In Vitro Antioxidant Activity of Dewberry (Rubus Caesius L. Var. Aquáticus Weihe. & Nees.) Leaf Extracts
In Vitro Antioxidant Activity of Dewberry (Rubus Caesius L. Var. Aquáticus Weihe. & Nees.) Leaf Extracts
In Vitro Antioxidant Activity of Dewberry (Rubus Caesius L. Var. Aquáticus Weihe. & Nees.) Leaf Extracts
2298/ABS150414109V
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
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
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):
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
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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