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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


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

1323
1324 Velikovi et al.

traditional medicine due to their anti-inflammatory of the Radovanjska and Crni Timok rivers (latitude:
(Shin et al., 2014), antiviral and antimicrobial effects N 43.846394; longitude: E 21.881641). The voucher
(Panizzi et al., 2002.) antiproliferative activity against specimen (No. 17086) has been deposited in the Her-
cancer cells (Martini et al., 2009), and antitumor and barium of the Institute of Botany and Botanical Gar-
wound-healing properties (George et al., 2014; Baeka den Jevremovac, Faculty of Biology, University of
et al., 2013). Belgrade (BEOU), Serbia.
Free radicals are implicated in many diseases in- Chemicals and reagents
cluding cancer, cardiovascular diseases, neural disor-
ders, Alzheimers disease, mild cognitive impairment,
All chemicals and reagents were of analytical grade.
Parkinsons disease, alcohol-induced liver disease, ul-
Organic solvents (methanol, ethanol, acetone), HCl
cerative colitis, aging and atherosclerosis (Alam et al.,
(concentrated hydrochloric acid), CH3COOH (gla-
2013).
cial acetic acid) and CCl 3COOH (trichloroacetic
Rubus spp. are a good source of antioxidants, con- acid) were purchased from Zorka Chemicals (abac,
taining an appreciable level of phenolic compounds Serbia). Gallic acid, 2,2-dyphenyl-1-picrylhydrazyl
(Lee et al., 2012). Phenols are a very diverse and large (DPPH), iron (III) chloride (FeCl36H2O), iron (II)
group of compounds, shown to have health benefits sulfate heptahydrate (FeSO47H2O), sodium acetate
for humans (Halliwell, 2006). The most studied group (CH 3COONa3H 2O) were obtained from Sigma
of phenols are flavonoids, which exhibit very impor- Chemicals Co., St. Louis, MO, USA. Folin-Cio-
tant biological activities: anti-inflammatory, anti-aller- calteu phenol reagent was purchased from Merck,
gic, antiviral and anticancer. Several research groups Darmstadt, Germany. Sodium carbonate anhydrous
have considered the relation between total phenol and (Na2CO3), potassium acetate (C2H3KO2), potassium
flavonoid content and antioxidant activity. However, peroxidisulphate (K2O8S2) and L(+)-ascorbic acid (Vi-
some of them found positive correlation and some tamin C) were purchased from AnalaR Normapur,
found no correlation between phenolic content and VWR, Geldenaaksebaan, Leuven, Belgium. Alu-
antioxidant properties (Conforti et al., 2011). minum nitrate nonahydrate [Al(NO3)39H2O] and
2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) were pur-
The antioxidant capacity of different Rubus spe- chased from Fluka Chemie AG, Buchs, Switzerland.
cies has been observed (Deighton et al., 2000; Kh- [2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic]
knen et al., 2001; Valcheva-Kuzmanova et al., 2006; acid (ABTS) and quercetin hydrate were obtained
Katalinic et al., 2006; Jabloska-Ry et al., 2009; from TCI Europe NV, Boerenveldsweg, Belgium.
Buricova et al., 2011; Kolbas et Reshetnikov, 2011;
Conforti et al., 2011; Fu et al., 2015). The aim of this Extract preparation
work was to determine the total phenol and flavonoid
content and examine the antioxidant properties of R. Plant material was dried for two weeks in shade in a
caesius var. aquticus, using four in vitro procedures well-aerated room. Dry leaves were ground to powder
(DPPH, ABTS, FRAP and TRC), which, to the best of and extracted with four different solvents: methanol,
our knowledge, has not been done before. 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-
MATERIALS AND METHODS cubation in the dark. After sonication and filtration
Plant material through Whatman No 1 filter paper, the solvent was
removed by vacuum evaporator. Prepared samples
Dewberry (R. caesius var. aquticus) leaves were col- were stored in the fridge at +4C until use.
lected in summer 2012 near Zajear at the confluence
Antioxidant activity of dewberry leaf extracts 1325

Determination of total phenolic content (TPC) slight modifications, and results were expressed as
IC50 values (g/mL).
The total phenolic content (TPC) was determined ac-
cording to the procedure described by Singleton and The ferric reducing antioxidant power (FRAP) assay
Rossi (1965).
The total phenolic content was measured at 740 The ferric reducing antioxidant power (FRAP) was
nm by JENWAY 6306 UV/vis spectrophotometer determined according to Benzie and Strain (1996)
and calculated from the calibration curve of gallic with minor modifications. The method is based on
acid (GA) (10-100 mg/L) and expressed as mg of gal- the reduction of a ferric 2,4,6-tripyridyl-s-triazine
lic acid equivalents (GAE) per g of dry extract. The complex (Fe3+-TPTZ) by antioxidants to the ferrous
results were taken from triplicate measurements. form (Fe2+-TPTZ), which leads to absorbance in-
crease and which was measured by PERKIN ELMER
Determination of total flavonoid content (TFC) LAMBDA BIO UV/VIS spectrophotometer at 595 nm
against solvent. Aqueous solutions of FeSO47H2O
were used for the calibration curve and results are
The quantitative analysis of flavonoids present in R.
expressed as mol Fe2+ equivalents per mg of dry ex-
caesius var. aquticus leaf extracts was estimated as
tract. All measurements were taken in triplicate.
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 The total reducing capacity (TRC)
quercetin equivalents per gram of dry extract.
The total reducing capacity (TRC) was estimated ac-
DPPH radical scavenging capacity cording to the procedure described by Oyaizu (1986),
and results are calculated with the following equation
Eq (2):
DPPH radical scavenging capacity was assessed ac-
cording to the method described by Blois (1958). This Reducing power (%) = % (2)
method is designed to evaluate the decrease of 2,2-di-
phenyl-1-picryllhydrazyl (DPPH) free radicals in the where AS is the absorbance of the samples and AC is
presence of plant extracts with antioxidant properties. the absorbance of the control (containing distilled
water instead of extract).
Inhibition of DPPH free radicals was calculated
using Eq. (1):

Percentage of inhibition (%) (1) Results and Discussion

where AC is the absorbance of the control solution The yield of extraction


containing all reagents except extract and AS is the
absorbance in the presence of the tested extract solu- The yields of dried extract of Rubus caesius var.
tion, respectively. IC50 values (g/mL) were calculated aquticus leaves are presented in Table 1. The highest
by regression equation obtained from the relation be- value was obtained for aqueous extract and methanol,
tween the concentration of samples and percentage of
inhibition of DPPH radicals. Table 1. Extraction yields (Y) of blackberry leaves in different solvent.
Solvent Y (%)
ABTS+ radical scavenging capacity methanol 7.42
ethanol 2.15
The ABTS radical scavenging capacity was estimated acetone 0.58
as suggested by Miller and Rice-Evans (1997), with water 11.66
1326 Velikovi et al.

then ethanol and the lowest yields for acetone. The R. fruticosus, R. caesius, R. nessensis and R. idaeus,
solvent, temperature and sonication time affected ex- estimated by Folin Ciocalteu assay, highly correlated
traction yields. A positive influence of temperature with antioxidant activity. In the same work, a high
and sonication time on extraction yield was proved. correlation was confirmed between total anthocyanin
The contribution to yield was higher for temperature content and ABTS values, as well. Moreover, the con-
than for sonication time as shown previously (Iva- tribution of phenols and flavonoids to the antioxidant
novic et al., 2014). In this study, all extracts were pre- activity of R. hirsutus was greater than the contribu-
pared under the same conditions. tion of anthocyanins and vitamin C.

Total phenol and flavonoid content Fu et al. (2015) showed that the amounts of phenols
and flavonoids in berries depend on their size. Small
The total phenol and flavonoid content in the tested fruits are rich in phenolic and flavonoid compounds,
samples is presented in Table 2. In this study, it was medium-sized fruits in ascorbic acid, while large
confirmed that higher phenolic content contributes fruits are rich in anthocyanins. Diverse berry genera
to lower IC50 values, which means higher antioxidant differ significantly in total phenol content.
activity. Khknen et al. (2001) investigated the phytochemi-
The acetone extract was the most enriched with phe- cal content and antioxidant activity of selected Rubus
nols (366.27 mg GAE/g of dry extract), followed by species and correlations between them. Comparative
aqueous (309.99 mg GAE/g of dry extract), which analyses of R. chamaemorus and R. idaeus showed
could explain the highest antioxidant activity of these that flavonols, hydroxycinnamic and hydroxyben-
extracts. The largest amount of flavonoids was also zoic acids are present in R. chamaemorus in greater
found in the acetone (75.83 mg QE/g of dry extract) amounts than in R. idaeus, while the opposite is the
and the lowest in the aqueous extract (10.73 mg QE/g case with phenols, anthocyanins and ellagitannins.
of dry extract). Despite, R. chamaemorus revealed higher antioxidant
activity than R. idaeus.
These results suggest that the high antioxidant ac-
tivity of the acetone extract is due to some phenolic A positive correlation was found between total an-
compounds, including flavonoids. On the other hand, thocyanin content and total phenol content (Ivanovic
the high antioxidant activity of the aqueous extract et al., 2014). According to these authors, the active
is probably due to some phenolic compounds, but compounds in blackberry extracts were glycosides of
not flavonoids. Literature data indicate that the high cyanidin (glucoside, xyloside, rutinoside). The pres-
antioxidant activity found in Rubus species is relat- ence of 3-glucoside, 3-rutinoside, 3-xyloside and 3-O-
ed to phenol, anthocyanin and tannin compounds. -(6-malonyl-glucoside) of cyanidin and 5 cyanidin
According to Kolbas and Reshetnikov (2011), total 3-O--(6-(3-hydroxyl-3-methylglutaroyl)-glucoside
phenol content in aqueous and ethanol extracts of was confirmed in blackberry extracts (Jordheim et
Table 2. Total phenol and total flavonoid contents and antioxidant properties of R. caesius var. aquticus extracts.
TPC14 TFC24 DPPH4 ABTS+4 FRAP34 TRC4
(mg GAE/g dw) (mg QE/g dw) IC50(g/mL) IC50(g/mL) (mol Fe+2/mg dw) RP50 (mg/mL)
methanol 280.268.01 44.771.03 18.940.45 11.920.56 5.310.18 /
ethanol 263.743.90 37.940.42 16.880.31 9.321.06 3.000.33 2.630.005
acetone 366.270.50 75.832.01 11.320.01 6.090.25 3.231.21 0.630.008
water 309.991.42 10.730.11 15.920.15 6.510.05 3.800.01 4.150.71
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.
Antioxidant activity of dewberry leaf extracts 1327

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 Fig. 1. Correlation between the level of DPPH and R. caesius var.
aquticus extract concentrations for different solvents. Linear
remains the highest, while anthocyanin content sig- correlation coefficients are for methanol, ethanol, acetone and
nificantly increases. The relatively high antioxidant aqueous extracts: R2 = 0.9777, R2 = 0.9895, R2 = 0.9977 and R2 =
activity measured by hydroxyl radical antioxidant 0.9987, respectively.
capacity (HORAC) assay is explained by synergistic
effects between phenolic components other than an- FRAP and TRC assays are based on reducing re-
thocyanins, probably ellagitannins. actions where iron is reduced by antioxidant species
from ferric (Fe+3) to ferrous (Fe+2) ion. These reac-
Antioxidant activity tions can be monitored by the increase in the absor-
bance, which is accompanied by a color change from
In this study we examined the in vitro antioxidant ac- yellow to blue. The higher is the absorbance, the high-
tivity of four leaf extracts of R. caesius var. aquticus. er is the antioxidant activity of the tested samples.
The different testing methods provided different ex- The total reducing ability obtained by FRAP assay
perimental environments (pH of the mixture, radicals is expressed in mol Fe+2 equivalents per mg of dry
produced in the mixture, solvent of the system, etc.), extract and obtained values were in range from 3.00
which resulted in different ranking orders of IC 50 mol Fe+2/mg dw for the ethanol extract to 5.31 mol
values. This is the reason why antioxidant properties Fe+2/mg dw for the methanol. Total reducing capac-
should be tested by more than one procedure to get a ity according to Oyaizu et al. (1986) is expressed in
more realistic and complete picture of the antioxidant the concentration (mg/mL) of a sample that shows
profile of tested extracts (Prior et al., 2005). 50% of total reducing power. The lowest value was
obtained for the acetone extract 0.63 mg/mL and the
The antioxidant properties of R. caesius var. highest for the aqueous extract 4.15 mg/mL. The
aquticus were evaluated according to four differ- results obtained for TRC again point out the highest
ent procedures and are presented in Table 2. For the antioxidant activity of acetone extract.
DPPH assay, the positive correlation between all test-
ed samples and increase of concentration is shown in In this work, the contribution of phenols and
Fig. 1. The lowest IC50 values were obtained for ac- flavonoids to the antioxidant properties of the tested
etone and aqueous extracts 11.32 g/mL and 15.92 extracts was considered, and presented by Pearsons
g/mL, respectively, followed by ethanol, and the correlation coefficient in Table 3. It was shown that
highest value was for methanol (18.94 g/mL). The total phenolic content highly correlated with DPPH
same order in IC50 values was obtained by the ABTS and ABTS values (r = 0.8998 and 0.7843, respec-
assay. The lowest and approximately equal IC50 values tively), while total flavonoids highly correlated with
were for acetone 6.09 g/mL and aqueous 6.51 g/mL TRC values (r = 0.9999). A moderate correlation was
extracts, then ethanol 9.32 g/mL, and the highest found between total phenol content and TRC values
was for methanol 11.92 g/mL. and between total flavonoid content and DPPH val-
1328 Velikovi et al.

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 ABTS FRAP TRC
Var. aquticus IC50(g/mL) IC50(g/mL) EQ (mol Fe+2/mg dw) RP50 (mg/mL)
Total phenol content
0.8998 0.7843 0.2834 0.6151
(mg GAE/g dw)
Total flavonoid content
0.5645 0.0566 0.1446 0.9999
(mg QE/g dw)

ues. Phenols, as well as flavonoids, weakly correlated properties and chemical composition of Brazilian
with FRAP values. berries, and found a high positive correlation be-
tween antioxidant capacity and total phenolic con-
Ivanovic et al. (2014) found a moderate corre- tent. Moreover, strong positive correlation was shown
lation between DPPH and FRAP assays (r=0.582), between total antioxidant capacity (TEAC) and total
and a weak correlation between FRAP values and monomeric anthocyanin content, and between DPPH
cyanidin content (r=0.171) for blackberry extracts. and total flavonoid content. In the same study, Rubus
In the same study, a high correlation was established spp. revealed the highest antioxidant activity among
between FRAP values and total tannin content. In ad- tested species, which was attributed to the highest to-
dition, the ability of tannins to reduce ferric (Fe+3) tal phenol content.
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 CONCLUSIONS
antioxidant procedures.
Phenolic compounds are major contributors to the
Several papers describe the antioxidant properties antioxidant properties of dewberry (R. caesius var.
of other Rubus species, usually raspberry R. idaeus aquticus) leaves. In future work, determination of
L. and blackberry R. ulmifolius. The high antioxidant the chemical composition of secondary metabolites in
activity of R. ulmifolius leaves shown in these works dewberry should clarify and explain which of the bio-
was ascribed to the activities of caffeic acid, ferulic active compounds provides the highest contribution
acid, caffeic quinic esters, quercetin-3-O-glucuronide, to antioxidant activity. The in vivo antioxidant activity
kaemferol-3-O-glucuronide and ellagic acid (Barros should also be tested, because dewberry fruits (fresh
et al., 2010). Bobinaite et al. (2012) tested the antioxi- or processed as juices, jams and jellies) and leaves (as
dant properties of 17 raspberry cultivars and showed teas) are consumed worldwide as part of a healthy
a strong correlation between total phenolic content diet; they could be used in pharmaceutical, food and
and antioxidant activity, as well as between ellagic cosmetic industries.
acid and antioxidant activity. According to the same
study, the contribution of total anthocyanin content
to the antioxidant activity was very low, which was Acknowledgments: This research was supported by the
also confirmed by Weber et al. (2008). Ministry of Education, Science and Technological Develop-
ment (Grant. No. 173029)
Notable antioxidant activity of R. adenotrichus
Authors contributions: Ivona Velikovi, the main author,
fruits was also ascribed to ellagitannins and not
contributed with original data and the design of the re-
anthocyanins (Acosta-Montoya et al., 2010). On search, conceived the project, organized and analyzed data
the other hand, Reyes-Carmona et al. (2005) found and wrote the manuscript. Slavica Gruji and Ana Dami
strong correlation between total anthocyanin content contributed by designing research, data analyses and re-
and antioxidant activity. Rios de Souza et al. (2014) viewed several drafts of the manuscript. Petar D. Marin was
investigated the bioactive compounds, antioxidant the main supervisor of the research project and reviewed
Antioxidant activity of dewberry leaf extracts 1329

several drafts of the manuscript. Professor Krivoej deter- Fazzio, A., Plastina, P., Meijerink, J., Witkamp, R.F. and B. Gabri-
mined plant material. All authors read and approved the ele (2013). Comparative analyses of seeds of wild fruits of
final manuscript. Rubus and Sambucus species from Southern Italy: fatty
acid composition of the oil, total phenolic content, antioxi-
Conflict of interest disclosure: We certify that there is no dant and anti-inflammatory properties of the methanolic
conflict of interest with any financial organization regard- extracts. Food Chem. 140, 817-824.
Fu, Y., Zhou, X., Chen, S., Sun, Y., Shen, Y. and X. Ye (2015).
ing the material discussed in the manuscript.
Chemical composition and antioxidant activity of Chinese
wild raspberry (Rubus hirsutus Thunb.). Food. Sci.Technol.
60, 1262-1268.
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