, R.O., Glende, E.A., Dolak, J.A., Waller, R.L., 1989. Mechanism of carbon
Istanbul. tetrachloride toxicity. Pharmacol. Ther. 43, 139–154.
Celep, E., Aydın, A., Yesilada, E., 2012. A comparative study on the in vitro Robles-Sánchez, M., Astiazarán-García, H., Martín-Belloso, O., Gorinstein, S.,
antioxidant potentials of three edible fruits: cornelian cherry, Japanese Alvarez-Parrilla, E., De la Rosa, L., Yepiz-Plascencia, G., 2011. Influence of
persimmon and cherry laurel. Food Chem. Toxicol. 50, 3329–3335. whole and fresh-cut mango intake on plasma lipids and antioxidant capacity of
Chanwitheesuk, A., Teerawutgulrag, A., Kilburn, J.D., Rakariyatham, N., 2007. healthy adults. Food Res. Int. 44, 1386–1391.
Antimicrobial gallic acid from Caesalpinia mimosoides Lamk. Food Chem. 100, Savikin, K., Zdunic, G., Jankovic, T., Stanojkovic, T., Juranic, Z., Menkovic, N., 2009. In
1044–1048. vitro cytotoxic and antioxidative activity of Cornus mas and Cotinus coggygria.
Chen, Y., Huang, B., He, J., Han, L., Zhan, Y., Wang, Y., 2011. In vitro and in vivo Nat. Prod. Res. 23, 1731–1739.
antioxidant effects of the ethanolic extract of Swertia chirayita. J. Verma, A.R., Vijayakumar, M., Mathela, C.S., Rao, C.V., 2009. In vitro and in vivo
Ethnopharmacol. 136, 309–315. antioxidant properties of different fractions of Moringa oleifera leaves. Food
Ersoy, N., Bağcı, Y., Gök, V., 2011. Antioxidant properties of 12 cornelian cherry fruit Chem. Toxicol. 47, 2196–2201.
types (Cornus mas L.) selected from Turkey. Sci. Res. Essays 6, 98–102. Yesßilada, E., Sezik, E., Honda, G., Takaishi, Y., Takeda, Y., Tanaka, T., 1999. Traditional
Feillet-Coudray, C., Rock, E., Coudray, C., Grzelkowska, K., Azais-Braesco, V., medicine in Turkey IX: folk medicine in north-west Anatolia. J. Ethnopharmacol.
Dardevet, D., Mazur, A., 1999. Lipid peroxidation and antioxidant status in 64, 195–210.
experimental diabetes. Clin. Chim. Acta 284, 31–43.