Arch. Biol. Sci., Belgrade, 67(4), 1323-1330, 2015 DOI:10.

2298/ABS150414109V

in vitro antioxidant activity of dewberry (Rubus caesius l. var.

aquticus WEIhe. & NEES.) leaf extracts

Ivona Velikovi1,*, Slavica Gruji1, Ana Dami1, Zoran Krivoej2 and Petar D. Marin1

1
University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden Jevremovac, Belgrade, Serbia
2
University of Pritina, Department of Biology, Faculty of Natural Sciences, Kosovska Mitrovica, Serbia

*Corresponding author: ivona@bio.bg.ac.rs

Abstract: In this study, the antioxidant properties and total phenolic and flavonoid contents of Rubus caesius L. var. aquti-
cus Weihe & Nees leaf extracts were examined. The radical scavenging capacity of four leaf extracts (methanol, ethanol,
acetone and aqueous) was estimated against 2,2-diphenyl-1-picrylhydrazil (DPPH) and 2,2-azino-bis(3-ethylbenzthiazoline-
6-sulfonic acid) (ABTS) assays. Total antioxidant capacity was tested by ferric reducing ability of plasma (FRAP) and total
reducing capacity (TRC) procedures. Total contents of phenols and flavonoids were also determined. Total phenol content
in the extracts was determined using Folin-Ciocalteu reagent and amounts ranged between 263.74 for ethanol to 366.27
mg gallic acid (GA)/g for acetone extracts. The amounts of flavonoids varied from 10.73 mg quercetin equivalent (QE)/g
for aqueous to 75.83 mg QE/g for acetone extract. The highest antioxidant activity against DPPH and ABTS+ radicals
was that of the acetone extract. Values for FRAP varied between 3.00 mol Fe+2/mg for the ethanol extract and 5.31 mol
Fe+2/mg for the methanol extract. The results obtained for total reducing capacity indicate that the acetone extract has the
highest antioxidant potential.

Key words: Rubus caesius var. aquticus; extracts; antioxidant activity; phenols; flavonoids

Received: April 14, 2015; Revised: May 6, 2015; Accepted: May 21, 2015

INTRODUCTION It is a wild-growing plant and widely cultivated as a

Several economically important species come from
the Rosaceae family including many edible fruits, or-
namental trees and shrubs. In addition, members of
Rosaceae are also used in traditional medicine be-
cause of their antimicrobial, anticonvulsant, muscle-
relaxant and radical scavenging activity (Martini et
al., 2009).
The genus Rubus, which includes about 750 spe-
cies, is widespread on all continents except Antarctica
(Alice and Campbell, 1999). It comprises perennial
herbs or shrubs, usually with spiny stems and pin-
nate, digitate or pedate leaves and solitary flowers
or racemose or paniculate inflorescences. Fruits are
often a coherent head of 1-seeded drupelets (Heslop-
Harrison, 1968). Rubus caesius L. is shrub with more
or less spiny stems, rarely with glandular trichomes.
crop. R. caesius can be found in forests, as well as in
thickets, on wastelands or roadsides. In Serbia, it is
widespread on hills and mountains (Tati, 1972). R.
caesius var. aquticus can be found near aquatic habi-
tats (Nyarady, 1956) and, as far as we know, this is the
first time it was found in the Republic of Serbia.
Previous phytochemical studies proved the pres-
ence of different structural secondary metabolites:
proanthocyanidins (Wada et al., 2011), anthocyanins
(Kubota et al., 2012), phenolics (Fazio et al., 2013;
Jabloska-Ry et al., 2009; Valcheva-Kuzmanova et al.,
2006; Lee et al., 2012; Fu et al., 2015), flavonoids and
tannins (Gudej and Tomczyk, 2004).
Dewberries are consumed worldwide, not only
because of their taste, but also for their health ben-
efits (Lee, 2012). Dewberry leaves have been used in

1323
1324 Velikovi et al.
traditional medicine due to their anti-inflammatory
(Shin et al., 2014), antiviral and antimicrobial effects
(Panizzi et al., 2002.) antiproliferative activity against
cancer cells (Martini et al., 2009), and antitumor and
wound-healing properties (George et al., 2014; Baeka
et al., 2013).
Free radicals are implicated in many diseases in-
cluding cancer, cardiovascular diseases, neural disor-
ders, Alzheimers disease, mild cognitive impairment,
Parkinsons disease, alcohol-induced liver disease, ul-
cerative colitis, aging and atherosclerosis (Alam et al.,
2013).
Rubus spp. are a good source of antioxidants, con-
taining an appreciable level of phenolic compounds
(Lee et al., 2012). Phenols are a very diverse and large
group of compounds, shown to have health benefits
for humans (Halliwell, 2006). The most studied group
of phenols are flavonoids, which exhibit very impor-
tant biological activities: anti-inflammatory, anti-aller-
gic, antiviral and anticancer. Several research groups
have considered the relation between total phenol and
flavonoid content and antioxidant activity. However,
some of them found positive correlation and some
found no correlation between phenolic content and
antioxidant properties (Conforti et al., 2011).
The antioxidant capacity of different Rubus spe-
cies has been observed (Deighton et al., 2000; Kh-
knen et al., 2001; Valcheva-Kuzmanova et al., 2006;
Katalinic et al., 2006; Jabloska-Ry et al., 2009;
Buricova et al., 2011; Kolbas et Reshetnikov, 2011;
Conforti et al., 2011; Fu et al., 2015). The aim of this
work was to determine the total phenol and flavonoid
content and examine the antioxidant properties of R.
caesius var. aquticus, using four in vitro procedures
(DPPH, ABTS, FRAP and TRC), which, to the best of
our knowledge, has not been done before.
MATERIALS AND METHODS
Plant material
Dewberry (R. caesius var. aquticus) leaves were col-
lected in summer 2012 near Zajear at the confluence
of the Radovanjska and Crni Timok rivers (latitude:
N 43.846394; longitude: E 21.881641). The voucher
specimen (No. 17086) has been deposited in the Her-
barium of the Institute of Botany and Botanical Gar-
den Jevremovac, Faculty of Biology, University of
Belgrade (BEOU), Serbia.
Chemicals and reagents
All chemicals and reagents were of analytical grade.
Organic solvents (methanol, ethanol, acetone), HCl
(concentrated hydrochloric acid), CH3COOH (gla-
cial acetic acid) and CCl 3COOH (trichloroacetic
acid) were purchased from Zorka Chemicals (abac,
Serbia). Gallic acid, 2,2-dyphenyl-1-picrylhydrazyl
(DPPH), iron (III) chloride (FeCl36H2O), iron (II)
sulfate heptahydrate (FeSO47H2O), sodium acetate
(CH 3COONa3H 2O) were obtained from Sigma
Chemicals Co., St. Louis, MO, USA. Folin-Cio-
calteu phenol reagent was purchased from Merck,
Darmstadt, Germany. Sodium carbonate anhydrous
(Na2CO3), potassium acetate (C2H3KO2), potassium
peroxidisulphate (K2O8S2) and L(+)-ascorbic acid (Vi-
tamin C) were purchased from AnalaR Normapur,
VWR, Geldenaaksebaan, Leuven, Belgium. Alu-
minum nitrate nonahydrate [Al(NO3)39H2O] and
2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) were pur-
chased from Fluka Chemie AG, Buchs, Switzerland.
[2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic]
acid (ABTS) and quercetin hydrate were obtained
from TCI Europe NV, Boerenveldsweg, Belgium.
Extract preparation
Plant material was dried for two weeks in shade in a
well-aerated room. Dry leaves were ground to powder
and extracted with four different solvents: methanol,
ethanol, acetone and distilled water. Five g of pow-
dered leaves was extracted with 50mL of solvent in
an ultrasonic bath for 1 h and again after 24 h of in-
cubation in the dark. After sonication and filtration
through Whatman No 1 filter paper, the solvent was
removed by vacuum evaporator. Prepared samples
were stored in the fridge at +4C until use.
 Antioxidant activity of dewberry leaf extracts
Determination of total phenolic content (TPC)
The total phenolic content (TPC) was determined ac-
cording to the procedure described by Singleton and
Rossi (1965).
The total phenolic content was measured at 740
nm by JENWAY 6306 UV/vis spectrophotometer
and calculated from the calibration curve of gallic
acid (GA) (10-100 mg/L) and expressed as mg of gal-
lic acid equivalents (GAE) per g of dry extract. The
results were taken from triplicate measurements.
Determination of total flavonoid content (TFC)
The quantitative analysis of flavonoids present in R.
caesius var. aquticus leaf extracts was estimated as
previously reported by Park et al. (1997). The flavo-
noid content in leaf extract was estimated spectropho-
tometrically at 415 nm and expressed as milligrams of
quercetin equivalents per gram of dry extract.
DPPH radical scavenging capacity
DPPH radical scavenging capacity was assessed ac-
cording to the method described by Blois (1958). This
method is designed to evaluate the decrease of 2,2-di-
phenyl-1-picryllhydrazyl (DPPH) free radicals in the
presence of plant extracts with antioxidant properties.
Inhibition of DPPH free radicals was calculated
using Eq. (1):
Percentage of inhibition (%) (1)
where AC is the absorbance of the control solution
containing all reagents except extract and AS is the
absorbance in the presence of the tested extract solu-
tion, respectively. IC50 values (g/mL) were calculated
by regression equation obtained from the relation be-
tween the concentration of samples and percentage of
inhibition of DPPH radicals.
ABTS+ radical scavenging capacity
The ABTS radical scavenging capacity was estimated
as suggested by Miller and Rice-Evans (1997), with
1325
slight modifications, and results were expressed as
IC50 values (g/mL).
The ferric reducing antioxidant power (FRAP) assay
The ferric reducing antioxidant power (FRAP) was
determined according to Benzie and Strain (1996)
with minor modifications. The method is based on
the reduction of a ferric 2,4,6-tripyridyl-s-triazine
complex (Fe3+-TPTZ) by antioxidants to the ferrous
form (Fe2+-TPTZ), which leads to absorbance in-
crease and which was measured by PERKIN ELMER
LAMBDA BIO UV/VIS spectrophotometer at 595 nm
against solvent. Aqueous solutions of FeSO47H2O
were used for the calibration curve and results are
expressed as mol Fe2+ equivalents per mg of dry ex-
tract. All measurements were taken in triplicate.
The total reducing capacity (TRC)
The total reducing capacity (TRC) was estimated ac-
cording to the procedure described by Oyaizu (1986),
and results are calculated with the following equation
Eq (2):
Reducing power (%) = % (2)
where AS is the absorbance of the samples and AC is
the absorbance of the control (containing distilled
water instead of extract).
Results and Discussion
The yield of extraction
The yields of dried extract of Rubus caesius var.
aquticus leaves are presented in Table 1. The highest
value was obtained for aqueous extract and methanol,
Table 1. Extraction yields (Y) of blackberry leaves in different solvent.
Solvent Y (%)
methanol 7.42
ethanol 2.15
acetone 0.58
water 11.66
1326
then ethanol and the lowest yields for acetone. The
solvent, temperature and sonication time affected ex-
traction yields. A positive influence of temperature
and sonication time on extraction yield was proved.
The contribution to yield was higher for temperature
than for sonication time as shown previously (Iva-
novic et al., 2014). In this study, all extracts were pre-
pared under the same conditions.
Total phenol and flavonoid content
The total phenol and flavonoid content in the tested
samples is presented in Table 2. In this study, it was
confirmed that higher phenolic content contributes
to lower IC50 values, which means higher antioxidant
activity.
The acetone extract was the most enriched with phe-
nols (366.27 mg GAE/g of dry extract), followed by
aqueous (309.99 mg GAE/g of dry extract), which
could explain the highest antioxidant activity of these
extracts. The largest amount of flavonoids was also
found in the acetone (75.83 mg QE/g of dry extract)
and the lowest in the aqueous extract (10.73 mg QE/g
of dry extract).
These results suggest that the high antioxidant ac-
tivity of the acetone extract is due to some phenolic
compounds, including flavonoids. On the other hand,
the high antioxidant activity of the aqueous extract
is probably due to some phenolic compounds, but
not flavonoids. Literature data indicate that the high
antioxidant activity found in Rubus species is relat-
ed to phenol, anthocyanin and tannin compounds.
According to Kolbas and Reshetnikov (2011), total
phenol content in aqueous and ethanol extracts of
Table 2. Total phenol and total flavonoid contents and antioxidant properties of R. caesius var. aquticus extracts.
TPC14 TFC24
(mg GAE/g dw) (mg QE/g dw)
methanol 280.268.01 44.771.03
ethanol 263.743.90 37.940.42
acetone 366.270.50 75.832.01
water 309.991.42 10.730.11
1
Total phenol content expressed as mg of gallic acid equivalents (GAE) per one gram of dry weight (dw); 2total flavonoid content expressed as mg of
quercetin equivalents (QE) per one gram of dry weight (dw); 3data were expressed as mol Fe+2 equivalents per one gram of dry weight (dw); 4data were
expressed as mean standard deviation.
Velikovi et al.
R. fruticosus, R. caesius, R. nessensis and R. idaeus,
estimated by Folin Ciocalteu assay, highly correlated
with antioxidant activity. In the same work, a high
correlation was confirmed between total anthocyanin
content and ABTS values, as well. Moreover, the con-
tribution of phenols and flavonoids to the antioxidant
activity of R. hirsutus was greater than the contribu-
tion of anthocyanins and vitamin C.
Fu et al. (2015) showed that the amounts of phenols
and flavonoids in berries depend on their size. Small
fruits are rich in phenolic and flavonoid compounds,
medium-sized fruits in ascorbic acid, while large
fruits are rich in anthocyanins. Diverse berry genera
differ significantly in total phenol content.
Khknen et al. (2001) investigated the phytochemi-
cal content and antioxidant activity of selected Rubus
species and correlations between them. Comparative
analyses of R. chamaemorus and R. idaeus showed
that flavonols, hydroxycinnamic and hydroxyben-
zoic acids are present in R. chamaemorus in greater
amounts than in R. idaeus, while the opposite is the
case with phenols, anthocyanins and ellagitannins.
Despite, R. chamaemorus revealed higher antioxidant
activity than R. idaeus.
A positive correlation was found between total an-
thocyanin content and total phenol content (Ivanovic
et al., 2014). According to these authors, the active
compounds in blackberry extracts were glycosides of
cyanidin (glucoside, xyloside, rutinoside). The pres-
ence of 3-glucoside, 3-rutinoside, 3-xyloside and 3-O-
-(6-malonyl-glucoside) of cyanidin and 5 cyanidin
3-O--(6-(3-hydroxyl-3-methylglutaroyl)-glucoside
was confirmed in blackberry extracts (Jordheim et
DPPH4 ABTS+4 FRAP34 TRC4
IC50(g/mL) IC50(g/mL) (mol Fe+2/mg dw) RP50 (mg/mL)
18.940.45 11.920.56 5.310.18 /
16.880.31 9.321.06 3.000.33 2.630.005
11.320.01 6.090.25 3.231.21 0.630.008
15.920.15 6.510.05 3.800.01 4.150.71
 Antioxidant activity of dewberry leaf extracts
al., 2011). The main compound in blackberry extracts
tested by Elisia et al. (2007) was cyanidin-glucoside,
which represented 87.5% of total anthocyanins present.
Acosta-Montoya et al. (2010) observed changes in the
phytochemical composition and antioxidant potential
of R. adenotrichus fruits during ripening. According
to these researchers, the main phenolic compounds
were ellagitannins (lambertianin C and sanguiin
H-6) and anthocyanins (cyaniding-3-glucoside and
cyaniding-3-(6-malonyl) glucoside). During ripen-
ing ellagitannin content significantly decreases, but
remains the highest, while anthocyanin content sig-
nificantly increases. The relatively high antioxidant
activity measured by hydroxyl radical antioxidant
capacity (HORAC) assay is explained by synergistic
effects between phenolic components other than an-
thocyanins, probably ellagitannins.
Antioxidant activity
In this study we examined the in vitro antioxidant ac-
tivity of four leaf extracts of R. caesius var. aquticus.
The different testing methods provided different ex-
perimental environments (pH of the mixture, radicals
produced in the mixture, solvent of the system, etc.),
which resulted in different ranking orders of IC 50
values. This is the reason why antioxidant properties
should be tested by more than one procedure to get a
more realistic and complete picture of the antioxidant
profile of tested extracts (Prior et al., 2005).
The antioxidant properties of R. caesius var.
aquticus were evaluated according to four differ-
ent procedures and are presented in Table 2. For the
DPPH assay, the positive correlation between all test-
ed samples and increase of concentration is shown in
Fig. 1. The lowest IC50 values were obtained for ac-
etone and aqueous extracts 11.32 g/mL and 15.92
g/mL, respectively, followed by ethanol, and the
highest value was for methanol (18.94 g/mL). The
same order in IC50 values was obtained by the ABTS
assay. The lowest and approximately equal IC50 values
were for acetone 6.09 g/mL and aqueous 6.51 g/mL
extracts, then ethanol 9.32 g/mL, and the highest
was for methanol 11.92 g/mL.
1327
Fig. 1. Correlation between the level of DPPH and R. caesius var.
aquticus extract concentrations for different solvents. Linear
correlation coefficients are for methanol, ethanol, acetone and
aqueous extracts: R2 = 0.9777, R2 = 0.9895, R2 = 0.9977 and R2 =
0.9987, respectively.
FRAP and TRC assays are based on reducing re-
actions where iron is reduced by antioxidant species
from ferric (Fe+3) to ferrous (Fe+2) ion. These reac-
tions can be monitored by the increase in the absor-
bance, which is accompanied by a color change from
yellow to blue. The higher is the absorbance, the high-
er is the antioxidant activity of the tested samples.
The total reducing ability obtained by FRAP assay
is expressed in mol Fe+2 equivalents per mg of dry
extract and obtained values were in range from 3.00
mol Fe+2/mg dw for the ethanol extract to 5.31 mol
Fe+2/mg dw for the methanol. Total reducing capac-
ity according to Oyaizu et al. (1986) is expressed in
the concentration (mg/mL) of a sample that shows
50% of total reducing power. The lowest value was
obtained for the acetone extract 0.63 mg/mL and the
highest for the aqueous extract 4.15 mg/mL. The
results obtained for TRC again point out the highest
antioxidant activity of acetone extract.
In this work, the contribution of phenols and
flavonoids to the antioxidant properties of the tested
extracts was considered, and presented by Pearsons
correlation coefficient in Table 3. It was shown that
total phenolic content highly correlated with DPPH
and ABTS values (r = 0.8998 and 0.7843, respec-
tively), while total flavonoids highly correlated with
TRC values (r = 0.9999). A moderate correlation was
found between total phenol content and TRC values
and between total flavonoid content and DPPH val-
1328
Table 3. Correlation between antioxidant activity (according to four different assays) and total phenol and flavonoid contents in R. caesius
var. aquticus extract expressed through Pearsons coefficient of regression.
Rubus caesius DPPH
Var. aquticus IC50(g/mL)
Total phenol content
0.8998
(mg GAE/g dw)
Total flavonoid content
0.5645
(mg QE/g dw)
ues. Phenols, as well as flavonoids, weakly correlated
with FRAP values.
Ivanovic et al. (2014) found a moderate corre-
lation between DPPH and FRAP assays (r=0.582),
and a weak correlation between FRAP values and
cyanidin content (r=0.171) for blackberry extracts.
In the same study, a high correlation was established
between FRAP values and total tannin content. In ad-
dition, the ability of tannins to reduce ferric (Fe+3)
to ferrous (Fe+2) ion was confirmed by Lopes (1999).
This could be an explanation for the discordance be-
tween FRAP values and values obtained by another
antioxidant procedures.
Several papers describe the antioxidant properties
of other Rubus species, usually raspberry R. idaeus
L. and blackberry R. ulmifolius. The high antioxidant
activity of R. ulmifolius leaves shown in these works
was ascribed to the activities of caffeic acid, ferulic
acid, caffeic quinic esters, quercetin-3-O-glucuronide,
kaemferol-3-O-glucuronide and ellagic acid (Barros
et al., 2010). Bobinaite et al. (2012) tested the antioxi-
dant properties of 17 raspberry cultivars and showed
a strong correlation between total phenolic content
and antioxidant activity, as well as between ellagic
acid and antioxidant activity. According to the same
study, the contribution of total anthocyanin content
to the antioxidant activity was very low, which was
also confirmed by Weber et al. (2008).
Notable antioxidant activity of R. adenotrichus
fruits was also ascribed to ellagitannins and not
anthocyanins (Acosta-Montoya et al., 2010). On
the other hand, Reyes-Carmona et al. (2005) found
strong correlation between total anthocyanin content
and antioxidant activity. Rios de Souza et al. (2014)
investigated the bioactive compounds, antioxidant
Velikovi et al.
ABTS FRAP TRC
IC50(g/mL) EQ (mol Fe+2/mg dw) RP50 (mg/mL)
0.7843 0.2834 0.6151
0.0566 0.1446 0.9999
properties and chemical composition of Brazilian
berries, and found a high positive correlation be-
tween antioxidant capacity and total phenolic con-
tent. Moreover, strong positive correlation was shown
between total antioxidant capacity (TEAC) and total
monomeric anthocyanin content, and between DPPH
and total flavonoid content. In the same study, Rubus
spp. revealed the highest antioxidant activity among
tested species, which was attributed to the highest to-
tal phenol content.
CONCLUSIONS
Phenolic compounds are major contributors to the
antioxidant properties of dewberry (R. caesius var.
aquticus) leaves. In future work, determination of
the chemical composition of secondary metabolites in
dewberry should clarify and explain which of the bio-
active compounds provides the highest contribution
to antioxidant activity. The in vivo antioxidant activity
should also be tested, because dewberry fruits (fresh
or processed as juices, jams and jellies) and leaves (as
teas) are consumed worldwide as part of a healthy
diet; they could be used in pharmaceutical, food and
cosmetic industries.
Acknowledgments: This research was supported by the
Ministry of Education, Science and Technological Develop-
ment (Grant. No. 173029)
Authors contributions: Ivona Velikovi, the main author,
contributed with original data and the design of the re-
search, conceived the project, organized and analyzed data
and wrote the manuscript. Slavica Gruji and Ana Dami
contributed by designing research, data analyses and re-
viewed several drafts of the manuscript. Petar D. Marin was
the main supervisor of the research project and reviewed
 Antioxidant activity of dewberry leaf extracts
several drafts of the manuscript. Professor Krivoej deter-
mined plant material. All authors read and approved the
final manuscript.
Conflict of interest disclosure: We certify that there is no
conflict of interest with any financial organization regard-
ing the material discussed in the manuscript.
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