Antioxidant Characterization of Oak Extracts Combining Spectrophotometric Assays and Chemometrics

Abstract

Antioxidant characteristics of leaves, twigs, and acorns from two Serbian oak species Quercus robur L. and Quercus petraea L. from Vojvodina province (northern Serbia) were investigated. 80% ethanol (in water) extracts were used for antiradical power (ARP) determinations against DPPH(•), (•)NO, and O2 (•-) radicals, ferric reducing antioxidant power (FRAP), total phenol, tannin, flavonoid, and proanthocyanidin contents. Permanganate reducing antioxidant capacity (PRAC) was determined using water extracts. Beside, mentioned parameters, soluble proteins, lipid peroxidation (LP), pigments and proline contents were also determined. The data of different procedures were compared and analyzed by multivariate techniques (correlation matrix calculation and principal component analysis (PCA)). PCA found that investigated organs of two different oak tree species possess similar antioxidant characteristics. The superior antioxidant characteristics showed oak leaves over twigs and acorns and seem to be promising source of antioxidants with possible use in industry and pharmacy.

Full text

Hindawi Publishing Corporation
e Scientic World Journal
Volume , Article ID , pages
http://dx.doi.org/.//
Research Article
Antioxidant Characterization of Oak Extracts Combining
Spectrophotometric Assays and Chemometrics
Boris M. PopoviT,1Dubravka Štajner,1RuDica Cdero,1Saša OrloviT,2and Zoran GaliT2
1FacultyofAgriculture,UniversityofNoviSad,TrgDositejaObradovi
´
ca 8, 21000 Novi Sad, Serbia
2InstituteofLowlandForestryandEnvironment,UniversityofNoviSad,Antona ˇ
Cehova 13, 21000 Novi Sad, Serbia
Correspondence should be addressed to Boris M. Popovi´
c; popovicb@polj.uns.ac.rs
Received  August ; Accepted  September 
Academic Editors: N. Ercal and Z. Gao
Copyright ©  Boris M. Popovi´
c et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Antioxidant characteristics of leaves, twigs, and acorns from two Serbian oak species Quercus robur L. and Quercus petraea L. from
Vojvodina province (northern Serbia) were investigated. % ethanol (in water) extracts were used for antiradical power (ARP)
determinations against DPPH∙,∙NO, and O2∙− radicals, ferric reducing antioxidant power (FRAP), total phenol, tannin, avonoid,
and proanthocyanidin contents. Permanganate reducing antioxidant capacity (PRAC) was determined using water extracts. Beside,
mentioned parameters, soluble proteins, lipid peroxidation (LP), pigments and proline contents were also determined. e data
of dierent procedures were compared and analyzed by multivariate techniques (correlation matrix calculation and principal
component analysis (PCA)). PCA found that investigated organs of two dierent oak tree species possess similar antioxidant
characteristics. e superior antioxidant characteristics showed oak leaves over twigs and acorns and seem to be promising source
of antioxidants with possible use in industry and pharmacy.
1. Introduction
Quercus trees, commonly known as oaks, belong to the
family Fagaceae. ey comprise  species worldwide [].
European oak corresponds well with these requirements and
is mainly represented by Quercus robur L. (pedunculate) and
Quercus petraea (Matt.) Liebl. (sessile oak). Oak wood is
valued for its mechanical properties and durability. It has
been widely used since prehistoric times []. Pedunculate is
the dominant tree species of natural forests in the area of
at Srem and also in whole region of Vojvodina, northern
Serbia. Besides pedunculate, sessile oak is the most valuable
oak in Serbia []. In the forestry fund of Serbia, there is
the signicant participation of sessile oak (.%) []. Oaks
are the major source of hardwood lumber and also they are
used for ornaments. e wood is durable and tough and also
attractively grained. It is especially valued in shipbuilding and
construction and for ooring, furniture, barrels, and veneer.
e bark of some oaks has been used in medicine, in tanning,
andfordyes.Inmythologyandreligion,theoakwasrevered
as a symbol of power []. In Serbia, oak is a sacred tree, used
in Serbian Christmas traditions.
Acorns, the fruit of oak trees, have long been employed as
a source of hog feed, tannin, oil, and especially food because
of the high content of carbohydrates, amino acids, proteins,
lipids, and various sterols [,]. Quercus acorns were mainly
used for making bread or as a substitute for coee. Oak
kernels were traditionally used in medicine, particularly
roastedonesasastringents,antidiarrhoeals,andantidotes[].
e acorns of Q. robur contain various biologically active
compounds with antioxidant activity (tannins, gallic and
ellagic acid, and dierent galloyl and hexahydroxydiphenoyl
derivatives) [,]. It was also known that the bark of dierent
Quercus speciescontainspolyphenolicconstituents.us,the
bark of Q. petraea contains both hydrolysable and condensed
tannins, avanols, and oligomeric proanthocyanidins [].
From the bark of Quercus robur more than  compounds
(catechins and oligomeric and polymeric proanthocyanidins)
have been isolated [].

e Scientic World Journal
Polyphenols are secondary metabolites of plants that are
generally involved in defense against ultraviolet radiation
and pathogens. In food, polyphenols contribute to the color,
avor, odor, bitterness, astringency, and oxidative stability.
Recent biomedical investigations connected to the polyphe-
nols and antioxidant activity of a number of herbals and
foodsshowthatpolyphenolssuchareavonoids,tannins,and
proanthocyanidins oer protection against development of
cancers, cardiovascular diseases, diabetes, osteoporosis and
neurodegenerative diseases [,].
Kim et al. []investigatedphenolicproleinleavesof
ve dierent Quercus species and approved presence of many
chemical constituents. is study demonstrated dierences
in phenolic compounds content in dierent parts of plants.
Quercus salicina Blume, for example, possesses high levels of
gentisic and chlorogenic acids as well as avonoids naringin
and rutin in the leaf. Brossa et al. []establishedthatmajor
constituents in holm oak (Quercus ilex L.) leaves are avanols
and avonols. According to Kamalak et al. [], oak leaves
from some Quercus species (Q. branti and Q. libari)may
have a high potential nutritive value for small ruminant
animals in terms of rumen and whole tract digestion. It is
established that content of phenolic compounds in Quercus
species highly depends on the stage of maturity and expresses
seasonal variation [,].
Sanchez-Burgos et al. [] established that aqueous extracts
from leaves from dierent white Quercus species (Quercus
resinosa, Quercus laeta, Quercus grisea, and Quercus obtusata)
displayed high radical scavenging activity against DPPH
and ∙OH radicals as well as antimicrobial activities and
antitopoisomerase activity only Q. resinosa leaves infusions.
Andrenˇ
sek et al. [] also pointed out that Q. robur cortex is
a promising plant material as the source of antioxidative and
antimicrobial activity.
Bearing in mind that antioxidant potential of two major
Serbian oaks Quercus robur L. and Quercus petraea L.has
not been studied well enough, especially their leaves and
twigs, the aim of this work was to investigate the in vitro
antioxidant and scavenging activities and also total phenol
(TPC), tannin (TAC), avonoid (FLC), Proanthocyanidin
(PAC), and proline contents as well as lipid peroxidation (LP)
in leaves, twigs, and acorns of these two Serbian oak tree
species.
2. Materials and Methods
2.1. Plant Material and Extraction Procedure. is paper
presents antioxidant characteristics of two Serbian oak
species Quercus robur L. and Quercus petraea L. from
Vojvodina province, in northern Serbia. During September
, twigs, leaves, and acorns were picked to make average
samples (from  trees per one replicate). ree independent
replicates were made for both species. All samples were dried
in open air in the dark.
Aer that, g of the dried sample was nely ground
into a ne powder in a mill and extracted with  mL of
water for  h at ∘C, followed by ltration. Prepared extract
wasusedforlipidperoxidation,solubleprotein,andPRAC
determination. For all ARP determinations and FRAP the
similar extraction tool was used, with % EtOH (in water) as
an extractant. For TPC and TAC acidic ethanol (. mol/dm3
HCl in EtOH) was used as an extractant. For determination
of DPPH∙,∙NO, and O2∙− ARP, % EtOH extracts were
evaporated to dryness and the dry residues were redissolved
again in % EtOH (in water) to obtain mass concentration
 mg/mL (for DPPH∙,∙NO and O2∙− ARP determination).
2.2. Lipid Peroxidation, Proline, Soluble Protein, and Pig-
ment Contents. Lipid peroxidation (LP) was estimated based
on thiobarbituric acid (TBA) reactivity. Samples were
evaluated for malondialdehyde (MDA) production using
a spectrophotometric assay. e extinction coecient of
, mol−1 cm−1 at  nm for the chromophore was used
to calculate the colour intensity of the MDA-TBA complex in
the supernatant [].
Proline accumulation was determined by the method
as described by Paquin and Lechasseur []. Proline was
determined aer extraction with sulphosalicylic acid, and
reaction with ninhydrin. A standard curve of proline was
used for calibration and was measured by its absorbance at
 nm.
Pigments were extracted with acetone and determined
spectrophotometrically using molar extinction coecients
according to von Wettstein []. Soluble protein content was
determined by the method of Bradford [].
2.3. Total Phenol, Tannin, Flavonoid, and Proanthocyani-
din Contents. Total polyphenols were determined by Folin-
Ciocalteu procedure []. e amount of total polyphenols
was calculated as a catechin equivalent from the calibration
curve of catechin standard solutions (covering the concentra-
tion range between . and . mg/mL) and expressed as mg
catechi/ g dr y plant material.
Total tannin content was determined by Folin-Ciocalteu
procedure as above, aer removal of tannins by their adsorp-
tion on insoluble matrix (polyvinylpolypyrrolidone, PVPP).
Calculated values were subtracted from total polyphenol
contents and total tannin contents expressed as mg cate-
chine/ g dry plant material.
Total avonoids were determined aer extraction of plant
material ( g) with extracting solvent methanol-water-acetic
acid ( :  : , V/V), according to Markham []. e
amount of avonoids was calculated as a rutin equivalent
from the calibration curve of rutin standard solutions and
expressed as mg rutin/ g of plant material.
Proanthocyanidins were determined by butanol-HCl
assay []. eir contents were expressed as mg leucoan-
thocyanidin/g of dry plant material, assuming that the
specic absorbance of leucoanthocyanidin was .
Allmeasurementsweredoneintriplicate.
2.4. FRAP. Total antioxidant capacity was estimated accord-
ing to the ferric reducing antioxidant power (FRAP) assay
[]. FRAP reagent was prepared by mixing acetate buer
( mM pH .), TPTZ (,,-tripyridyl-s-triazine) reagent
( mM in  mM HCl), and FeCl3⋅H2(mM)inratio

e Scientic World Journal 
::. Sample (L) was mixed with  mL of working
FRAP reagent and absorbance ( nm) was measured at 
minutes aer vortexing. FRAP value was calculated using
formula
FRAP value =sample (– min)
standard (– min) .()
 MFe
2+ wasusedasastandard;FRAPunit=M
Fe2+.
Total antioxidant capacity was expressed in FRAP units.
2.5. Permanganate Reducing Antioxidant Capacity. e
methodisbasedontheredoxreactionsbetweenthe
antioxidant sample and the potassium permanganate in
sulfuric acid media, leading to sample discoloration until
no colour is observed []. Variable amounts of samples
(-mL), depending on the intensity of the antioxidant
activity, were introduced in a  mL quartz vat containing
an oxidative mixture of . mL potassium permanganate
. M; . mL sulfuric acid  M, and (-)mL distilled
water. at moment was considered the zero time. e
spectrophotometer signal was then registered at  nm
until constant value. Subsequent decrease of potassium
permanganate concentration was determined based on a
previously prepared calibration curve. A calibration curve
was determined by preparing a series of six solutions
with dierent concentrations of potassium permanganate
and registering the absorbance for each of them. In order
to quantitatively compare the antioxidant activities, we
proposed the following formula:
50 =(standard)
(plant sample)⋅(standard)
(plant)⋅(standard)
(plant sample)⋅
(extract),()
where 50 is antioxidant activity expressed, reected in the
time until the sample induces a decrease of the oxidizing
agent [potassium permanganate] concentration up to one
half, compared against a standard [ascorbic acid] (mmol
equivalent standard/g plant), (plant sample)isthetimeuntilthe
sample induces a decrease of the permanganate concentra-
tion up to one half (min), (standard)is the time until the stan-
dard (ascorbic acid) induces a decrease of the permanganate
concentration up to one half (min), (standard)is standard
(ascorbic acid) concentration (mmol/mL) [. mmol/mL],
(plant)is weight (g) of the plant sample submitted to
extraction [ g], (plant sample)is volume of the plant extract
submitted to the analysis [. mL], (standard)is volume of
the standard submitted to the analysis [ mL], and (extract)is
volume (mL) of the obtained extract [ mL].
2.6. Radical Scavenging Determinations. DPPH∙-RSC assay
was based on measurement of the loss of DPPH (,-
diphenyl--picrylhydrazyl) color aer reaction with test com-
pounds []. e DPPH∙radical is one of the few stable
organic nitrogen radicals, which bears a deep purple color.
is assay is based on the measurement of the reducing ability
of antioxidants toward DPPH∙.eabilitycanbeevaluated
by measuring the decrease of its absorbance. e widely used
decoloration assay was rst reported by Brand-Williams et
al. [].Appropriatevolumeofeachextractwasmixedwith
 M DPPH∙in methanol making up nal volume of . mL.
e mixtures were shaken vigorously and were stored in dark
for  min at room temperature. e decrease of absorbance
of the reaction mixtures regarding the control was monitored
spectrophotometrically at  nm.
∙NO-RSC was evaluated by measuring the accumulation
of nitrite (formed by the reaction of NO with oxygen),
according to the Griess reaction []. NO was generated by
sodium nitroprusside in buered aqueous solution. Appro-
priate volume of each extract was mixed with fresh pre-
paredsolutionofsodiumnitroprusside(.mL,.Min
NaH2PO4-Na2HPO4buer, . M, pH .) and NaH2PO4-
Na2HPO4buer (. M, pH .) making nal volume of
. mL. ese mixtures were illuminated at  lx and ∘C
for  min. Aer illumination, each reaction mixture ( mL)
was mixed with Griess reagent (mL, .% N-(-naphtyl)-
ethylenediamine dihydrochloride (NEDA) in distilled water
and % sulfanilamide in % H3PO4). Reduction of nitrite
by the extracts was determined spectrophotometrically at
 nm, by measuring the decrease of absorbance of the
reaction mixtures regarding the control (containing the same
chemicals, except for the sample).
O2∙−-RSC assay was based on the capacity of crude
extracts to inhibit the photochemical reduction of nitro blue
tetrazolium (NBT) in the riboavin-light-NBT system [].
Each  mL of reaction mixture contained sodium phosphate
buer ( mM, pH .), methionine ( mM), riboavin
( M), EDTA ( M), NBT ( M), and extract solution.
Reaction systems were illuminated at  lx and ∘Cfor
 min. e increase in absorbance at  nm was monitored.
e scavenging capacity was expressed as reduction percent-
age of NBT absorbance induced by sample.
For each sample three replicates were carried out. RSC
was calculated by the following equation:
RSC =0−1
0⋅100, ()
where 0is control and 1is a sample solution absorbance.
e concentration (in the nal reaction media in each
method) that causes a decrease in the initial absorbance
(control) by % is dened as IC50.eIC
50 values for all
RSC determinations were determined by polynomial tting
of the inhibition values (RSC) using soware ORIGIN ..
e antioxidant capacity of the extracts was expressed as
antiradical power (ARP) and it was dened as
ARP = 1
IC50 ⋅100. ()
2.7. Statistical and PCA Analysis. Statistical comparisons
between samples were performed with Duncan t-test for
independent observations. Dierences were considered sig-
nicant at  < 0.05. e antioxidant test results were inves-
tigated with multivariate analysis. e correlation matrix was
calculated, giving the correlation coecients between each
pair of variables, that is, the analytical parameters tested.

e Scientic World Journal
Each term of the matrix is a number ranging from −to+:
the + or −sign indicates a positive or negative interdepen-
dence between variables (direction), and the absolute value
indicates the strength of the interdependence. Correlations
between dierent parameters were considered signicant at
 > 0.95( < 0.05). Autoscaling transformation of data for
phenolic parameters (TPC, TAC, FLC, and PAC) was done
using STATISTICA . and presented by graphic (Figure ).
3. Results and Discussion
3.1. Soluble Proteins, Proline, Pigment and MDA Contents.
Solubleproteincontentrangedfrom.(Q. robur twigs)
to . mg/g (Q. robur leaves); Proline content ranged from
. (Q. petraea acorns) to . g/g (Q. robur twigs); Chla
contentrangedfrom.(Q. robur acorns) to . mg/g
(Q. petraea leaves); Chlb content ranged from . (Q. robur
acorns) to . mg/g (Q. petraea leaves); Carotenoid content
ranged from . (Q. robur acorns) to . mg/g (Q. robur
leaves); MDA content ranged from . (Q. petraea acorns)
to . nmol/mg protein (Q. robur twigs), (Tables and ).
Signicant positive correlations were found between Chla
and Chlb ( = 0.9957), and also between carotenoids and
both chlorophylls ( = 0.84). Positive correlation was found
between proline content and MDA content ( = 0.4459).
Lipids are susceptible to oxidation and lipid peroxidation
products, such as MDA quantity, are potential biomarkers for
oxidative stress status in vivo. Proteins are also the direct tar-
get for Reactive Oxygen Species (ROS) because of their high
concentrations. eir oxidation may result in deamination,
decarboxylation, peptide backbone cleavage, cross-linking,
and many other chemical modications leading eventually to
inactivationof enzyme activity and accumulation within cells
and extracellular environment []. Furthermore, antioxidant
capacity and the ratio between reduced forms to oxidized
formsofmoleculesmaybealsousedasbiomarkersof
oxidative stress []. According to our results, the highest
accumulation of MDA was observed in twigs of both Quercus
species,wherethesolubleproteincontentwaslowestdueto
increased level of oxidative stress.
Carotenoids, along with phenolics, are responsible for
bright colours of plants and are also powerful antioxidants.
Carotenoids can protect membranes against damage by free
radicals and retard the ageing processes []. e highest
content of pigments, carotenoids, and chlorophylls was found
in the leaves of both, especially Q. petraea.Prolineisan
amino acid that acts as an antioxidant—it reduces free
radicals in plant cells [,]. Its production is a self-defense
mechanism. Plant’s proline levels are an indicator of both
the environment stress and the plant’s response. Proline does
not interfere with normal biochemical reactions but allows
the plants to survive under stress []. e accumulation of
proline in plant tissues is also a clear marker for environ-
mental stress, particularly in plants under drought conditions
andmayalsobepartofthestresssignalinuencingadaptive
responses []. Positive correlation between free proline
content and LP intensity conrms both antioxidant and
defense natures of this amino acid.
3.2. Total Phenol, Tannin, Flavonoid, and Proanthocyani-
din Contents. Total phenol content ranged from . (Q.
petraea twigs) to .mg catechin/g (Q. petraea leaves);
Tannin content ranged from . (Q. petraea twigs) to
. mg catechin/g (Q. petraea leaves); Flavonoid content
ranged from . (Q. petraea twigs) to . mg rutin/ g
(Q. petraea leaves); Proanthocyanidine content ranged
from . (Q. petraea twigs) to  mg rutin/ g (Q.
robur leaves) (Table ). Signicant positive correlations were
observed between all mentioned parameters. e highest
positive correlation was observed between TPC and TAC
( = 0.9955). All phenolic parameters were signicantly
positively correlated with carotenoid content. Total phenol,
tannin, and avonoid contents were also signicantly posi-
tively correlated with both chlorophylls (a and b). Phenolic
parameters were analyzed by a multivariate approach and
results are showed by line plot of multiple variables (Figure ).
All phenolic parameters are negatively but not signicantly
correlated with LP parameter.
Our results are in accordance with that obtained by
Raki´
cetal.[] who indicated that oak acorns from Q.
robur are material rich in polyphenols and tannins. We have
found similar results for Q. petraea acorns. Kamalak et al. []
evaluated nutritive values of browse leaves from ve oak
species, namely, Quercus branti,Quercus coccifera,Quer-
cus cerris,Quercus libari,andQuercus infectoria based on
their chemical composition. It was established that tannin
contentrangedfrom.to.mg/gmatterwhichisin
accordance with our results for Q. robur and Q. petraea.
Q. petraea and Q. robur arealsorichsourceofavonoids
and proanthocyanidins which are found in all plant organs,
especially in leaves. Flavonoid content found in leaves of Q.
petraea and Q. robur is in range of that found in Quercus
salicina Blume []. According to Salminen et al. ()
hydrolysable tannins were the dominant phenolic group in
leaves of all ages of Q. robur which is in well agreement with
our results. However, hydrolysable tannins and avonoid
glycosides showed highly variable seasonal patterns. Young
oakleavesweremuchricherinhydrolysabletanninsand
avonoid glycosides than old leaves, and vice versa for proan-
thocyanidins []. Although in smaller quantities, twigs also
contained all classes of polyphenols (Ta b l e  ). e obtained
results have provided further grounds for establishing Q.
robur and Q. petraea leaves, acorns, and twigs as a source for
functional food preparation.
3.3. FRAP and PRAC Methods. FRAP values ranged from
. (Q. robur twigs) to . FRAP units (Q. petraea
leaves); PRAC values ranged from . (Q. petraea twigs)
to . mmol ascorbate eq./g (Q. petraea acorns) (Ta b l e  ).
FRAP was signicantly positively correlated with phenolic
parameters (TPC, TAC, and FLC) and with pigment (Chla,
Chlb, and Car) contents. e highest positive correlation was
observed between FRAP and TAC ( = 0.9587). PRAC value
was positively correlated with O2∙−-ARP ( = 0.6196)and
negatively correlated with LP (= −0.8113).
Benzie and Strain [] introduced FRAP as a simple
and automated test measuring the ferric reducing ability

e Scientic World Journal 
T : Total phenol, tannin, avonoid, proantocyanidine, chlorophyll a and b, carotenoid, and proline contents in oak twigs, leaves, and
acorns of two Serbian oak species Quercus robur L. and Quercus petraea L.
Plant organ Locality TPC
(mg catechin/g)
TAC
(mg catechin/g)
FLC
(mg rutin/ g)
PAC
(mg leukocyani-
dine/ g)
Chla
(mg/g)
Chlb
(mg/g)
Car
(mg/g)
Pro.
(g/g)
Twigs Q. robur .a.a.a.a.a.a.a.a
Q. petraea .   b.b.a.b.a.b.b.b
Leaves Q. robur .c.c.bc.b.c.c.c
Q. petraea .d.d.c.d.c.d.d.d
Acorns Q. robur .e.e.a.e.d.e.e.ad
Q. petraea .e.e.d.a.a.a.ab .e
∗Values with the same letter, in each colon, are not signicantly dierent according to Duncan test (𝑃 < 0.05).
∗∗TPC: total phenolic content; TAC: tannin content; FLC: avonoid content; PAC: proanthocyanidin content; Chla and Chlb: chlorophyll a and b contents;
Car: carotenoid content; Pro: proline content.
T : Protein content, DPPH, NO, and O2∙−-antiradical powers, permanganate reducing antioxidant capacity, ferric reducing antioxidant
power, and lipid peroxidation in oak twigs, leaves, and acorns of two Serbian oak species Quercus robur L. and Quercus petraea L.
Plant
organ Locality Prot.
(mg/g)
DPPH-ARP
((/IC)⋅)
NO-ARP
((/IC)⋅)
O2∙−-ARP
((/IC)⋅)
PRAC
(A)
FRAP (FRAP
units)
LP
(nmol/mg prot.)
Twigs Q. robur .a.a.a.a.a.a.a
Q. petraea .b.b.b.b.a.a.b
Leaves Q. robur .c.    c.c.b.b.b.c
Q. petraea .d.d.d.c.c.c.c
Acorns Q. robur .e.d.e.d.d.d.d
Q. petraea .b.e.e.e.e.e.d
∗Values with the same letter, in each colon, are not signicantly dierent according to Duncan test (𝑃 < 0.05).
∗∗Prot.: proteins; ARP: antiradical power; ARP = ((/IC)⋅); IC: the concentration of an sample at which % inhibition off ree radical activityis obs erved;
PRAC: permanganate reducing antioxidant capacity; A: antioxidant activity reected in time until the sample induces a decrease of the oxidizing agent
(potassium permanganate) up to one half, compared against a standard (ascorbic acid); A = mmol ascorbate eq./g; FRAP: ferric reducing antioxidant power;
FRAPunit=𝜇mol/dmFe+; LP: lipid peroxidation.
of plasma and a novel method for assessing “antioxidant
power.” Ferric to ferrous ion reduction at low pH causes a
colored ferrous-tripyridyltriazine complex to form. Accord-
ingtoMaqsoodandBenjakul[] tannic acid showed
higher FRAP value in comparison with other investigated
phenolics including catechin which is in agreement with
our observation that the highest positive correlation was
found between FRAP and TAC parameter. Same reaction
mechanism based on electron transfer explains high positive
correlations between FRAP and phenolic parameters (TPC,
TAC, and FLC ) [ ]. According to FRAP method, antioxidant
capacity of oak samples, especially leaves, was relatively
high comparing with other plants [–]. FRAP value
was more or less positively correlated with all investigated
parameters excluding LP, where negative correlation was
found (−.).
PRAC method was rstly introduced by Cacig and Szabo
[] as a simple spectrophotometric method for evaluation
of antioxidant capacity and later compared with other total
antioxidant capacity methods []. Unlike the oak leaves
where the highest FRAP was found, oak acorns showed the
highest PRAC. High positive correlations with O2∙−-AR P
indicate that the same structures are probably responsible for
superoxide scavenging and for reduction of permanganate
in acidic media. e highest negative correlations of both
parameters with LP also indicate that scavengers of super-
oxide and substances with high reducing potential against
permanganate are mostly responsible for suppression of LP.
3.4. Antiradical Power Determinations. DPPH-ARP ranged
from . (Q. petraea twigs) to . (Q. robur acorns); NO-
ARP ranged from . (Q. robur acorns) to . (Q. robur
leaves); O2∙−-ARP ranged f rom . (Q. robur twigs) to .
(Q. robur acorns) (Tab l e  ). DPPH-ARP and NO-ARP were
positively correlated with all phenolic parameters (TPC, TAC,
FLC, and PAC), pigments (Chla, Chlb, and Car), and FRAP.
In contrast to them, O2∙−-ARP showed rather less positive
correlations with phenolic parameters, pigments, and FRAP
but relatively high positive correlation with PRAC (.).
Among investigated ARP parameters, only O2∙−-ARP sh owed
signicant negative correlation with LP (= −0.8360).
All three ARP power determinations against DPPH,
O2∙−, and NO radicals generally proceed also via hydrogen
atom transfer or electron transfer mechanism depending on
present antioxidant structure, pH, dielectric constant of the
solvent, and so forth []. DPPH and O2∙−-ARP were corre-
lated with each other ( = 0.6776), while correlations with
NO-ARP were much lower. Signicant positive correlation of

e Scientic World Journal
−1.0 −0.5 0.0 0.5 1.0
−1.0
−0.5
0.0
0.5
1.0
Factor 1: 58.33%
Factor 2: 21.51%
Prot.
LP
Pro.
NO-ARP
Chla
Chlb
FLC
TPC
DPPH-ARP
PRAC
FRAP
TAC
PAC
Car
O2
−ARP
F : Graph of loading plot of antioxidant markers for Serbian
oak species Quercus robur L. and Quercus petraea L. Prot.: proteins;
ARP: antiradical power; PRAC: permanganate reducing antioxidant
capacity; FRAP: ferric reducing antioxidant power; LP: lipid per-
oxidation; TPC: total phenolic content; TAC: tannin content; FLC:
avonoid content; PAC: Proanthocyanidin content. Chla and Chlb:
chlorophyll a and b contents; car: Carotenoid content; Pro: proline
content. Parameters with close interdependence and correlation are
close to each other and vice versa.
NO-ARP and carotenoids ( = 0.8525) indicated signicance
of carotenoid antioxidants for NO scavenging. Sindhu et al.
[] also established that carotenoids lutein and zeaxanthin
showed stronger antiradical potential against NO than DPPH
and O2∙− radicals. e highest DPPH- and O2∙−-ARP showed
acorns and the highest NO-ARP showed leaves Q. robur.
Rivas-Arreola et al. []alsoinvestigatedantioxidantactivity
of oak (Q. sideroxyla,Q. Eduardii,andQ. resinosa)leaves
infusions against free radicals and obtained similar results
for radical scavenger capacities. Oak twigs expressed lower
DPPH, O2∙−,andNOARPincomparisonwithleavesand
acorns but also relatively high antiradical potential.
3.5. PCA Analysis. e original data set was renormalized by
an autoscaling transformation (data not shown) and dierent
parameters were analyzed by a multivariate approach. e
loadings plot is presented by Figure  and the scores plot by
Figure . e scree plot (data not shown) indicates that the
rst two principal components account for .% of the total
variance (PC = . and PC = .).
As reported in the loadings plot (Figure ), antiradical
power parameters (DPPH∙,∙NO, and O2∙−-ARP), FRAP,
polyphenol (TPC, TAC, FLC, and PAC), protein, and pigment
contents are positioned closely due to the signicant positive
correlations among them. PRAC and O2∙− ARP are partially
isolated and located opposite to LP as a parameter of oxi-
dative stress. Positive correlations were also found among
−6 −5 −4 −3 −2 −1 01234
−4
−3
−2
−1
0
1
2
3
4
5
Factor 2: 21.51%
Factor 1: 58.33%
Twigs QR
Twigs QPLeaves QR
Leaves QP
Acorns QR
Acorns QP
F:GraphofscoresplotforSerbianoakspeciesQuercus
robur L. (QR) and Quercus petraea L. (QP). Quercus robur L. (QR)
and Quercus samples that are close to each other possess similar
antioxidant statuses.
DPPH-ARP and proteins and also between ∙NO-ARP and
proline content.
PCA found three dierent clusters of oak samples based
on antioxidant characteristics: leaves, acorns, and twigs from
investigated species are grouped (Figure ). Leaves dier
from twigs and acorns predominantly by Factor  (where
the major contributors are polyphenols and FRAP). And the
dierence between twigs and acorns is based on Factor 
(where the major contributors are LP, PRAC, and O2∙−-ARP).
Opposite direction of LP on one side and PRAC O2∙−-ARP
on another side indicates that the major contributors against
lipid peroxidation are components which are antioxidants
which can easily reduce permanganate and also scavenge
O2∙−-radicals. Very close interdependence was observed
between leaves and twigs from both species, but twigs from
two Quercus species showed slightly higher dierences. Line
plot of multiple variables aer autoscaling transformation
of polyphenolic content parameters (TPC, TAC, FLC, and
PAC ) w a s s h o w n i n Figure . It is obvious that polyphenolic
parameters for leaves are separated and are greater than the
same parameters for acorns and twigs.
4. Conclusion
is investigation pointed out antioxidant potential of both
Serbian oak species (Q. robur and Q. petraea). Leaves from
both oak species possessed high contents of total phenols,
tannins, avonoids, proanthocyanidins, and pigment con-
tents. Antiradical power parameters were also very high for
oak leaves and LP intensity was relatively low. Ferric reducing
antioxidant capacity was the highest in oak leaf extracts,
especially for Q. petraea. Among investigated leaf extracts
which are signicant source of phenolic compounds, oak
acorns showed also high antioxidant potential and the lowest
LP intensity. Antioxidant capacity values including DPPH∙,
∙NO and O2∙−-ARP, and FRAP showed high positive correla-
tions among themselves and also with polyphenol parameters

e Scientic World Journal 
−1.5
−1.0
−0.5
0.0
0.5
1.0
1.5
2.0
Twigs QR
Twigs QP
Leaves QR
Leaves QP
Acorns QR
Acorns QP
TPC TAC FLC PAC
Line plot of multiple variables
F : Line plot of multiple variables aer autoscaling transfor-
mation of phenolic content parameters (TPC, TAC, FLC, and PAC).
Quercus robur L. (QR) and Quercus petraea L. (QP). TPC: Total
phenolic content; TAC: Tannin content; FLC: Flavonoid content;
PAC: Proanthocyanidin content.
(TPC, TAC, FLC, and PAC), protein, and pigment contents.
Permanganate PRAC and O2∙−-ARP were selected as the
best antioxidant markers for oak trees because of the highest
negative correlations with LP intensity. Considering high
antioxidant potential of investigated organs of Serbian oak
species (Q. robur and Q. petraea), besides acorns, oak leaves,
and even twigs, could be recommended as source of natural
antioxidants and promising source of pharmaceuticals with
possible use in industry and pharmacy.
Abbreviations
PCA: Principal component analysis
RSC: Radical scavenging capacity
ARP: Antiradical power
PRAC: Permanganate reducing antioxidant
capacity
FRAP: Ferric reducing antioxidant power
LP: Lipid peroxidation
TPC: Total phenolic content
TAC: Tannin content
FLC: Flavonoid content
PAC: Proanthocyanidin content
Chla and Chlb: Chlorophyll a and b contents
Car: Carotenoid content
Pro: Proline content.
Acknowledgment
is research is part of Project no. III which is nan-
cially supported by the Ministry of Science, Technologies and
Development of the Republic of Serbia.
References
[] J. A. Sanchez-Burgos, M. V. Ramirez-Maresb, M. M. Larrosac et
al., “Antioxidant, antimicrobial, antitopoisomerase and gastro-
protective eect of herbal infusions from four Quercus species,”
Industrial Crops and Products,vol.,pp.–,.
[] K. Haneca, K. C. Katarina ˇ
Cufar, and H. Beeckman, “Oaks,
tree-rings and wooden cultural heritage: a review of the main
characteristics and applications of oak dendrochronology in
Europe,” Journal of Archaeological Science,vol.,no.,pp.–,
.
[] R. Cvjeti´
canin, R. O. Koˇ
sanin, and M. Novakovi´
c, “Ekoloˇ
ske
jedinice ˇ
suma hrasta kitnjakau istraˇ
zivanim sastojinama
severoistoˇ
cne Srbije,” ˇ
Sumarstvo,vol.,pp.–,.
[] M. Kneˇ
zevi´
c, V. Babi´
c, Z. Gali´
c, and O. Koˇ
sanin, “Osobine
zemljiˇ
sta u ˇ
sumama hrasta kitnjaka (Quercetum montanum
typicum ˇ
Cer. et Jov. ) na podruˇ
cju Fruˇ
ske gore,” Glasnik
ˇ
Sumarskog Fakulteta,vol.,pp.–,.
[] http://www.about-oak-trees.com/index.htm.
[] I.M.G.LopesandM.G.Bernardo-Gil,“Characterisationof
acorn oils extracted by hexane and by supercritical carbon
dioxide,” European Journal of Lipid Science and Technology,vol.
, no. , pp. –, .
[] M. Le´
on-Camacho, I. Viera-Alcaide, and I. M. Vicario, “Acorn
(Quercus spp.) fruit lipids: saponiable and unsaponiable
fractions: a detailed study,” JournaloftheAmericanOilChemists’
Society,vol.,no.,pp.–,.
[] S. Raki´
c, S. Petrovi´
c, J. Kuki´
c et al., “Inuence of thermal
treatment on phenolic compounds and antioxidant properties
of oak acorns from Serbia,” Food Chemistry,vol.,no.,pp.
–, .
[] S. Raki´
c, D. Povrenovi´
c, V. Teˇ
sevi´
c, M. Simi´
c, and R. Maleti´
c,
“Oak acorn, polyphenols and antioxidant activity in functional
food,” Journal of Food Engineering,vol.,no.,pp.–,
.
[] E. Pallenbach, E. Scholz, M. K¨
onig, and H. Rimpler, “Proantho-
cyanidins from Quercus petraea bark,” Planta Medica,vol.,
no.,pp.–,.
[] Z. A. Kuliev, A. D. Vdovin, N. D. Abdullaev, A. B.
Makhmatkulov, and V. M. Malikov, “Study of the catechins and
proanthocyanidins of Quercus robur,” Chemistry of Natural
Compounds,vol.,no.,pp.–,.
[] K. B. Pandey and S. I. Rizvi, “Plant polyphenols as dietary
antioxidants in human health and disease,” Oxidative Medicine
and Cellular Longevity,vol.,no.,pp.–,.
[]Y.Cai,Q.Luo,M.Sun,andH.Corke,“Antioxidantactivity
and phenolic compounds of  traditional Chinese medicinal
plants associated with anticancer,” Life Sciences,vol.,no.,
pp.–,.
[] J. J. Kim, B. K. Ghimire, H. C. Shin et al., “Comparison of
phenolic compounds content in indeciduous Quercus species,”
JournalofMedicinalPlantsResearch,vol.,no.,pp.–
, .
[] R. Brossa, I. Casals, M. Pint´
o-Marijuan, and I. Fleck, “Leaf
avonoid content in Quercus ilex L. resprouts and its seasonal
variation,” Tre es,vol.,no.,pp.–,.
[] A.Kamalak,O.Canbolat,O.Ozay,andS.Aktas,“Nutritivevalue
of oak (Quercus spp.) leaves,” Small Ruminant Research,vol.,
no. -, pp. –, .
[] H. P. S. Makkar, R. K. Dawra, and B. Singh, “Tannin levels
in leaves of some oak species at dierent stages of maturity,”
JournalofScienceofFoodandAgriculture,vol.,no.,pp.–
, .

e Scientic World Journal
[] S. Andrenˇ
sek, B. Simonovska, I. Vovk, P. Fyhrquist, H. Vuorela,
and P. Vuorela, “Antimicrobial and antioxidative enrichment of
oak (Quercus robur) bark by rotation planar extraction using
ExtraChrom,” International Journal of Food Microbiology,vol.
,no.,pp.–,.
[] T.B.Ng,F.Liu,andZ.T.Wang,“Antioxidativeactivityofnatural
products from plants,” Life Sciences,vol.,no.,pp.–,
.
[] R. Paquin and P. Lechasseur, “Observations sur une m´
ethode de
dosage de la proline libre dans les extraits de plantes,” Canadian
Journal of Botany,vol.,no.,pp.–,.
[] D. von Wettstein, “Chlorophyll-letale und der submikroskopis-
che Formwechsel der Plastiden,” Experimental Cell Research,
vol. , no. , pp. –, .
[] M. M. Bradford, “A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the principle
of protein dye binding ,” Analytical Biochemistry,vol.,no.-,
pp.–,.
[] A. Hagerman, I. Harvey-Mueller, and H. P. S. Makker, Quan-
tication of Tannins in the Foliage—A Laboratory Manual,
FAO/IAEA, Vienna, Austria, .
[] K. R. Markham, “Flavones, avonols and their glycosides,” in
Methods in Plant Biochemistry,P.M.DeyandJ.B.Harborne,
Eds., Academic Press, London, UK, .
[] I. F. F. Benzie and J. J. Strain, “Ferric reducing/antioxidant power
assay: direct measure of total antioxidant activity of biological
uids and modied version for simultaneous measurement
of total antioxidant power and ascorbic acid concentration,”
Methods in Enzymology,vol.,pp.–,.
[] S. I. Cacig and M. I. Szabo, “Spectrophotometric method for
the study of the antioxidant activity applied on Ziziphus jujoba
and Hydrangea paniculata aqueous extracts,” in Zbornik Matice
srpske za prirodne nauke (Proceedings for Natural Sciences),pp.
–, Matica Srpska, Novi Sad, Serbia, .
[] J. C. Esp´
ın, C. Soler-Rivas, and H. J. Wichers, “Characterization
of the total free radical scavenger capacity of vegetable oils
and oil fractions using ,-diphenyl--picrylhydrazyl radical,”
Journal of Agricultural and Food Chemistry,vol.,no.,pp.
–, .
[] W. Brand-Williams, M. E. Cuvelier, and C. Berset, “Use of a free
radical method to evaluate antioxidant activity,” LWT—Food
Science and Technology,vol.,no.,pp.–,.
[] L. C. Green, D. A. Wagner, J. Glogowski, P. L. Skipper, J. S.
Wishnok, and S. R. Tannenbaum, “Analysis of nitrate, nitrite,
and [N]nitrate in biological uids,” Analytical Biochemistry,
vol. , no. , pp. –, .
[] N. Dasgupta and B. De, “Antioxidant activity of Piper betle L.
leaf extract in vitro,” Food Chemistry, vol. , no. , pp. –,
.
[] M. J. Davies, “e oxidative environment and protein damage,”
Biochimica et Biophysica Acta,vol.,no.,pp.–,.
[] M.Kemp,Y.M.Go,andD.P.Jones,“Nonequilibriumther-
modynamics of thiol/disulde redox systems: a perspective on
redox systems biology,” Free Radical Biology and Medicine,vol.
, no. , pp. –, .
[   ] O . V. B u l d a , V. V. R a s s a d i n a , H . N . A l e k s e i c h u k , a n d N .
A. Laman, “Spectrophotometric measurement of carotenes,
xanthophylls, and chlorophylls in extracts from plant seeds,”
Russian Journal of Plant Physiology,vol.,no.,pp.–,
.
[] P. P. Saradhi, A. Alia, S. Arora, and K. V. S. K. Prasad,
“Proline accumulates in plants exposed to UV radiation and
protects them against UV induced peroxidation,” Biochemical
and Biophysical Research Communications,vol.,no.,pp.
–, .
[] N. Krishnan, M. B. Dickman, and D. F. Becker, “Proline
modulates the intracellular redox environment and protects
mammalian cells against oxidative stress,” Free Radical Biolog y
and Medicine,vol.,no.,pp.–,.
[] C. R. Stewart, “Proline accumulation: biochemical aspects,” in
Physiology and Biochemistry of Drought Resistance in Plants,L.
G. Paleg and D. Aspinall, Eds., pp. –, .
[] A. Maggio, S. Miyazaki, P. Veronese et al., “Does proline
accumulation play an active role in stress-induced growth
reduction?” Plant Journal,vol.,no.,pp.–,.
[] S. Raki´
c, R. Maleti´
c, M. Perunovi´
c, and G. Svrzi´
c, “Inuence of
thermal treatment on tannin content and antioxidation eect
of oak acorn Quercus cerris extract,” Journal of Agricultural
Sciences,vol.,pp.–,.
[] J. I. Kim, H. Y. Kim, S. G. Kim, K. T. Lee, I. H. Ham, and W. K.
Whang, “Antioxidant compounds from Quercus salicina Blume
stem,” Archives of Pharmacal Research,vol.,no.,pp.–,
.
[] J. P. Salminen, T. Roslin, M. Karonen, J. Sinkkonen, K. Pih-
laja, and P. Pulkkinen, “Seasonal variation in the content of
hydrolyzable tannins, avonoid glycosides, and proanthocyani-
dins in oak leaves,” Journal of Chemical Ecology,vol.,no.,
pp. –, .
[] S. Maqsood and S. Benjakul, “Comparative studies of four
dierent phenolic compounds on in vitro antioxidative activity
and the preventive eect on lipid oxidation of sh oil emulsion
and sh mince,” Food Chemistry,vol.,no.,pp.–,.
[] R. L. Prior, X. Wu, and K. Schaich, “Standardized methods
for the determination of antioxidant capacity and phenolics
in foods and dietary supplements,” Journal of Agricultural and
Food Chemistry,vol.,no.,pp.–,.
[] D. ˆ
Stajner, B. M. Popovi´
c, J. ˆ
Canadanovi´
c-Brunet, and G.
Anaˆ
ckov, “Exploring Equisetum arvense L., Equisetum ramo-
sissimum L. and Equisetum telmateia L. as sources of natural
antioxidants,” Phy totherapy Research,vol.,no.,pp.–,
.
[] D. ˇ
Stajner, B. M. Popovi´
c, A. Kapor, P. Boˇ
za, and M. ˇ
Stajner,
“Antioxidant and scavenging capacity of Anacamptis pyrim-
idalis L.—Pyrimidal orchid from Vojvodina,” Phytotherapy
Research,vol.,no.,pp.–,.
[] D. ˇ
Stajner, B. M. Popovi´
c, D. ´
Cali´
c-Dragosavac, Ð. Malenˇ
ci´
c,
and S. Zdravkovi´
c-Kora´
c, “Comparative study on Allium
schoenoprasum cultivated plant and Allium schoenoprasum
tissue culture organs antioxidant status,” Phytotherapy Research,
vol.,no.,pp.–,.
[] B. M. Popovi´
c, D. ˇ
Stajner, K. Slavko, and B. Sandra, “Antioxidant
capacity of cornelian cherry (Cornus mas L.)—comparison
between permanganate reducing antioxidant capacity and other
antioxidant methods,” Food Chemistry,vol.,no.,pp.–
,  .
[] E.R.Sindhu,K.C.Preethi,andR.Kuttan,“Antioxidantactivity
of carotenoid lutein in vitro and in vivo,” Indian Journal of
Experimental Biology,vol.,no.,pp.–,.
[] M. J. Rivas-Arreola, N. E. Rocha-Guzm´
an,J.A.Gallegos-Infante
et al., “Antioxidant activity of oak (Quercus) leaves infusions
against free radicals and their cardioprotective potential,” Pak-
istan Journal of Biological Sciences,vol.,no.,pp.–,
.