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Scavenging Capacity of Superoxide Radical and Screening of Antimicrobial Activity of Castanea sativa Mill. Extracts

  • Faculty of Technology, University Novi Sad, Serbia

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Scavenging capacity of superoxide radical and screening of antimicrobial activity of Castanea sativa Mill. extracts. Czech J. Food Sci., 28: 61–68. The superoxide radical scavenging and antimicrobial activity were examined of sweet chestnuts: seeds, peeled chest-nut, brown seed coat, leaves, catkin, spiny burs, and chestnut bark as well as of Lovran's marrone leaves and grafted Italian marrone cultivar. Parts of chestnut were extracted under the same conditions with 50% ethanol as extractant and afterwards the dry extracts of the examined samples were obtained. The total phenolics and flavonoids contents were determined using standard spectrophotometrical methods. The capacity of the investigated extracts to remove radicals was evaluated by EPR method. The most efficient in scavenging
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Czech J. Food Sci. Vol. 28, 2010, No. 1: 61–68
Scavenging Capacity of Superoxide Radical and Screening
of Antimicrobial Activity of Castanea sativa Mill. Extracts
1, Z ZEKOV
2, I MUJIĆ
3, S VIDOV
2, G NIKOL
1 and N TRUT
1Department of Pharmacy, Medical Faculty, University of Niš, N, Serbia;
2Faculty of Technology, University of Novi Sad, Novi Sad, Serbia; 3Colegium Fluminense
Polytechnic of Rijeka, Rijeka, Croatia
Ž J., Z Z., M I., V S., C D., L Ž., N G., T N.
(2010): Scavenging capacity of superoxide radical and screening of antimicrobial activity of Castanea
sativa Mill. extracts. Czech J. Food Sci., 28: 61–68.
The superoxide radical scavenging and antimicrobial activity were examined of sweet chestnuts: seeds, peeled chest-
nut, brown seed coat, leaves, catkin, spiny burs, and chestnut bark as well as of Lovran’s marrone leaves and grafted
Italian marrone cultivar. Parts of chestnut were extracted under the same conditions with 50% ethanol as extractant
and afterwards the dry extracts of the examined samples were obtained. The total phenolics and flavonoids contents
were determined using standard spectrophotometrical methods. The capacity of the investigated extracts to remove
radicals was evaluated by EPR method. The most efficient in scavenging O2
radicals proved to be the extracts of leaves
of grafted Italian marrone (RI = 86%) and of Lovran's marrone cultivar (RI = 80%). Catkin, leaves, chestnut bark, and
spiny burs extracts demonstrated the highest antimicrobial activity. Very significant and significant correlations were
established between the antimicrobial activity of extracts and O2
radicals scavenging in all samples examined. The ex-
tracts of Castanea Sativa Mill. are important sources of components active in reducing the level of oxidative stress.
Keywords: antimicrobial activity; antioxidant; superoxide radical; sweet chestnut; total flavonoids; total phenolics
Reactive oxygen species (ROS), including super-
oxide radicals (O2
), hydrogen peroxide (H2O2),
hydroxyl radicals (OH), and singlet oxygen (1O2),
are generated as byproducts of normal metabolism
(W & J 2000). Both positive and negative
biological functions are ascribed to superoxide
), while the hydroxyl radical (OH) is exclusively
negative due to its high reactivity (H &
G 1999). The food-derived antioxidants
such as vitamins and phenolic phytochemicals have
received growing attention because they are known
to function as chemopreventive agents against
oxidative damage. To counteract the deleterious
effects of ROS, phenolic compounds, naturally
distributed in plants, are effective (C 1998).
They are also powerful reducing agents (S-
 1970).
Polyphenols have many favourable effects on hu-
man health, such as the inhibition of the low density
proteins oxidisation (F et al. 1993), thereby
decreasing the heart disease risks (W &
E 1997). They also have anti-inflammatory
activity and anti-carcinogenic properties (M-
 et al. 1999). On the other hand, the activity
of these compounds as food lipid antioxidants is
well known (R-E et al. 1997), and this fact
Vol. 28, 2010, No. 1: 61–68 Czech J. Food Sci.
promoted studies of extracts from different plants
containing them. Polyphenols are considered to
be plant chemical defenses against pathogens and
herbivores, and these compounds can exert det-
rimental effects in a multitude of ways (B
et al. 1989).
The resistance to antimicrobial agents has be-
come an increasingly important and pressing
global problem. For example, methicillin-resis-
tant Staphylococcus aureus, which was present
at low levels a decade ago, now accounts for 50%
of all S. aureus isolates (C & L 2005).
Microorganisms have unfavourable effects on the
quality, safety, and shelf life of foods. Synthetic
antioxidants are widely used in the food industry,
however, they also possess toxic and carcinogenic
effects. Thereby, the interest in finding natural
antioxidants, without undesirable side effects, has
greatly increased (Bet al. 2004). Generally,
it is accepted that phytochemicals are less potent
anti-infective than the agents of microbial origin,
i.e. antibiotics (Y 1991).
Chestnuts can be: used as a vegetable, steamed,
boiled, puréed, used in stuffing for meat, used as
a main component of soups, ground into flour for
confectionery use. The wood is used for timber,
paper, or fuel. Although it has already been dem-
onstrated that chestnut fruits (R et al. 2007)
and leaves (R et al. 1981) contain phenolic
compounds, little is known about their antioxi-
dant potential. It was recently demonstrated that
C. sativa leaves extract possesses a pronounced in
vitro antibacterial effect (Bet al. 2000).
So far, little is known about the possible appli-
cation of chestnut in diet and therapy. With the
intention to find new natural sources of active
compounds, we studied the ability of different
extracts of Castanea sativa Miller to scavenge
superoxide (O2
) radicals, and also their antimi-
crobial activities.
Chemicals. Ethanol and methanol were obtained
from Zorka Šabac (Serbia). Folin-Ciocalteu reagent
was purchased from Sigma Chemical Co. (St. Louis,
USA). Gallic acid and (+) catechin hydrate were
obtained from Fluka AG, Chemische Fabric (St.
Gallen, Switzerland). DEPMPO was purchased
from Alexis Biochemical (Lausen, Switzerland).
Hypoxantine and xantine oxidase were obtained
from Sigma Chemicals Co. (St Louis, USA). Other
chemicals and solvents used were of the highest
analytical grade. Spectrophotometrical measur-
ing was done using spectrophotometer Hewlett
Packard 8452. For the extraction, the ultrasonic
bathroom Branson model b-220 SmithKline Co.,
Shelton, USA (50/60 Hz, 125 W) was used. The
EPR spectra were recorded on Varian E104-A EPR
(Pal Alto, USA) spectrometer.
Sample preparation. Chestnut samples were col-
lected in the area of the Una-Sana canton (B&H),
in 2006. The investigation included three most
predominant cultivars: sweet chestnut, Lovran’s
marrone, and grafted Italian marrone cultivar. Scan
investigations were done with sweet chestnut, while
with the other two cultivars only leaves were col-
lected. There are many different types of chestnut,
varying in shape and colour of fruit. One of them
is Lovran’s marun” which is appreciated for the
high quality of its fruit. The word “marron” usually
signifies a large sort of domestic chestnut obtained
by inoculation. In Italy, marron means a particular
Castanea sativa cultivar of excellent quality.
The fruits were harvested in the chestnut ripen-
ing season, between the middle of September and
the end of October. The results given for the seed
refer to milled seed without burs. Separate parts
of seed were investigated, like peeled chestnut
(hand-peeled) and brown seed coat. In addition,
leaves, catkin, spiny burs, and bark of tree were
The samples were milled for the analysis in a
laborator y homogenisator. The mean particle
diameter of the investigated samples was deter-
mined. To 50 g of the sample, 250 ml of 50% ethanol
was added (sample solvent ratio was 1:5; w/v).
The extraction was carried out using ultrasound
(30 min). After the mass checking and possible
solvent addition, the liquid extract was obtained
by filtration through Whatman Grade 4 filter
paper. A defined volume of the liquid extract was
taken and the solvent was completely removed by
evaporation under vacuum at the temperature of
40°C. In this way, dry extracts of the investigated
chestnut samples were obtained, and the yields of
the dry extracts were calculated. All dry extract
samples were kept in the fridge.
Total phenolics. Total phenolics were determined
in the dry extracts by Folin-Ciocalteu procedure
(S & R 1965; K et al.
1999). For the preparation of the calibration curve,
0.1 ml aliquots of 0.037, 0.072, 0.108, 0.144, and
Czech J. Food Sci. Vol. 28, 2010, No. 1: 61–68
0.180 mg/ml of ethanolic gallic acid solutions were
mixed with 7.9 ml of H2O, 0.5 ml of Folin-Ciocaleu
reagent, and 1.5 ml of 20% sodium carbonate. The
blank was distilled water instead of 0.1 ml of the
investigated sample. The absorption was read after
2 h at the temperature of 20°C at 765 nm and the
calibration cur ve was constructed. 0.1 ml of the
methanolic plant extract (1 mg/ml) was mixed
with the same reagents as described above, and
after 2 h the absorption was measured for the
determination of plant phenolics. The content
of total phenolics compounds in the investigated
plant methanolic extracts was expressed as g of
gallic acid equivalents (GAE) per 100 g of the dry
extract sample (%; w/w), i.e. % GAE.
Total flavonoids. Total flavonoids content was
measured by means of the aluminium chloride
colorimetric assay (M 1989). An aliquot
(1 ml) of 0.037, 0.074, 0.112, 0.149, 0.186 mg/ml
methanolic catechin solutions or methanolic plant
extracts (1 mg/ml) was added into 10 ml volumetric
flask containing 1 ml of H2O. Then 0.3 ml of 5%
NaNO2 was added and after 5 min, 0.3 ml of 10%
AlCl3, was added. At 6th min, 2 ml of 1M NaOH
was added and the total volume was made up to
10 ml with H2O. The solution was well mixed and
the absorbance was measured against the prepared
blank at 510 nm. Total flavonoids were expressed
as g of catechin equivalents (CE) per 100 g of the
dry extract sample (%; w/w), i.e. % CE.
Scavenging of O2
radical. The ability of Cas-
tanea sativa Mill. extracts to scavenge superoxide
radicals was tested using Hypoxantine/Xantine
oxidase (HX/XO) reaction as an “O2
The capacity of the investigated extracts to re-
move radicals is evaluated by the difference be-
tween the amplitudes of the EPR signals of the
trapped radicals in radical-generating systems,
with or without addition of the investigated extract.
The results are presented as the relative inhibition
(RI), which represents the relative decrease of the
radical production:
RI = 100 × [peak amplitude (radical-generating
system) – peak amplitude (radical-generating
system + extract)]/[peak intensity (radical-
generating system)]
Superoxide radicals were generated in a hy-
poxanthine/xanthine-oxidase system (HX/XO)
consisting of hypoxanthine – 1.6mM, and xanthine
oxidase – 1.6 IU/ml (Sigma-Aldrich, St. Louis, USA)
dissolved in bidistilled 18Mdeionised water.
The final concentration of DEPMPO was 28mM.
DEPMPO reacts with O2
to form DEPMPO/OOH
adducts (Figure 1). The final concentration of the
extracts was 0.2 mg/ml. The incubation time was
2 minutes. The samples with no extract served as
controls. The final concentration of the extracts
(previously dissolved in water) was 0.2 mg/ml.
EPR spectroscopy. EPR spectra were recorded
at room temperature using a Varian E104-A EPR
spectrometer operating at X-band (9.51 GHz) with
the following settings: modulation amplitude 2 G;
modulation frequency 100 kHz; microwave power
10 mW; time constant 0.032 s; field centre 3410 G;
scan range 200 G. The spectra were recorded us-
ing EW software (Scientific Software, Blooming-
ton, USA). The samples were drawn into 10 cm
long gas-permeable Teflon tubes (wall thickness
0.025 mm and internal diameter 0.6 mm; Zeus
Industries, Raritan, USA). The measurements
were performed using quartz capillaries in which
Teflon tubes were placed. The recordings proceded
2 min after the beginning of the reaction, with the
recording time of 4 minutes.
Antimicrobial activity.The disc-diffusion meth-
od was used as a screening test for antibacterial
activity. Filter paper discs (6 mm in diameter)
impregnated with sample solutions were placed
on Mueller Hinton agar plates (Difco, Detroit,
USA), which had been inoculated with the test
organisms. From the primary isolation medium,
2–3 colonies of the investigated microorganisms
were taken with a flamed loop, suspended in Muel-
ler Hinton broth (Merck, Darmstadt, Germany),
and subsequently incubated at 37°C. The suspen-
sion for both inoculations was prepared from
the broth cultures. The number of cells in 1 ml
of suspension for inoculation measured with a
McFarland nephelometer was 1 × 107 CF U/ml.
1 ml of this suspension was homogenised with 9
ml of melted (45°C) Mueller Hinton poured into
Petri dishes . For screening, sterile 6 mm discs
(HiMedia®, Mumbai, India) were impregnated
with 10 µl of 10 mg/ml of C. sativa Mill. extracts
diluted in 30% ethanol. After incubation for 48 h in
a thermostat at 37°C, the inhibition zone diameters
(ZI, including disc) were measured and expressed
in mm with 0.1 mm accuracy and the effect was
calculated as a mean of triplicate tests. The pres-
ence of the inhibition zone indicated the activity
of the tested extracts against bacteria: Sarcina
lutea (ATCC 9341), Staphylococcus aureus (ATCC
Vol. 28, 2010, No. 1: 61–68 Czech J. Food Sci.
25923), Bacillus cereus (ATCC 10876), Lactococcus
lactis ssp. lactis (B-4449), Micrococcus pyrogenes
var. albus (isolated from natural environment) (all
Gram-positive), Proteus mirabilis (ATCC 35659
Gram-negative), and Salmonella typhimuri (ATCC
14028 Gram-negative). Penicillin (10 units/disc)
and amoxicillin (25 µg/disc) obtained from Bio-
analyse Co., Ltd. (Ankara, Turkey) were used as
reference standards. In parallel with the antimi-
crobial investigation of C. sativa extracts, pure
solvent was also tested, however, it did not exhibit
any antimicrobial activity (data not shown). The
bacteria were obtained from the stock cultures of
Microbiology Laboratory, Faculty of Technology,
University of Novi Sad.
Statistical analysis. All experiments were per-
formed at least in triplicates. The results are pre-
sented as the mean values ± SD. Statistical analysis
was carried out using Statistica 6.0 (StatSoft Inc,
Tulsa, USA). Pearson correlation test was conducted
to determine the correlations between the variables.
Significant levels were defined at P ≤ 0.05.
The yields of dry extracts of sweet chestnut
(Table 1), expressed in % (w/w), i.e. g/100 g of a
sample, ranged from 1.82% for spiny burs to 12.79%
for peeled sweet chestnut.
The highest content of total phenolic compounds
(3.28% GAE) was found in the extracts of sweet
chestnut catkin, while the lowest one (0.42% GAE)
was obtained with sweet chestnut seeds. Total
flavonoid content ranged from 0.09% CE in peeled
sweet chestnut to 0.69% CE in chestnut bark of
sweet chestnut.
The parts of chestnut with the highest content
of polyphenols included catkin, chestnut bark,
leaves, and brown seed coat. The lowest content
of polyphenols was detected in seeds and peeled
chestnut, because chestnut is fruit rich in car-
bohydrates (especially starch) and low in the fat
content (E et al. 2006). Chestnuts have an
average starch content of 22.3 g/100 g of raw ed-
ible portion. This fact places chestnut fruit among
the main sources of starch (P et al.
The ability of the chestnut extracts to scavenge
radicals was tested using EPR spin-trapping tech-
nique. This technique enables the detection of
different ROS species by EPR spectroscopy quali-
tatively (different species can be distinguished)
and with greater sensitivity (B & M
One of the most commonly used spin traps,
5,5-dimethylpyrroline-N-oxide (DMPO), is the
most versatile and commonly used spin trap
for measuring and identifying oxygen radicals.
However, it has several fundamental limitations.
Table 1. e yield of dry extract, total phenolics and total flavonoids contents
Extracts Yield of dry extract
(% w/w)
Total phenolics content
(% expressed as GAE)
Total flavonoids content
(% expressed as CE)
Sweet chestnut
Seeds 7.09 ± 0.091 0.42 ± 0.067 0.17 ± 0.008
Peeled chestnut 12.79 ± 0.092 0.59 ± 0.029 0.09 ± 0.003
Brown seed coat 3.30 ± 0.083 1.19 ± 0.126 0.65 ± 0,021
Catkin 10.04 ± 0.046 3.28 ± 0.154 0.60 ± 0.031
Leaf 4.94 ± 0.038 1.40 ± 0.011 0.26 ± 0.008
Chestnut bark 3.40 ± 0.089 1.70 ± 0.097 0.69 ± 0.056
Spiny burs 1.82 ± 0.066 0.49 ± 0.023 0.13 ± 0.017
Lovran’s marrone
Leaf 7.03 ± 0.063 2.43 ± 0.056 0.61 ± 0.024
Grafted Italian marrone
Leaf 6.18 ± 0.045 1.71 ± 0.065 0.42 ± 0.039
Czech J. Food Sci. Vol. 28, 2010, No. 1: 61–68
One major limitation is the short half-life of the
DMPO-superoxide adduct, DMPO-OOH, in aque-
ous biological media. The half life of DMPO-OOH
in buffer solutions at pH 7 is approximately 50 s
(Ret al. 1997). On the other hand, the
spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyr-
roline N-oxide (DEPMPO) is very efficient for the
detection of O2
. It forms a superoxide adduct
with a half-life of almost 15 min while DEPMPO/
OOH is about 10 times higher compared to the
respective DMPO adduct (B1978). In
comparison to DMPO, DEPMPO is suitable for
a more accurate detection of O2
. The amount of
oxygen within the investigated system must also
be taken into account when dealing with oxygen
consuming systems (B et al. 2008). When,
experiments with the systems consuming oxygen
are performed in quartz capillaries or flat cells,
both the production of oxygen radical species and
the rate of conversion of DEPMPO adducts vary
with the changes of pO2, thus making the observed
system very difficult to analyse (B  M
2005). Since the adduct conversion appears to be
rather slow compared to the radical generation,
DEPMPO spin-trap can be efficiently used for the
detection of oxygen-centred radicals produced by
the systems in vivo (Met al. 2005).
Figure 1 shows a characteristic EPR signal ob-
tained in the X/XO system using DEPMPO. Table 2
shows that all the extracts investigated exhibited a
significant capacity for O2
radicals scavenging.
The most efficient in O2
radicals scavenging
proved to be the extracts of leaves of grafted Italian
marrone (RI = 86%) and Lovran's marrone cultivar
(RI = 80%). A high antioxidant activity was also
expressed by the extract of spiny burs (RI = 78%).
Those extracts possessed high contents of total
phenolics and total flavonoids (Table 1). For the
investigated extracts, the following order of the
strength to scavenge O2
radical was obtained:
leaves > catkin > spiny burs > chestnut bark >
brown seed coat > seeds > peeled chestnut.
Unlike the synthetic antioxidants, which are
phenolic compounds with varying degrees of al-
kyl substitution, the natural antioxidants can be
phenolic compounds (flavonoids, phenolic acids,
and tannins), nitrogen-containing compounds,
carotenoids, tocopherols, or ascorbic acid and its
derivatives (Vet al. 1998).
In out previous research (Ž et al. 2008),
we investigated the capacity of sweet chestnut ex-
Table 2. RI of O2
radical production in the X/XO system
induced by C. sativa extracts
Extracts RI (%)
Sweet chestnut
Seeds 67 ± 0.05
Peeled chestnut 54 ± 0.01
Brown seed coat 70 ± 0.03
Catkin 70 ± 0.04
Leaf 73 ± 0.06
Chestnut bark 77 ± 0.07
Spiny burs 79 ± 0.03
Lovran’s marrone
Leaf 80 ± 0.03
Grafted Italian marrone
Leaf 86 ± 0.04
Figure 1. EPR signal of DEPMPO adducts obtained in the X/XO system. Open circle marks the line of DEPMPO/OOH
signal whose amplitude was measured. A weak signal of DEPMPO/OH adduct can also be observed, however, it does
not affect the amplitude or shape of two central lines of DEPMPO/OOH signal (B et al. 2007)
Figure. 1. EPR signal of DEPMPO adducts obtained in the X/XO system. Open circle marks
line of DEPMPO/OOH signal which amplitude was measured. Weak signal of DEPMPO/OH
adduct can also be observed, however it does not affect amplitude nor shape of two central
lines of DEPMPO/OOH signal (BAýIûet al. 2007).
3330 3370 3410 3450 3490
Magnetic field (Gauss)
Vol. 28, 2010, No. 1: 61–68 Czech J. Food Sci.
tracts concerning the removal of lipid peroxidation.
It was found that all the extracts protected liposomes
from lipid peroxidation. Also, the extracts could be
used as protective reagents due to the obstruction
of oxidative stress provoked by OH and H2O2 and,
at the same time, they were extremely resistant to
the radical effects of this degradation process. For
these reasons, the effect of lipid peroxidation on
their scavenging capacity is minimal. Especially,
these extracts are very efective scavengers of su-
peroxide radicals in all respects.
The results of antimicrobial activity determina-
tion of 30% (v/v) ethanol extracts of C. sativa and
standards are shown in Table 3.
All the investigated extracts showed antibacterial
activity. The highest activity of the extracts was
found against M. pyrogenes var. albus, S. aureus,
and S. typhimurium. Catkin and leaves extracts
showed the highest antimicrobial activity. A high
activity was also exhibited by the extracts of chest-
nut bark, spiny burs, and brown seed coat. On the
other hand, the seeds and peeled chestnut (with
the exception of L. lactis ssp. lactis), revealed no
antimicrobial activity.
The antimicrobial activity of the analysed extracts
wa s exhibited in the following order: catkin >
leaves > chestnut bark > spiny burs > brown seed
coat >>> seeds and peeled chestnut.
The results obtained in the determination of
total phenolics, total flavonoids, antioxidant and
antimicrobial activities of chestnut extracts are
shown in Tables 1–3, and were analysed by means
of the correlation analysis. The values of the cor-
relation coefficients (r), which express the strength
of the relationship between two variables, are
given in Table 4.
The total phenolic content correlated signifi-
cantly with total flavonoids (r = 0.73). A very
significant correlation (P < 0.01) was obtained
between the antimicrobial activity of the extracts
and O2
radical scavenging with all the s even
bacterial strains investigated. The total phenolic
content correlated significantly with the antimi-
crobial activity of the extracts toward S. lutea
(r = 0.76) and S. aureus (r = 0.78). A significant
linear correlation (P < 0.05) was determinated
between the total flavonoids and antimicrobial
activity (S. aureus) (r = 0.71).
Table 3. Antimicrobial activity of C. sativa extracts and reference standards inhibition zone in diameter (mm)
around the discs (6 mm)
Test microorganisms
lactis ssp.
var. albus
Sweet chestnut
Seeds 10.67 ± 1.53
Peeled chestnut
Brown seed coat 9.67 ± 0.58 7.33 ± 0.58 9.33 ± 0.58 10 ± 0 15 ± 2.5 12.33 ± 1.15
Catkin 15.33 ± 0.58 13.66 ± 0.58 10 ± 0 11.33 ± 0.47 10.33 ± 0.72 15.5 ± 2.59 17.82 ± 0.78
Leaf 14 ± 4.89 11 ± 1 10 ± 0 12 ± 0 14.5 ± 2.5 14.33 ± 1.15 13.67 ± 0.88
Chestnut bark 14.67 ± 0.58 9.33 ± 0.58 10 ± 0 11 ± 0 12.33 ± 1.53 13.67 ± 0.58 11.65 ± 0.55
Spiny burs 9.33 ± 0.58 10 ± 0 10 ± 1 11.33 ± 0.47 10 ± 1 14.16 ± 1.25 15.83 ± 1.82
Lovran’s marrone
Leaf 16 ± 3.46 13 ± 1 10.33 ± 0.58 12.66 ± 0.47 11.5 ± 0.84 16.33 ± 0.58 17.83 ± 1.19
Grafted Italian marrone
Leaf 14 ± 1.73 9 ± 1 9.67 ± 0.58 11.33 ± 0.47 14 ± 1.73 14.33 ± 1.15 12.47 ± 0.61
Amoxicillin 27.3 ± 1.15 55.0 ± 1.0 29.0 ± 0 26.33 ± 0.57 8 ± 0 36 ± 0.58 16 ± 0
Penicillin 30.3 ± 2.25 37.7 ± 0.49 34.0 ± 0 34.33 ± 0.57 12 ± 0.58 52 ± 0 15 ± 0
Czech J. Food Sci. Vol. 28, 2010, No. 1: 61–68
The extracts of leaf, catkin, brown seed coat,
spiny burs, and chestnut bark of C. sativa can be
used as natural antioxidants with the application
in diet and therapy.
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lactis ssp.
var. albus
S. lutea 0.95**
B. cereus 0.94** 0.94**
P. mirabilis 0.95** 0.95** 0.99**
L. lactis ssp. lactis NS NS 0.69* 0.68*
M. pyrogenes
var. albus 0.96** 0.97** 0.99** 0.99** NS
S. typhimurium 0.82** 0.89** 0.77* 0.80** NS 0.82**
Total phenolics 0.78* 0.76* NS NS NS NS NS
Total flavonoids 0.71* NS NS NS NS NS NS 0.73*
of O2
radical 0.74* 0.68* 0.77* 0.78* 0.73* 0.77* 0.67* NS NS
*correlation is significant at 0.05 level; **correlation is significant at 0.01 level; NS – no significant correlation (P > 0.05)
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Received for publication July 14, 2009
Accepted after corrections December 19, 2009
Corresponding Author:
Prof. Dr. J V Ž, University of Nis, Medical Faculty, Department of Pharmacy,
Bulevar Zorana Đinđića 81, 18 000 Niš, Serbia
tel.: + 381 18 511 359, e-mail:
... Pearson correlation test was conducted to determine correlations between the variables. Significant level was set at p ≤ 0.05 [16]. ...
... There is a direct relation between antioxidant activity and the reducing power. Furthermore, the direct relationship between reducing power and antioxidant activity has also been correlated with extract concentration and extraction method [6,16]. ...
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Purpose: Cynara scolymus is a medicinal plant frequently used in traditional medicine for stomach and liver diseases. The purpose of the study was to identify the most suitable extraction solvent for maximum antioxidant and antimicrobial effect of fluidized bed extracts. Methods: The extracts were obtained by conventional maceration and fluidized bed extraction using 25, 50, 75 and 97 % v/v ethanol as solvent. The antioxidant effect of the extracts was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The quantities of phenols and flavonoids, and the reducing power of the extracts were also determined. The antimicrobial activity of the extracts was tested against different microbial strains using agar well diffusion method. The minimum inhibiting concentration (MIC) of the fluidized bed extract was also determined. Results: The ethanol extract showed the highest antioxidant activity as well as yielded the largest quantity of polyphenolic compounds. For the extract obtained by fluidized bed technique, the optimum concentration of 10 mg/ml gave maximum antioxidant activity of 65.15 %. The MIC values obtained using the freeze-dried extract ranged from 5.0 mg/ml – 15.0 mg/ml. The extracts showed significant inhibitory activity against the tested strains of Listeria innocua CMGB 218, Bacillus cereus CMGB 215 with MIC of 5 mg/ml but showed MIC of 15 mg/ml for the other strains. Conclusion: The results indicate that the freeze-dried extract from Cynara scolymus is capable of yielding nutritional supplements with antioxidant and antimicrobial activities.
... Plant by-products could also be used as antimicrobial agents and some studies have demonstrated their antimicrobial activity (Rauha et al., 2000;Pereira et al., 2007;Kavak et al., 2010;Zivković et al., 2010). The tendency of the consumers to avoid products prepared with preservatives of chemical origin together with the increased resistance to antibiotics (Rauha et al., 2000;Oliveira et al., 2007) is promoting the interest in using natural antimicrobial compounds, especially extracted from plants (Zhu et al., 2004). ...
Walnut green husk is an agro-forest waste generated in the walnut (Juglans regia L.) harvest that could be valued as a source of natural compounds with antioxidant and antimicrobial properties. At this respect, the effect of the solvent (water, methanol, ethanol and 50% aqueous solutions of methanol and ethanol) on the extraction yields and extracts bioactive properties was analysed. Total phenols content of the extracts was determined by the Folin–Ciocalteau method. Extract antioxidant activity was evaluated using the reducing power assay and by the ability of the extracts to scavenge the DPPH radical. The scavenging effect of the aqueous extracts on the nitric oxide radical was also evaluated. The highest extraction yield was achieved with water (44.11%) and high bioactive potential was shown by the samples extracted with water/ethanol (1:1) (84.46 mg GAE/g extract; EC50 = 0.95 mg/mL for reducing power and EC50 = 0.33 mg/mL for DPPH assay). All the antioxidant properties analysed showed a concentration-dependent activity. The antimicrobial activity of the aqueous extracts was assessed and showed ability to inhibit the growth of Gram positive bacteria. The results obtained demonstrated the potential of the walnut green husk as an economical source of antioxidant and antimicrobial agents.
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This study investigated the effects of dietary supplementation with chestnut tannins (CNT) on metabolic and antioxidant status of prepartum cows along with their colostrum quality. Pregnant multiparous Holstein cows were paired according to parity and body condition score, and assigned either to a diet supplemented with 20 g/d of commercially available product containing chestnut tannins (CNT, n=20) or to an unsupplemented control diet (CON, n=20) for the last 25±2 d of pregnancy. Serum metabolite, insulin and antioxidant capacity indices were measured in blood samples taken at d 25 and d 5 before expected parturition. Chemical composition and IgG concentration were determined in colostrum samples collected from the first milking postpartum. The addition of CNT led to lower BUN (P=0.02) and consequently higher serum glucose (P=0.02) and insulin (P<0.01) concentrations which were associated with lower circulating NEFA (P<0.01) and BHBA (P<0.01) in CNT group than those of CON. The serum paraoxonase 1 (PON 1) activity and total antioxidant capacity (TAOC) were higher at -5 d in CNT than in CON (P<0.01, P=0.03; respectively). Close-up CNT improved lactose percentage and IgG concentration (P=0.03, P=0.04; respectively) and tended to improve percentage of protein and SNF (Solid Not Fat) in primary colostrum (P=0.06, respectively), without affecting colostrum fat and total solid (P=0.98, P=0.43; respectively). Supplementation of CNT in the diet during close-up period did not have adverse effects on metabolic profiles prepartum. Instead, this feeding regimen was more beneficial to antioxidant capacity and colostrum quality than feeding the control diet.
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Permanent attention of researchers has been focused on the use of natural antioxidants to inhibit lipid peroxidation, or to protect the damage provoked by free radicals. Extracts of sweet chestnut, Lovran's Marrone cultivar and grafted Italian "Marrone" cultivar obtained in two years (2006. and 2007.), were investigated for their capacity to remove lipid peroxidation in liposomes exposed to hydroxyl radical, and for their total phenolics and flavonoids content. Selected tissues of sweet chestnut (Castanea sativa Mill.) fruit, leaf, catkin, spiny burs, young and old bark of chestnut tree have been extracted under the same conditions using 50% ethanol and afterwards dry extracts of examined samples have been gained. All extracts, excluding those with low content of both, total phenolics and flavonoids, showed the ability to protect liposomes from lipid peroxidation. We find practical importance of results in the fact that extract of spiny burs could potentially have beneficial effects in diet of hypertensive patients.
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The "French paradox" (apparent compatibility of a high fat diet with a low incidence of coronary atherosclerosis) has been attributed to the regular drinking of red wine. However, the alcohol content of wine may not be the sole explanation for this protection. Red wine also contains phenolic compounds, and the antioxidant properties of these may have an important role. In in-vitro studies with phenolic substances in red wine and normal human low-density lipoprotein (LDL) we found that red wine inhibits the copper-catalysed oxidation of LDL. Wine diluted 1000-fold containing 10 mumol/L total phenolics inhibited LDL oxidation significantly more than alpha-tocopherol. Our findings show that the non-alcoholic components of red wine have potent antioxidant properties toward oxidation of human LDL.
We present a brief account of the difficulties involved in detection of oxygen free radicals in plants and give a rationale for using the EPR spin trapping technique in such studies. Comparative analysis of characteristics of different spin traps is given, having in mind their suitability in trapping oxygen-centered free radicals. Certain technical aspects of EPR experiments related to successful trapping of free radicals are discussed. Previous studies of trapping of oxygen radicals in plants are reviewed in terms of how efficient the experimental approach employed has been in their detection and how this influences conclusions about the mechanisms of their production. In addition, we analyze the potential of spin labels in the analysis of free radical production in plants and demonstrate that the combination of EPR spin traps and spin labels is extremely efficient for this purpose.
The choice of the spin-trap that is to be applied in any EPR study represents the crossroad between a comprehensive investigation and an "ordinary" quantification of production of radicals. So, the scope of our study was to compare the performance of different spin-traps for qualitative analysis of radical-generating systems, and their ability to recognize previously unnoticed radicals. In addition, we present a brief account of the difficulties involved in the detection of oxygen-centered radicals in chemical and biological systems accompanied by the rationale for using the EPR spin-trapping technique in quantitative studies of such reactive species. Certain technical aspects of EPR experiments related to efficient trapping of free radicals in biochemical systems are also discussed. As an example we present here results obtained using EPR spectroscopy and the spin-trap DEPMPO, which show that the Fenton reaction, as well as various biological systems generate a previously unappreciated hydrogen (*H) atom.
1. Oxygen is a toxic gas - an introductionto oxygen toxicity and reactive species 2. The chemistry of free radicals and related 'reactive species' 3. Antioxidant defences Endogenous and Diet Derived 4. Cellular responses to oxidative stress: adaptation, damage, repair, senescence and death 5. Measurement of reactive species 6. Reactive species can pose special problems needing special solutions. Some examples. 7. Reactive species can be useful some more examples 8. Reactive species can be poisonous: their role in toxicology 9. Reactive species and disease: fact, fiction or filibuster? 10. Ageing, nutrition, disease, and therapy: A role for antioxidants?
There is currently much interest in phytochemicals as bioactive components of food. The roles of fruit, vegetables and red wine in disease prevention have been attributed, in part, to the antioxidant properties of their constituent polyphenols (vitamins E and C, and the carotenoids). Recent studies have shown that many dietary polyphenolic constituents derived from plants are more effective antioxidants in vitro than vitamins E or C, and thus might contribute significantly to the protective effects in vivo. It is now possible to establish the antioxidant activities of plant-derived flavonoids in the aqueous and lipophilic phases, and to assess the extent to which the total antioxidant potentials of wine and tea can be accounted for by the activities of individual polyphenols.