V in vitro antioxidant activity of dewberry ( rubus caesius l . var . aquáticus weihe . & neeS . ) leaf extractS

In this study, the antioxidant properties and total phenolic and flavonoid contents of Rubus caesius L. var. aquáticus 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-ethylbenzthiazoline6-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 μmol Fe+2/mg for the ethanol extract and 5.31 μmol Fe+2/mg for the methanol extract. The results obtained for total reducing capacity indicate that the acetone extract has the highest antioxidant potential.


introdUction
Several economically important species come from the Rosaceae family including many edible fruits, ornamental trees and shrubs.In addition, members of Rosaceae are also used in traditional medicine because of their antimicrobial, anticonvulsant, musclerelaxant and radical scavenging activity (Martini et al., 2009).
The genus Rubus, which includes about 750 species, is widespread on all continents except Antarctica (Alice and Campbell, 1999).It comprises perennial herbs or shrubs, usually with spiny stems and pinnate, 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.
It is a wild-growing plant and widely cultivated as a 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 (Tatić, 1972).R. caesius var.aquáticus can be found near aquatic habitats (Nyarady, 1956) and, as far as we know, this is the first time it was found in the Republic of Serbia.
Dewberries are consumed worldwide, not only because of their taste, but also for their health benefits (Lee, 2012).Dewberry leaves have been used in 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 including cancer, cardiovascular diseases, neural disorders, Alzheimer's disease, mild cognitive impairment, Parkinson's disease, alcohol-induced liver disease, ulcerative colitis, aging and atherosclerosis (Alam et al., 2013).
Rubus spp.are a good source of antioxidants, containing 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 important biological activities: anti-inflammatory, anti-allergic, 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).

Plant material
Dewberry (R. caesius var.aquáticus) leaves were collected in summer 2012 near Zaječar 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 Herbarium of the Institute of Botany and Botanical Garden "Jevremovac", Faculty of Biology, University of Belgrade (BEOU), Serbia.

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 powdered leaves was extracted with 50mL of solvent in an ultrasonic bath for 1 h and again after 24 h of incubation 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 +4°C until use.

determination of total phenolic content (tPc)
The total phenolic content (TPC) was determined according 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 gallic 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.aquáticus leaf extracts was estimated as previously reported by Park et al. (1997).The flavonoid content in leaf extract was estimated spectrophotometrically 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 according to the method described by Blois (1958).This method is designed to evaluate the decrease of 2,2-diphenyl-1-picryllhydrazyl (DPPH) free radicals in the presence of plant extracts with antioxidant properties.

Percentage of inhibition (%) (1)
where A C is the absorbance of the control solution containing all reagents except extract and A S is the absorbance in the presence of the tested extract solution, respectively.IC 50 values (µg/mL) were calculated by regression equation obtained from the relation between 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 slight modifications, and results were expressed as IC 50 values (µg/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 (Fe 3+ -TPTZ) by antioxidants to the ferrous form (Fe 2+ -TPTZ), which leads to absorbance increase and which was measured by PERKIN ELMER LAMBDA BIO UV/VIS spectrophotometer at 595 nm against solvent.Aqueous solutions of FeSO 4 ×7H 2 O were used for the calibration curve and results are expressed as µmol Fe 2+ equivalents per mg of dry extract.All measurements were taken in triplicate.

the total reducing capacity (trc)
The total reducing capacity (TRC) was estimated according to the procedure described by Oyaizu (1986), and results are calculated with the following equation Eq (2): where A S is the absorbance of the samples and A C 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.aquáticus leaves are presented in then ethanol and the lowest yields for acetone.The solvent, temperature and sonication time affected extraction 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 (Ivanovic et al., 2014).In this study, all extracts were prepared 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 IC 50 values, which means higher antioxidant activity.
The acetone extract was the most enriched with phenols (366.27mg 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 activity 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 related to phenol, anthocyanin and tannin compounds.
According to Kolbas and Reshetnikov (2011), total phenol content in aqueous and ethanol extracts of 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 contribution of phenols and flavonoids to the antioxidant activity of R. hirsutus was greater than the contribution 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.Kähkönen et al. (2001) investigated the phytochemical 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 hydroxybenzoic 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.

antioxidant activity
In this study we examined the in vitro antioxidant activity of four leaf extracts of R. caesius var.aquáticus.
The different testing methods provided different experimental 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.aquáticus were evaluated according to four different procedures and are presented in Table 2.For the DPPH assay, the positive correlation between all tested samples and increase of concentration is shown in Fig. 1.The lowest IC 50 values were obtained for acetone and aqueous extracts − 11.32 µg/mL and 15.92 µg/mL, respectively, followed by ethanol, and the highest value was for methanol (18.94 µg/mL).The same order in IC 50 values was obtained by the ABTS assay.The lowest and approximately equal IC 50 values were for acetone 6.09 µg/mL and aqueous 6.51 µg/mL extracts, then ethanol 9.32 µg/mL, and the highest was for methanol 11.92 µg/mL.FRAP and TRC assays are based on reducing reactions where iron is reduced by antioxidant species from ferric (Fe +3 ) to ferrous (Fe +2 ) ion.These reactions can be monitored by the increase in the absorbance, which is accompanied by a color change from yellow to blue.The higher is the absorbance, the higher is the antioxidant activity of the tested samples.The total reducing ability obtained by FRAP assay is expressed in µmol Fe +2 equivalents per mg of dry extract and obtained values were in range from 3.00 µmol Fe +2 /mg dw for the ethanol extract to 5.31 µmol Fe +2 /mg dw for the methanol.Total reducing capacity 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 Pearson's 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, respectively), 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- ues.Phenols, as well as flavonoids, weakly correlated with FRAP values.Ivanovic et al. (2014) found a moderate correlation 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 addition, 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 between 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 antioxidant 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 properties and chemical composition of Brazilian berries, and found a high positive correlation between antioxidant capacity and total phenolic content.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 total phenol content.conclUSionS Phenolic compounds are major contributors to the antioxidant properties of dewberry (R. caesius var.aquáticus) leaves.In future work, determination of the chemical composition of secondary metabolites in dewberry should clarify and explain which of the bioactive 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.
al., 2011).The main compound in blackberry extracts tested byElisia 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 ripening ellagitannin content significantly decreases, but remains the highest, while anthocyanin content significantly increases.The relatively high antioxidant activity measured by hydroxyl radical antioxidant capacity (HORAC) assay is explained by synergistic effects between phenolic components other than anthocyanins, probably ellagitannins.

acknowledgments:
This research was supported by the Ministry of Education, Science and TechnologicalDevelopment (Grant.No. 173029)    authors' contributions: Ivona Veličković, the main author, contributed with original data and the design of the research, conceived the project, organized and analyzed data and wrote the manuscript.Slavica Grujić and Ana Džamić contributed by designing research, data analyses and reviewed several drafts of the manuscript.Petar D. Marin was the main supervisor of the research project and reviewed

Table 1 .
The highest value was obtained for aqueous extract and methanol,

table 1 .
Extraction yields (y) of blackberry leaves in different solvent.

table 2 .
Total phenol and total flavonoid contents and antioxidant properties of R. caesius var.aquáticus extracts.

table 3 .
Correlation between antioxidant activity (according to four different assays) and total phenol and flavonoid contents in R. caesius var.aquáticus extract expressed through Pearson's coefficient of regression.drafts of the manuscript.Professor Krivošej determined 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 regarding the material discussed in the manuscript. several