# Antioxidant Characterization of Oak Extracts Combining Spectrophotometric

Authors: Boris M. Popović; Dubravka Štajner; Ružica Ždero; Saša Orlović; Zoran Galić
Journal: The Scientific World Journal
 (2013)

---

## Abstract

Antioxidant characteristics of leaves, twigs, and acorns from two Serbian oak speciesQuercus 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 D
P
P
H
•, N
O
•, and O
2
∙
- 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.


---

## Body

## 1. Introduction

Quercus trees, commonly known as oaks, belong to the family Fagaceae. They comprise 450
species worldwide [1]. 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 [2]. Pedunculate is the dominant
tree species of natural forests in the area of flat Srem and also in whole region of
Vojvodina, northern Serbia. Besides pedunculate, sessile oak is the most valuable oak in
Serbia [3]. In the forestry fund of Serbia, there is
the significant participation of sessile oak (7.28%) [4]. Oaks are the major source of hardwood lumber and also they are used for
ornaments. The wood is durable and tough and also attractively grained. It is especially
valued in shipbuilding and construction and for flooring, furniture, barrels, and veneer.
The bark of some oaks has been used in medicine, in tanning, and for dyes. In mythology and
religion, the oak was revered as a symbol of power [5]. 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 [6, 7]. Quercus acorns were mainly used for
making bread or as a substitute for coffee. Oak kernels were traditionally used in medicine,
particularly roasted ones as astringents, antidiarrhoeals, and antidotes [8]. The acorns of Q. robur contain
various biologically active compounds with antioxidant activity (tannins, gallic and ellagic
acid, and different galloyl and hexahydroxydiphenoyl derivatives) [8, 9]. It was also known that the
bark of different Quercus species contains polyphenolic constituents. Thus,
the bark of Q. petraea contains both hydrolysable and condensed tannins,
flavanols, and oligomeric proanthocyanidins [10].
From the bark of Quercus robur more than 20 compounds (catechins and
oligomeric and polymeric proanthocyanidins) have been isolated [11].Polyphenols are secondary metabolites of plants that are generally involved in defense
against ultraviolet radiation and pathogens. In food, polyphenols contribute to the color,
flavor, odor, bitterness, astringency, and oxidative stability. Recent biomedical
investigations connected to the polyphenols and antioxidant activity of a number of herbals
and foods show that polyphenols such are flavonoids, tannins, and proanthocyanidins offer
protection against development of cancers, cardiovascular diseases, diabetes, osteoporosis
and neurodegenerative diseases [12, 13].Kim et al. [14] investigated phenolic profile in
leaves of five different Quercus species and approved presence of many
chemical constituents. This study demonstrated differences in phenolic compounds content in
different parts of plants. Quercus salicinaBlume, for example, possesses
high levels of gentisic and chlorogenic acids as well as flavonoids naringin and rutin in
the leaf. Brossa et al. [15] established that major
constituents in holm oak (Quercus ilex L.) leaves are flavanols and
flavonols. According to Kamalak et al. [16], oak
leaves from some Quercusspecies (Q. brantiand 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 Quercusspecies highly depends on the stage of maturity and
expresses seasonal variation [15, 17].Sanchez-Burgos et al. [1] established that aqueous
extracts from leaves from different white Quercusspecies (Quercus
resinosa, Quercus laeta, Quercus grisea,andQuercus obtusata)
displayed high radical scavenging activity against DPPH and OH
• radicals as well as antimicrobial activities and
antitopoisomerase activity only Q. resinosa leaves infusions.
Andrenšek et al. [18] 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 oaksQuercus
robur L. and Quercus petraeaL.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), flavonoid (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

This paper presents antioxidant characteristics of two Serbian oak speciesQuercus robur L. and Quercus petraeaL. from Vojvodina
province, in northern Serbia. During September 2011, twigs, leaves, and acorns were picked
to make average samples (from 20 trees per one replicate). Three independent replicates
were made for both species. All samples were dried in open air in the dark.After that, 20 g of the dried sample was finely ground into a fine powder in a
mill and extracted with 500 mL of water for 24 h at 25°C, followed by
filtration. Prepared extract was used for lipid peroxidation, soluble protein, and PRAC
determination. For all ARP determinations and FRAP the similar extraction tool was used,
with 80% EtOH (in water) as an extractant. For TPC and TAC acidic ethanol
(0.1 mol/dm3 HCl in EtOH) was used as an extractant. For determination
of DPPH
•, NO
•, and O
2
•
- ARP, 80% EtOH extracts were evaporated to dryness and
the dry residues were redissolved again in 80% EtOH (in water) to obtain mass
concentration 25 mg/mL (for DPPH
•, NO
• and O
2
•
- ARP determination).


### 2.2. Lipid Peroxidation, Proline, Soluble Protein, and Pigment Contents

Lipid peroxidation (LP) was estimated based on thiobarbituric acid (TBA) reactivity.
Samples were evaluated for malondialdehyde (MDA) production using a spectrophotometric
assay. The extinction coefficient of
153,000 mol−1 cm−1 at 532 nm
for the chromophore was used to calculate the colour intensity of the MDA-TBA complex in
the supernatant [19].Proline accumulation was determined by the method as described by Paquin and Lechasseur
[20]. Proline was determined after extraction
with sulphosalicylic acid, and reaction with ninhydrin. A standard curve of proline was
used for calibration and was measured by its absorbance at 532 nm.Pigments were extracted with acetone and determined spectrophotometrically using molar
extinction coefficients according to von Wettstein [21]. Soluble protein content was determined by the method of Bradford [22].


### 2.3. Total Phenol, Tannin, Flavonoid, and Proanthocyanidin Contents

Total polyphenols were determined by Folin-Ciocalteu procedure [23]. The amount of total polyphenols was calculated as a catechin
equivalent from the calibration curve of catechin standard solutions (covering the
concentration range between 0.1 and 1.0 mg/mL) and expressed as mg
catechi/100 g dry plant material.Total tannin content was determined by Folin-Ciocalteu procedure as above, after removal
of tannins by their adsorption on insoluble matrix (polyvinylpolypyrrolidone, PVPP).
Calculated values were subtracted from total polyphenol contents and total tannin contents
expressed as mg catechine/100 g dry plant material.Total flavonoids were determined after extraction of plant material (1 g) with
extracting solvent methanol-water-acetic acid (140 : 50 : 10,
V/V), according to Markham [24]. The amount of
flavonoids was calculated as a rutin equivalent from the calibration curve of rutin
standard solutions and expressed as mg rutin/100 g of plant material.Proanthocyanidins were determined by butanol-HCl assay [23]. Their contents were expressed as mg leucoanthocyanidin/100 g of dry
plant material, assuming that the specific absorbance of leucoanthocyanidin was 460.All measurements were done in triplicate.


### 2.4. FRAP

Total antioxidant capacity was estimated according to the ferric reducing antioxidant
power (FRAP) assay [25]. FRAP reagent was prepared
by mixing acetate buffer (300 mM pH 3.6), TPTZ (2,4,6-tripyridyl-s-triazine)
reagent (10 mM in 40 mM HCl), and FeCl3·6H20
(20 mM) in ratio 3 : 1 : 1. Sample
(100 μL) was mixed with 3 mL of working FRAP
reagent and absorbance (593 nm) was measured at 4 minutes after vortexing. FRAP
value was calculated using formula (1)
FRAP
value
=
Δ
A
sample
(
0–4 min
)
Δ
A
standard
(0–4 min)
. 100 μM Fe2+ was used
as a standard; 1 FRAP unit = 100 μM Fe2+.Total antioxidant capacity was expressed in FRAP units.


### 2.5. Permanganate Reducing Antioxidant Capacity

The method is based on the redox reactions between the antioxidant sample and the
potassium permanganate in sulfuric acid media, leading to sample discoloration until no
colour is observed [26]. Variable amounts of
samples (V-mL), depending on the intensity of the antioxidant
activity, were introduced in a 30 mL quartz vat containing an oxidative mixture of
1.5 mL potassium permanganate 0.01 M; 3.5 mL sulfuric acid
2 M, and (20-V) mL distilled water. That moment was considered the
zero time. The spectrophotometer signal was then registered at 535 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 different 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:
(2)
A
50
=
t
(
standard
)
t
(
plant
sample
)
·
c
(
standard
)
m
(
plant
)
·
V
(
standard
)
V
(
plant
sample
)
·
V
(
extract
)
, where A
50 is antioxidant activity expressed, reflected 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), t
(
plant
sample
) is the time until the sample induces a decrease of the
permanganate concentration up to one half (min), t
(
standard
) is the time until the standard (ascorbic acid) induces a
decrease of the permanganate concentration up to one half (min), c
(
standard
) is standard (ascorbic acid) concentration (mmol/mL)
[0.01 mmol/mL], m
(
plant
) is weight (g) of the plant sample submitted to extraction
[4 g], V
(
plant
sample
) is volume of the plant extract submitted to the analysis
[0.2 mL], V
(
standard
) is volume of the standard submitted to the analysis
[1 mL], and V
(
extract
) is volume (mL) of the obtained extract [40 mL].


### 2.6. Radical Scavenging Determinations

DPPH
•-RSC assay was based on measurement of the loss of DPPH
(2,2-diphenyl-1-picrylhydrazyl) color after reaction with test compounds [27]. The DPPH
• radical is one of the few stable organic nitrogen radicals,
which bears a deep purple color. This assay is based on the measurement of the reducing
ability of antioxidants toward DPPH
•. The ability can be evaluated by measuring the decrease of
its absorbance. The widely used decoloration assay was first reported by Brand-Williams et
al. [28]. Appropriate volume of each extract was
mixed with 90 μM DPPH
• in methanol making up final volume of 3.0 mL. The
mixtures were shaken vigorously and were stored in dark for 30 min at room
temperature. The decrease of absorbance of the reaction mixtures regarding the control was
monitored spectrophotometrically at 515 nm.NO
•-RSC was evaluated by measuring the accumulation of nitrite
(formed by the reaction of NO with oxygen), according to the Griess reaction [29]. NO was generated by sodium nitroprusside in
buffered aqueous solution. Appropriate volume of each extract was mixed with fresh
prepared solution of sodium nitroprusside (0.5 mL, 0.01 M in
NaH2PO4-Na2HPO4 buffer, 0.067 M, pH
7.4) and NaH2PO4-Na2HPO4 buffer
(0.067 M, pH 7.4) making final volume of 1.0 mL. These mixtures were
illuminated at 3000 lx and 25°C for 10 min. After illumination, each
reaction mixture (1 mL) was mixed with Griess reagent (1 mL, 0.1%
N-(1-naphtyl)-ethylenediamine dihydrochloride (NEDA) in distilled water and 1%
sulfanilamide in 5% H3PO4). Reduction of nitrite by the
extracts was determined spectrophotometrically at 546 nm, by measuring the decrease
of absorbance of the reaction mixtures regarding the control (containing the same
chemicals, except for the sample).O
2
•
--RSC assay was based on the capacity of crude extracts to
inhibit the photochemical reduction of nitro blue tetrazolium (NBT) in the
riboflavin-light-NBT system [30]. Each 3 mL
of reaction mixture contained sodium phosphate buffer (50 mM, pH 7.8), methionine
(13 mM), riboflavin (2 μM), EDTA
(100 μM), NBT (75 μM), and
extract solution. Reaction systems were illuminated at 3000 lx and 25°C for
10 min. The increase in absorbance at 560 nm was monitored. The scavenging
capacity was expressed as reduction percentage of NBT absorbance induced by sample.For each sample three replicates were carried out. RSC was calculated by the following
equation:(3)
RSC
=
(
A
0
-
A
1
A
0
)
·
100
, where A
0 is control and A
1 is a sample solution absorbance. The concentration (in the
final reaction media in each method) that causes a decrease in the initial absorbance
(control) by 50% is defined as IC50. The IC50 values for all
RSC determinations were determined by polynomial fitting of the inhibition values (RSC)
using software ORIGIN 9.1. The antioxidant capacity of the extracts was expressed as
antiradical power (ARP) and it was defined as (4)
ARP
=
(
1
IC
50
)
·
100
.


### 2.7. Statistical and PCA Analysis

Statistical comparisons between samples were performed with Duncant-test for independent observations. Differences were considered
significant at P
<
0.05. The antioxidant test results were investigated with
multivariate analysis. The correlation matrix was calculated, giving the correlation
coefficients between each pair of variables, that is, the analytical parameters tested.
Each term of the matrix is a number ranging from −1 to +1: the + or − sign
indicates a positive or negative interdependence between variables (direction), and the
absolute value indicates the strength of the interdependence. Correlations between
different parameters were considered significant at r
>
0.95 (P
<
0.05). Autoscaling transformation of data for phenolic
parameters (TPC, TAC, FLC, and PAC) was done using STATISTICA 9.1 and presented by graphic
(Figure 2).


## 2.1. Plant Material and Extraction Procedure

This paper presents antioxidant characteristics of two Serbian oak speciesQuercus robur L. and Quercus petraeaL. from Vojvodina
province, in northern Serbia. During September 2011, twigs, leaves, and acorns were picked
to make average samples (from 20 trees per one replicate). Three independent replicates
were made for both species. All samples were dried in open air in the dark.After that, 20 g of the dried sample was finely ground into a fine powder in a
mill and extracted with 500 mL of water for 24 h at 25°C, followed by
filtration. Prepared extract was used for lipid peroxidation, soluble protein, and PRAC
determination. For all ARP determinations and FRAP the similar extraction tool was used,
with 80% EtOH (in water) as an extractant. For TPC and TAC acidic ethanol
(0.1 mol/dm3 HCl in EtOH) was used as an extractant. For determination
of DPPH
•, NO
•, and O
2
•
- ARP, 80% EtOH extracts were evaporated to dryness and
the dry residues were redissolved again in 80% EtOH (in water) to obtain mass
concentration 25 mg/mL (for DPPH
•, NO
• and O
2
•
- ARP determination).


## 2.2. Lipid Peroxidation, Proline, Soluble Protein, and Pigment Contents

Lipid peroxidation (LP) was estimated based on thiobarbituric acid (TBA) reactivity.
Samples were evaluated for malondialdehyde (MDA) production using a spectrophotometric
assay. The extinction coefficient of
153,000 mol−1 cm−1 at 532 nm
for the chromophore was used to calculate the colour intensity of the MDA-TBA complex in
the supernatant [19].Proline accumulation was determined by the method as described by Paquin and Lechasseur
[20]. Proline was determined after extraction
with sulphosalicylic acid, and reaction with ninhydrin. A standard curve of proline was
used for calibration and was measured by its absorbance at 532 nm.Pigments were extracted with acetone and determined spectrophotometrically using molar
extinction coefficients according to von Wettstein [21]. Soluble protein content was determined by the method of Bradford [22].


## 2.3. Total Phenol, Tannin, Flavonoid, and Proanthocyanidin Contents

Total polyphenols were determined by Folin-Ciocalteu procedure [23]. The amount of total polyphenols was calculated as a catechin
equivalent from the calibration curve of catechin standard solutions (covering the
concentration range between 0.1 and 1.0 mg/mL) and expressed as mg
catechi/100 g dry plant material.Total tannin content was determined by Folin-Ciocalteu procedure as above, after removal
of tannins by their adsorption on insoluble matrix (polyvinylpolypyrrolidone, PVPP).
Calculated values were subtracted from total polyphenol contents and total tannin contents
expressed as mg catechine/100 g dry plant material.Total flavonoids were determined after extraction of plant material (1 g) with
extracting solvent methanol-water-acetic acid (140 : 50 : 10,
V/V), according to Markham [24]. The amount of
flavonoids was calculated as a rutin equivalent from the calibration curve of rutin
standard solutions and expressed as mg rutin/100 g of plant material.Proanthocyanidins were determined by butanol-HCl assay [23]. Their contents were expressed as mg leucoanthocyanidin/100 g of dry
plant material, assuming that the specific absorbance of leucoanthocyanidin was 460.All measurements were done in triplicate.


## 2.4. FRAP

Total antioxidant capacity was estimated according to the ferric reducing antioxidant
power (FRAP) assay [25]. FRAP reagent was prepared
by mixing acetate buffer (300 mM pH 3.6), TPTZ (2,4,6-tripyridyl-s-triazine)
reagent (10 mM in 40 mM HCl), and FeCl3·6H20
(20 mM) in ratio 3 : 1 : 1. Sample
(100 μL) was mixed with 3 mL of working FRAP
reagent and absorbance (593 nm) was measured at 4 minutes after vortexing. FRAP
value was calculated using formula (1)
FRAP
value
=
Δ
A
sample
(
0–4 min
)
Δ
A
standard
(0–4 min)
. 100 μM Fe2+ was used
as a standard; 1 FRAP unit = 100 μM Fe2+.Total antioxidant capacity was expressed in FRAP units.


## 2.5. Permanganate Reducing Antioxidant Capacity

The method is based on the redox reactions between the antioxidant sample and the
potassium permanganate in sulfuric acid media, leading to sample discoloration until no
colour is observed [26]. Variable amounts of
samples (V-mL), depending on the intensity of the antioxidant
activity, were introduced in a 30 mL quartz vat containing an oxidative mixture of
1.5 mL potassium permanganate 0.01 M; 3.5 mL sulfuric acid
2 M, and (20-V) mL distilled water. That moment was considered the
zero time. The spectrophotometer signal was then registered at 535 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 different 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:
(2)
A
50
=
t
(
standard
)
t
(
plant
sample
)
·
c
(
standard
)
m
(
plant
)
·
V
(
standard
)
V
(
plant
sample
)
·
V
(
extract
)
, where A
50 is antioxidant activity expressed, reflected 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), t
(
plant
sample
) is the time until the sample induces a decrease of the
permanganate concentration up to one half (min), t
(
standard
) is the time until the standard (ascorbic acid) induces a
decrease of the permanganate concentration up to one half (min), c
(
standard
) is standard (ascorbic acid) concentration (mmol/mL)
[0.01 mmol/mL], m
(
plant
) is weight (g) of the plant sample submitted to extraction
[4 g], V
(
plant
sample
) is volume of the plant extract submitted to the analysis
[0.2 mL], V
(
standard
) is volume of the standard submitted to the analysis
[1 mL], and V
(
extract
) is volume (mL) of the obtained extract [40 mL].


## 2.6. Radical Scavenging Determinations

DPPH
•-RSC assay was based on measurement of the loss of DPPH
(2,2-diphenyl-1-picrylhydrazyl) color after reaction with test compounds [27]. The DPPH
• radical is one of the few stable organic nitrogen radicals,
which bears a deep purple color. This assay is based on the measurement of the reducing
ability of antioxidants toward DPPH
•. The ability can be evaluated by measuring the decrease of
its absorbance. The widely used decoloration assay was first reported by Brand-Williams et
al. [28]. Appropriate volume of each extract was
mixed with 90 μM DPPH
• in methanol making up final volume of 3.0 mL. The
mixtures were shaken vigorously and were stored in dark for 30 min at room
temperature. The decrease of absorbance of the reaction mixtures regarding the control was
monitored spectrophotometrically at 515 nm.NO
•-RSC was evaluated by measuring the accumulation of nitrite
(formed by the reaction of NO with oxygen), according to the Griess reaction [29]. NO was generated by sodium nitroprusside in
buffered aqueous solution. Appropriate volume of each extract was mixed with fresh
prepared solution of sodium nitroprusside (0.5 mL, 0.01 M in
NaH2PO4-Na2HPO4 buffer, 0.067 M, pH
7.4) and NaH2PO4-Na2HPO4 buffer
(0.067 M, pH 7.4) making final volume of 1.0 mL. These mixtures were
illuminated at 3000 lx and 25°C for 10 min. After illumination, each
reaction mixture (1 mL) was mixed with Griess reagent (1 mL, 0.1%
N-(1-naphtyl)-ethylenediamine dihydrochloride (NEDA) in distilled water and 1%
sulfanilamide in 5% H3PO4). Reduction of nitrite by the
extracts was determined spectrophotometrically at 546 nm, by measuring the decrease
of absorbance of the reaction mixtures regarding the control (containing the same
chemicals, except for the sample).O
2
•
--RSC assay was based on the capacity of crude extracts to
inhibit the photochemical reduction of nitro blue tetrazolium (NBT) in the
riboflavin-light-NBT system [30]. Each 3 mL
of reaction mixture contained sodium phosphate buffer (50 mM, pH 7.8), methionine
(13 mM), riboflavin (2 μM), EDTA
(100 μM), NBT (75 μM), and
extract solution. Reaction systems were illuminated at 3000 lx and 25°C for
10 min. The increase in absorbance at 560 nm was monitored. The scavenging
capacity was expressed as reduction percentage of NBT absorbance induced by sample.For each sample three replicates were carried out. RSC was calculated by the following
equation:(3)
RSC
=
(
A
0
-
A
1
A
0
)
·
100
, where A
0 is control and A
1 is a sample solution absorbance. The concentration (in the
final reaction media in each method) that causes a decrease in the initial absorbance
(control) by 50% is defined as IC50. The IC50 values for all
RSC determinations were determined by polynomial fitting of the inhibition values (RSC)
using software ORIGIN 9.1. The antioxidant capacity of the extracts was expressed as
antiradical power (ARP) and it was defined as (4)
ARP
=
(
1
IC
50
)
·
100
.


## 2.7. Statistical and PCA Analysis

Statistical comparisons between samples were performed with Duncant-test for independent observations. Differences were considered
significant at P
<
0.05. The antioxidant test results were investigated with
multivariate analysis. The correlation matrix was calculated, giving the correlation
coefficients between each pair of variables, that is, the analytical parameters tested.
Each term of the matrix is a number ranging from −1 to +1: the + or − sign
indicates a positive or negative interdependence between variables (direction), and the
absolute value indicates the strength of the interdependence. Correlations between
different parameters were considered significant at r
>
0.95 (P
<
0.05). Autoscaling transformation of data for phenolic
parameters (TPC, TAC, FLC, and PAC) was done using STATISTICA 9.1 and presented by graphic
(Figure 2).


## 3. Results and Discussion

### 3.1. Soluble Proteins, Proline, Pigment and MDA Contents

Soluble protein content ranged from 52.45 (Q. robur twigs) to
427.0 mg/g (Q. robur leaves); Proline content ranged from 0.041
(Q. petraea acorns) to 0.083 μg/g
(Q. robur twigs); Chla content ranged from 0.016 (Q.
robur acorns) to 0.229 mg/g (Q. petraea leaves); Chlb
content ranged from 0.017 (Q. robur acorns) to 0.164 mg/g
(Q. petraea leaves); Carotenoid content ranged from 0.009 (Q.
robur acorns) to 0.175 mg/g (Q. robur leaves); MDA
content ranged from 2.023 (Q. petraea acorns) to 67.18 nmol/mg
protein (Q. robur twigs), (Tables 1 and 2). Significant positive
correlations were found between Chla and Chlb (r
=
0.9957), and also between carotenoids and both chlorophylls
(r
=
0.84). Positive correlation was found between proline content
and MDA content (r
=
0.4459).Table 1
Total phenol, tannin, flavonoid, proantocyanidine, chlorophyll a and b, carotenoid,
and proline contents in oak twigs, leaves, and acorns of two Serbian oak speciesQuercus robur L. and Quercus petraeaL.
Plant organ
Locality
TPC (mg catechin/g)
TAC (mg catechin/g)
FLC (mg rutin/100 g)
PAC (mg leukocyani-dine/100 g)
Chla (mg/g)
Chlb (mg/g)
Car (mg/g)
Pro. (μg/g)
Twigs
Q. robur
12.86a
9.094a
22.06a
148.9a
0.033a
0.027a
0.029a
0.083a
Q. petraea
7.44b
4.667b
19.12a
79.04b
0.035a
0.042b
0.018b
0.053b
Leaves
Q. robur
32.37c
22.47c
290.2b
1102c
0.115b
0.091c
0.175c
0.067c
Q. petraea
35.52d
26.24d
306.1c
757.6d
0.229c
0.164d
0.147d
0.075d
Acorns
Q. robur
16.25e
11.76e
26.17a
612.4e
0.016d
0.017e
0.009e
0.077ad
Q. petraea
15.90e
12.65e
73.81d
163.6a
0.026a
0.032a
0.023ab
0.041e
*Values with the same letter, in each colon, are not
significantly different according to Duncan test (P
<
0.05).
**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.Table 2
Protein content, DPPH, NO, andO
2
•
--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 petraeaL.
Plant organ
Locality
Prot. (mg/g)
DPPH-ARP ((1/IC50)·100)
NO-ARP ((1/IC50)·100)
O
2
•
--ARP ((1/IC50)·100)
PRAC (A50)
FRAP (FRAP units)
LP (nmol/mg prot.)
Twigs
Q. robur
52.45a
5.874a
0.358a
2.174a
0.016a
141.54a
67.18a
Q. petraea
103.9b
4.039b
0.241b
3.571b
0.010a
178.5a
28.08b
Leaves
Q. robur
427.0c
7.628c
0.531c
3.448b
0.304b
873.8b
11.01c
Q. petraea
159.6d
8.779d
0.400d
4.785c
0.235c
1252.3c
11.96c
Acorns
Q. robur
352.1e
9.066d
0.188e
5.359d
0.411d
370.0d
3.282d
Q. petraea
95.60b
6.734e
0.202e
4.098e
0.543e
614.6e
2.023d
*Values with the same letter, in each colon, are not
significantly different according to Duncan test (P
<
0.05).
**Prot.: proteins; ARP: antiradical power; ARP =
((1/IC50)·100); IC50: the concentration of an sample
at which 50% inhibition of free radical activity is observed; PRAC:
permanganate reducing antioxidant capacity; A50: antioxidant activity
reflected in time until the sample induces a decrease of the oxidizing agent
(potassium permanganate) up to one half, compared against a standard (ascorbic
acid); A50 = mmol ascorbate eq./g; FRAP: ferric reducing antioxidant
power; 1 FRAP unit = 100 μmol/dm3
Fe2+; LP: lipid peroxidation.Lipids are susceptible to oxidation and lipid peroxidation products, such as MDA
quantity, are potential biomarkers for oxidative stress statusin vivo.
Proteins are also the direct target for Reactive Oxygen Species (ROS) because of their
high concentrations. Their oxidation may result in deamination, decarboxylation, peptide
backbone cleavage, cross-linking, and many other chemical modifications leading eventually
to inactivation of enzyme activity and accumulation within cells and extracellular
environment [31]. Furthermore, antioxidant capacity
and the ratio between reduced forms to oxidized forms of molecules may be also used as
biomarkers of oxidative stress [32]. According to
our results, the highest accumulation of MDA was observed in twigs of both
Quercus species, where the soluble protein content was lowest due to
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 [33]. The
highest content of pigments, carotenoids, and chlorophylls was found in the leaves of
both, especially Q. petraea. Proline is an amino acid that acts as an
antioxidant—it reduces free radicals in plant cells [34, 35]. 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 [36]. The accumulation of proline in plant tissues is also a clear
marker for environmental stress, particularly in plants under drought conditions and may
also be part of the stress signal influencing adaptive responses [37]. Positive correlation between free proline content and LP intensity
confirms both antioxidant and defense natures of this amino acid.


### 3.2. Total Phenol, Tannin, Flavonoid, and Proanthocyanidin Contents

Total phenol content ranged from 7.44 (Q. petraea twigs) to
35.52 mg catechin/g (Q. petraea leaves); Tannin content
ranged from 4.667 (Q. petraea twigs) to 26.24 mg catechin/g
(Q. petraea leaves); Flavonoid content ranged from 19.12 (Q.
petraea twigs) to 306.1 mg rutin/100 g (Q.
petraea leaves); Proanthocyanidine content ranged from 79.04 (Q.
petraea twigs) to 1102 mg rutin/100 g (Q.
robur leaves) (Table 1). Significant
positive correlations were observed between all mentioned parameters. The highest positive
correlation was observed between TPC and TAC (r
=
0.9955). All phenolic parameters were significantly positively
correlated with carotenoid content. Total phenol, tannin, and flavonoid contents were also
significantly positively 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 3). All phenolic parameters are
negatively but not significantly correlated with LP parameter.Our results are in accordance with that obtained by Rakić et al. [38] 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. [16] evaluated nutritive values of browse leaves from five oak species,
namely, Quercus branti, Quercus coccifera,
Quercus cerris, Quercus libari, and Quercus
infectoriabased on their chemical composition. It was established that tannin
content ranged from 14.9 to 47.9 mg/g matter which is in accordance with our
results for Q. robur andQ. petraea. Q.
petraea andQ. robur are also rich source of flavonoids and
proanthocyanidins which are found in all plant organs, especially in leaves. Flavonoid
content found in leaves of Q. petraeaandQ. robur is in
range of that found in Quercus salicina Blume [39]. According to Salminen et al. (2004) 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 flavonoid glycosides
showed highly variable seasonal patterns. Young oak leaves were much richer in
hydrolysable tannins and flavonoid glycosides than old leaves, and vice
versa for proanthocyanidins [40].
Although in smaller quantities, twigs also contained all classes of polyphenols (Table
1). The obtained results have provided further
grounds for establishing Q. roburandQ. petraea leaves,
acorns, and twigs as a source for functional food preparation.


### 3.3. FRAP and PRAC Methods

FRAP values ranged from 141.54 (Q. robur twigs) to 1252.3 FRAP units
(Q. petraea leaves); PRAC values ranged from 0.010 (Q.
petraea twigs) to 0.543 mmol ascorbate eq./g (Q.
petraea acorns) (Table 2). FRAP was
significantly positively correlated with phenolic parameters (TPC, TAC, and FLC) and with
pigment (Chla, Chlb, and Car) contents. The highest positive correlation was observed
between FRAP and TAC (r
=
0.9587). PRAC value was positively correlated with
O
2
•
--ARP (r
=
0.6196) and negatively correlated with LP
(r
=
-
0.8113).Benzie and Strain [25] introduced FRAP as a simple
and automated test measuring the ferric reducing ability 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. According to Maqsood and
Benjakul [41] 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) [42]. According to FRAP method, antioxidant capacity of
oak samples, especially leaves, was relatively high comparing with other plants [43–45].
FRAP value was more or less positively correlated with all investigated parameters
excluding LP, where negative correlation was found (−0.5448).PRAC method was firstly introduced by Cacig and Szabo [26] as a simple spectrophotometric method for evaluation of antioxidant capacity
and later compared with other total antioxidant capacity methods [46]. Unlike the oak leaves where the highest FRAP was found, oak acorns
showed the highest PRAC. High positive correlations with O
2
•
--ARP indicate that the same structures are probably
responsible for superoxide scavenging and for reduction of permanganate in acidic media.
The highest negative correlations of both parameters with LP also indicate that scavengers
of superoxide and substances with high reducing potential against permanganate are mostly
responsible for suppression of LP.


### 3.4. Antiradical Power Determinations

DPPH-ARP ranged from 4.039 (Q. petraea twigs) to 9.066 (Q.
robur acorns); NO-ARP ranged from 0.188 (Q. robur acorns) to
0.531 (Q. robur leaves); O
2
•
--ARP ranged from 2.174 (Q. robur twigs) to
5.359 (Q. robur acorns) (Table 2). 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,
O
2
•
--ARP showed rather less positive correlations with phenolic
parameters, pigments, and FRAP but relatively high positive correlation with PRAC
(0.6196). Among investigated ARP parameters, only O
2
•
--ARP showed significant negative correlation with LP
(r
=
-
0.8360).All three ARP power determinations against DPPH,O
2
•
-, 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 [46]. DPPH and O
2
•
--ARP were correlated with each other
(r
=
0.6776), while correlations with NO-ARP were much lower.
Significant positive correlation of NO-ARP and carotenoids (r
=
0.8525) indicated significance of carotenoid antioxidants for NO
scavenging. Sindhu et al. [47] also established
that carotenoids lutein and zeaxanthin showed stronger antiradical potential against NO
than DPPH and O
2
•
- radicals. The highest DPPH- and O
2
•
--ARP showed acorns and the highest NO-ARP showed leaves
Q. robur. Rivas-Arreola et al. [48] also investigated antioxidant activity of oak (Q.
sideroxyla, Q. Eduardii, and Q. resinosa)
leaves infusions against free radicals and obtained similar results for radical scavenger
capacities. Oak twigs expressed lower DPPH, O
2
•
-, and NO ARP in comparison with leaves and acorns but also
relatively high antiradical potential.


### 3.5. PCA Analysis

The original data set was renormalized by an autoscaling transformation (data not shown)
and different parameters were analyzed by a multivariate approach. The loadings plot is
presented by Figure1 and the scores plot by Figure
2. The scree plot (data not shown) indicates that
the first two principal components account for 79.84% of the total variance (PC1 =
58.33 and PC2 = 21.51).Figure 1
Graph of loading plot of antioxidant markers for Serbian oak speciesQuercus
robur L. and Quercus petraeaL. Prot.: proteins; 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.
Parameters with close interdependence and correlation are close to each other and vice
versa.Figure 2
Graph of scores plot for Serbian oak speciesQuercus robur L. (QR)
and Quercus petraeaL. (QP). Quercus robur L. (QR)
andQuercus samples that are close to each other possess similar
antioxidant statuses.Figure 3
Line plot of multiple variables after autoscaling transformation of phenolic content
parameters (TPC, TAC, FLC, and PAC).Quercus robur L. (QR) and
Quercus petraeaL. (QP). TPC: Total phenolic content; TAC: Tannin
content; FLC: Flavonoid content; PAC: Proanthocyanidin content.As reported in the loadings plot (Figure1),
antiradical power parameters (DPPH
•, NO
•, and O
2
•
--ARP), FRAP, polyphenol (TPC, TAC, FLC, and PAC), protein,
and pigment contents are positioned closely due to the significant positive correlations
among them. PRAC and O
2
•
- ARP are partially isolated and located opposite to LP as a
parameter of oxidative stress. Positive correlations were also found among DPPH-ARP and
proteins and also between NO
•-ARP and proline content.PCA found three different clusters of oak samples based on antioxidant characteristics:
leaves, acorns, and twigs from investigated species are grouped (Figure2). Leaves differ from twigs and acorns predominantly
by Factor 1 (where the major contributors are polyphenols and FRAP). And the difference
between twigs and acorns is based on Factor 2 (where the major contributors are LP, PRAC,
and O
2
•
--ARP). Opposite direction of LP on one side and PRAC
O
2
•
--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 O
2
•
--radicals. Very close interdependence was observed between
leaves and twigs from both species, but twigs from two Quercus species
showed slightly higher differences. Line plot of multiple variables after autoscaling
transformation of polyphenolic content parameters (TPC, TAC, FLC, and PAC) was shown in
Figure 3. It is obvious that polyphenolic
parameters for leaves are separated and are greater than the same parameters for acorns
and twigs.


## 3.1. Soluble Proteins, Proline, Pigment and MDA Contents

Soluble protein content ranged from 52.45 (Q. robur twigs) to
427.0 mg/g (Q. robur leaves); Proline content ranged from 0.041
(Q. petraea acorns) to 0.083 μg/g
(Q. robur twigs); Chla content ranged from 0.016 (Q.
robur acorns) to 0.229 mg/g (Q. petraea leaves); Chlb
content ranged from 0.017 (Q. robur acorns) to 0.164 mg/g
(Q. petraea leaves); Carotenoid content ranged from 0.009 (Q.
robur acorns) to 0.175 mg/g (Q. robur leaves); MDA
content ranged from 2.023 (Q. petraea acorns) to 67.18 nmol/mg
protein (Q. robur twigs), (Tables 1 and 2). Significant positive
correlations were found between Chla and Chlb (r
=
0.9957), and also between carotenoids and both chlorophylls
(r
=
0.84). Positive correlation was found between proline content
and MDA content (r
=
0.4459).Table 1
Total phenol, tannin, flavonoid, proantocyanidine, chlorophyll a and b, carotenoid,
and proline contents in oak twigs, leaves, and acorns of two Serbian oak speciesQuercus robur L. and Quercus petraeaL.
Plant organ
Locality
TPC (mg catechin/g)
TAC (mg catechin/g)
FLC (mg rutin/100 g)
PAC (mg leukocyani-dine/100 g)
Chla (mg/g)
Chlb (mg/g)
Car (mg/g)
Pro. (μg/g)
Twigs
Q. robur
12.86a
9.094a
22.06a
148.9a
0.033a
0.027a
0.029a
0.083a
Q. petraea
7.44b
4.667b
19.12a
79.04b
0.035a
0.042b
0.018b
0.053b
Leaves
Q. robur
32.37c
22.47c
290.2b
1102c
0.115b
0.091c
0.175c
0.067c
Q. petraea
35.52d
26.24d
306.1c
757.6d
0.229c
0.164d
0.147d
0.075d
Acorns
Q. robur
16.25e
11.76e
26.17a
612.4e
0.016d
0.017e
0.009e
0.077ad
Q. petraea
15.90e
12.65e
73.81d
163.6a
0.026a
0.032a
0.023ab
0.041e
*Values with the same letter, in each colon, are not
significantly different according to Duncan test (P
<
0.05).
**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.Table 2
Protein content, DPPH, NO, andO
2
•
--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 petraeaL.
Plant organ
Locality
Prot. (mg/g)
DPPH-ARP ((1/IC50)·100)
NO-ARP ((1/IC50)·100)
O
2
•
--ARP ((1/IC50)·100)
PRAC (A50)
FRAP (FRAP units)
LP (nmol/mg prot.)
Twigs
Q. robur
52.45a
5.874a
0.358a
2.174a
0.016a
141.54a
67.18a
Q. petraea
103.9b
4.039b
0.241b
3.571b
0.010a
178.5a
28.08b
Leaves
Q. robur
427.0c
7.628c
0.531c
3.448b
0.304b
873.8b
11.01c
Q. petraea
159.6d
8.779d
0.400d
4.785c
0.235c
1252.3c
11.96c
Acorns
Q. robur
352.1e
9.066d
0.188e
5.359d
0.411d
370.0d
3.282d
Q. petraea
95.60b
6.734e
0.202e
4.098e
0.543e
614.6e
2.023d
*Values with the same letter, in each colon, are not
significantly different according to Duncan test (P
<
0.05).
**Prot.: proteins; ARP: antiradical power; ARP =
((1/IC50)·100); IC50: the concentration of an sample
at which 50% inhibition of free radical activity is observed; PRAC:
permanganate reducing antioxidant capacity; A50: antioxidant activity
reflected in time until the sample induces a decrease of the oxidizing agent
(potassium permanganate) up to one half, compared against a standard (ascorbic
acid); A50 = mmol ascorbate eq./g; FRAP: ferric reducing antioxidant
power; 1 FRAP unit = 100 μmol/dm3
Fe2+; LP: lipid peroxidation.Lipids are susceptible to oxidation and lipid peroxidation products, such as MDA
quantity, are potential biomarkers for oxidative stress statusin vivo.
Proteins are also the direct target for Reactive Oxygen Species (ROS) because of their
high concentrations. Their oxidation may result in deamination, decarboxylation, peptide
backbone cleavage, cross-linking, and many other chemical modifications leading eventually
to inactivation of enzyme activity and accumulation within cells and extracellular
environment [31]. Furthermore, antioxidant capacity
and the ratio between reduced forms to oxidized forms of molecules may be also used as
biomarkers of oxidative stress [32]. According to
our results, the highest accumulation of MDA was observed in twigs of both
Quercus species, where the soluble protein content was lowest due to
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 [33]. The
highest content of pigments, carotenoids, and chlorophylls was found in the leaves of
both, especially Q. petraea. Proline is an amino acid that acts as an
antioxidant—it reduces free radicals in plant cells [34, 35]. 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 [36]. The accumulation of proline in plant tissues is also a clear
marker for environmental stress, particularly in plants under drought conditions and may
also be part of the stress signal influencing adaptive responses [37]. Positive correlation between free proline content and LP intensity
confirms both antioxidant and defense natures of this amino acid.


## 3.2. Total Phenol, Tannin, Flavonoid, and Proanthocyanidin Contents

Total phenol content ranged from 7.44 (Q. petraea twigs) to
35.52 mg catechin/g (Q. petraea leaves); Tannin content
ranged from 4.667 (Q. petraea twigs) to 26.24 mg catechin/g
(Q. petraea leaves); Flavonoid content ranged from 19.12 (Q.
petraea twigs) to 306.1 mg rutin/100 g (Q.
petraea leaves); Proanthocyanidine content ranged from 79.04 (Q.
petraea twigs) to 1102 mg rutin/100 g (Q.
robur leaves) (Table 1). Significant
positive correlations were observed between all mentioned parameters. The highest positive
correlation was observed between TPC and TAC (r
=
0.9955). All phenolic parameters were significantly positively
correlated with carotenoid content. Total phenol, tannin, and flavonoid contents were also
significantly positively 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 3). All phenolic parameters are
negatively but not significantly correlated with LP parameter.Our results are in accordance with that obtained by Rakić et al. [38] 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. [16] evaluated nutritive values of browse leaves from five oak species,
namely, Quercus branti, Quercus coccifera,
Quercus cerris, Quercus libari, and Quercus
infectoriabased on their chemical composition. It was established that tannin
content ranged from 14.9 to 47.9 mg/g matter which is in accordance with our
results for Q. robur andQ. petraea. Q.
petraea andQ. robur are also rich source of flavonoids and
proanthocyanidins which are found in all plant organs, especially in leaves. Flavonoid
content found in leaves of Q. petraeaandQ. robur is in
range of that found in Quercus salicina Blume [39]. According to Salminen et al. (2004) 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 flavonoid glycosides
showed highly variable seasonal patterns. Young oak leaves were much richer in
hydrolysable tannins and flavonoid glycosides than old leaves, and vice
versa for proanthocyanidins [40].
Although in smaller quantities, twigs also contained all classes of polyphenols (Table
1). The obtained results have provided further
grounds for establishing Q. roburandQ. petraea leaves,
acorns, and twigs as a source for functional food preparation.


## 3.3. FRAP and PRAC Methods

FRAP values ranged from 141.54 (Q. robur twigs) to 1252.3 FRAP units
(Q. petraea leaves); PRAC values ranged from 0.010 (Q.
petraea twigs) to 0.543 mmol ascorbate eq./g (Q.
petraea acorns) (Table 2). FRAP was
significantly positively correlated with phenolic parameters (TPC, TAC, and FLC) and with
pigment (Chla, Chlb, and Car) contents. The highest positive correlation was observed
between FRAP and TAC (r
=
0.9587). PRAC value was positively correlated with
O
2
•
--ARP (r
=
0.6196) and negatively correlated with LP
(r
=
-
0.8113).Benzie and Strain [25] introduced FRAP as a simple
and automated test measuring the ferric reducing ability 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. According to Maqsood and
Benjakul [41] 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) [42]. According to FRAP method, antioxidant capacity of
oak samples, especially leaves, was relatively high comparing with other plants [43–45].
FRAP value was more or less positively correlated with all investigated parameters
excluding LP, where negative correlation was found (−0.5448).PRAC method was firstly introduced by Cacig and Szabo [26] as a simple spectrophotometric method for evaluation of antioxidant capacity
and later compared with other total antioxidant capacity methods [46]. Unlike the oak leaves where the highest FRAP was found, oak acorns
showed the highest PRAC. High positive correlations with O
2
•
--ARP indicate that the same structures are probably
responsible for superoxide scavenging and for reduction of permanganate in acidic media.
The highest negative correlations of both parameters with LP also indicate that scavengers
of superoxide and substances with high reducing potential against permanganate are mostly
responsible for suppression of LP.


## 3.4. Antiradical Power Determinations

DPPH-ARP ranged from 4.039 (Q. petraea twigs) to 9.066 (Q.
robur acorns); NO-ARP ranged from 0.188 (Q. robur acorns) to
0.531 (Q. robur leaves); O
2
•
--ARP ranged from 2.174 (Q. robur twigs) to
5.359 (Q. robur acorns) (Table 2). 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,
O
2
•
--ARP showed rather less positive correlations with phenolic
parameters, pigments, and FRAP but relatively high positive correlation with PRAC
(0.6196). Among investigated ARP parameters, only O
2
•
--ARP showed significant negative correlation with LP
(r
=
-
0.8360).All three ARP power determinations against DPPH,O
2
•
-, 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 [46]. DPPH and O
2
•
--ARP were correlated with each other
(r
=
0.6776), while correlations with NO-ARP were much lower.
Significant positive correlation of NO-ARP and carotenoids (r
=
0.8525) indicated significance of carotenoid antioxidants for NO
scavenging. Sindhu et al. [47] also established
that carotenoids lutein and zeaxanthin showed stronger antiradical potential against NO
than DPPH and O
2
•
- radicals. The highest DPPH- and O
2
•
--ARP showed acorns and the highest NO-ARP showed leaves
Q. robur. Rivas-Arreola et al. [48] also investigated antioxidant activity of oak (Q.
sideroxyla, Q. Eduardii, and Q. resinosa)
leaves infusions against free radicals and obtained similar results for radical scavenger
capacities. Oak twigs expressed lower DPPH, O
2
•
-, and NO ARP in comparison with leaves and acorns but also
relatively high antiradical potential.


## 3.5. PCA Analysis

The original data set was renormalized by an autoscaling transformation (data not shown)
and different parameters were analyzed by a multivariate approach. The loadings plot is
presented by Figure1 and the scores plot by Figure
2. The scree plot (data not shown) indicates that
the first two principal components account for 79.84% of the total variance (PC1 =
58.33 and PC2 = 21.51).Figure 1
Graph of loading plot of antioxidant markers for Serbian oak speciesQuercus
robur L. and Quercus petraeaL. Prot.: proteins; 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.
Parameters with close interdependence and correlation are close to each other and vice
versa.Figure 2
Graph of scores plot for Serbian oak speciesQuercus robur L. (QR)
and Quercus petraeaL. (QP). Quercus robur L. (QR)
andQuercus samples that are close to each other possess similar
antioxidant statuses.Figure 3
Line plot of multiple variables after autoscaling transformation of phenolic content
parameters (TPC, TAC, FLC, and PAC).Quercus robur L. (QR) and
Quercus petraeaL. (QP). TPC: Total phenolic content; TAC: Tannin
content; FLC: Flavonoid content; PAC: Proanthocyanidin content.As reported in the loadings plot (Figure1),
antiradical power parameters (DPPH
•, NO
•, and O
2
•
--ARP), FRAP, polyphenol (TPC, TAC, FLC, and PAC), protein,
and pigment contents are positioned closely due to the significant positive correlations
among them. PRAC and O
2
•
- ARP are partially isolated and located opposite to LP as a
parameter of oxidative stress. Positive correlations were also found among DPPH-ARP and
proteins and also between NO
•-ARP and proline content.PCA found three different clusters of oak samples based on antioxidant characteristics:
leaves, acorns, and twigs from investigated species are grouped (Figure2). Leaves differ from twigs and acorns predominantly
by Factor 1 (where the major contributors are polyphenols and FRAP). And the difference
between twigs and acorns is based on Factor 2 (where the major contributors are LP, PRAC,
and O
2
•
--ARP). Opposite direction of LP on one side and PRAC
O
2
•
--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 O
2
•
--radicals. Very close interdependence was observed between
leaves and twigs from both species, but twigs from two Quercus species
showed slightly higher differences. Line plot of multiple variables after autoscaling
transformation of polyphenolic content parameters (TPC, TAC, FLC, and PAC) was shown in
Figure 3. It is obvious that polyphenolic
parameters for leaves are separated and are greater than the same parameters for acorns
and twigs.


## 4. Conclusion

This 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, flavonoids, proanthocyanidins, and
pigment contents. 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 significant source of phenolic compounds, oak acorns showed also high antioxidant
potential and the lowest LP intensity. Antioxidant capacity values including
DPPH
•, NO
• and O
2
•
--ARP, and FRAP showed high positive correlations among
themselves and also with polyphenol parameters (TPC, TAC, FLC, and PAC), protein, and
pigment contents. Permanganate PRAC and O
2
•
--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.



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*Source: 134656-2013-12-25.xml*