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DOI: 10.2298/APT1142271V BIBLID: 1450-7188 (2011) 42, 271-279
Original scientific paper

POLYPHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF THE FOUR

BERRY FRUITS POMACE EXTRACTS

Jelena J. Vulić*, Vesna T. Tumbas, SlaĊana M. Savatović, Sonja M. Đilas,

Gordana S. Ćetković and Jasna M. Ĉanadanović-Brunet

University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia

Four berry (bilberry, blackberry, strawberry and raspberry) fruits pomace, a waste
product coming from juice production, were extracted with 80% methanol containing
0.05% acetic acid. The highest content of antioxidant components, total polyphenols,
flavonoids and anthocyanins, was found in the bilberry pomace extract (1116.24,
1047.39 and 1279.49 mg/100g fresh pomace, respectively). The antioxidant activity of the
four berry pomace extracts (BPE) was determined using DPPH free radical scavenging
assay and reducing power. There was a high linear correlation between the IC50 and
content of anthocyanins (r2=0.98) and polyphenols (r2=0.85). Also, a good correlation
was found between the IC50 and the content of flavonoids. The strawberry pomace extract
showed the highest (RP0.5=0.35 mg/ml), while the blackberry pomace extract showed the
lowest (RP0.5=0.57 mg/ml) reducing power. The results showed that BPE can be a valu-
able source of natural ingrediences of the products in the food, cosmetical and phar-
maceutical industry.

KEY WORDS: berry fruits pomace, polyphenols, DPPH radical scavenging activity,
reducing power

INTRODUCTION

The berry fruits are rich in phenolic compounds such as phenolic acids (1, 2), flavo-
noids (3, 2) and anthocyanins (4). Also, small berries are rich sources of vitamin C. The
contents of flavonoids and phenolic acids in berries vary widely. Variation in the content
of phenolic compounds within one species is mainly due to the differences in the berry
varieties (5, 6) or in the growth conditions (7). Also, methodological differences (8, 9)
may contribute to the variability in the reported flavonoid and phenolic acid concentra-
tions. Discrepancies found may also partly be due to the differences in the maturity stage
of the fruits (10, 11). Anthocyanins constitute the main group of phenolic compounds in
berries. In raspberry and strawberry, the content of ellagic acid is reported to be high
(12), but not higher than that of anthocyanins.

* Corresponding author: Jelena J. Vuliš, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1,
21000 Novi Sad, Serbia, e-mail: jvulic@uns.ac.rs

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These bioactive compounds display potential health-promoting effects (13). The phe-
nolic compounds in berries have been reported to have antioxidant, anticancer, antiinfla-
mmatory, and antineurodegenerative biological properties (14). Because of the biological
properties associated with berry fruits, the identification of their antioxidant activity is
necessary for the evaluation of berry consumption on human health.
The berry fruits are not only available fresh but are also generally consumed frozen
and processed into juice, jam, ice cream and wine (15). After juice processing, berry fruit
pomace, the primary by-product remains unutilized. By-products of processing of fruit
and vegetables present a major disposal problem for the industry concerned, but they can
also be promising sources of compounds which may be used because of their technolo-
gical or nutritional properties and biological activity (16).
The objective of this study was to investigate the phenolic composition of berry fruits
pomace extracts (BPEs) obtained from bilberry (Vaccinium myrtillus L.), blackberry
(Rubus fruticosus L.), raspberry (Rubus idaeus L.) and strawberry (Fragaria vesca) fruit
pomace. Free radical scavenging activity of these BPEs was determinated against stable
2,2-diphenyl-1-picrylhidrazyl (DPPH) radicals. Also, the reducing power of the four ex-
tracts was evaluated.

EXPERIMENTAL

Chemicals and reagents

DPPH radical, Folin–Ciocalteu reagent, gallic acid, rutin and trichloroacetic acid, all
of analytical reagent grade were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). The other used chemicals and solvents, also of the highest analytical grade, were
obtained from „Zorka“ Šabac (Serbia).

Preparation of BPEs

Four berry fruits (bilberry, blackberry, strawberry and raspberry) were obtained from
a local store. They were washed in running water, juice was prepared using a domestic
food processor (Neo SK-400). After juice separation, the sample of the obtained pomaces
were stored at -20°C until analysis. Samples of investigated pomaces (20 g) were extrac-
ted three times: 60 min (160 ml), 30 min (80 ml) and 30 min (80 ml) with Heidolph
DIAX 900. The extraction was performed using 80% methanol aqueous solution with
0.05% acetic acid. The obtained extracts were combined and evaporated to dryness under
reduced pressure. The yields (Y) of the obtained bilberry, blackberry, strawberry and
raspberry extracts were: Y1= 10.65% (w/w), Y2= 13.10% (w/w), Y3= 8.50% (w/w), and
Y4= 8.75% (w/w), respectively.

Quantitative analysis of antioxidative components

Total polyphenolics. The amount of total soluble polyphenolics in extracts was de-
termined spectrophotometrically according to the Folin-Ciocalteu method (17). The reac-

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tion mixture was prepared by mixing 0.1 ml of water solution (concentration 1 mg/ml) of
extract, 7.9 ml of distilled water, 0.5 ml of Folin-Ciocalteu‟s reagent and 1.5 ml of 20%
sodium carbonate. After 2 h, the absorbance at 750 nm (UV-1800 spectrophotometer,
Shimadzu, Kyoto, Japan) was obtained against control that was prepared by replacing the
extract with distilled water. The results were calculated based on the calibration curve for
gallic acid and expressed as gallic acid equivalents in milligrams per 100 gram of fresh
pomace.
Total flavonoids. The total flavonoids content was measured using a colorimetric
assay (18). An aliquot (1 ml) of extracts (concentration 1 mg/ml) was added to 10 ml vo-
lumetric flask containing 4 ml of distilled H2O. To the flask, 0.3 ml of 5% NaNO2 was
added and 0.3 ml of 10% AlCl3 5 minutes later. After 6 minutes, 2 ml of 1M NaOH
solution was added and the total volume was made up to 10 ml with distilled H2O. The
solution was mixed well and the absorbance was measured at 510 nm against reagent
control containing distilled water instead of the extract. The total flavonoid content was
expressed as mg of rutin equivalents per 100 g of fresh pomace.
Total anthocyanins. Total monomeric plus polymerized anthocyanins in the extracts
were estimated spectrophotometrically using the pH single method (19). The extracts we-
re diluted with two buffer solutions of pH 1 and 4.5. The absorbance of each dilution was
measured at 510 nm (A510) and 700 nm (A700) against a distilled water control. The
total anthocyanin concentration was obtained from the equation:

Ctot(mg/l) = (Atot ×MW×DF×1000)/ε×L,

where Atot is calculated as Atot =A510–A700, ε is the molar absorbance coefficient of

cyanidin-3-glucoside (26900 l mol-1cm-1), MW is the molecular weight of cyanidin-3-
glucoside (449.2 g/mol), DF is dilution factor and L is the cell path length (1 cm). Total
anthocyanins content was expressed as mg of cyanidin-3-glucoside equivalents per 100 g
of fresh pomace.

Antioxidant assays

DPPH free radical scavenging assay. The antioxidant activity of berry extracts was
determined spectrophotometrically. The hydrogen atom or electron donation abilities of
the extracts was measured based on the bleaching of a purple-colored methanol solution
of the stable DPPH radical according to the method of Yen and Chen (20). Briefly, 1 ml
of solution of berry the extract in 95% methanol was added to 3 ml of methanol and 1 ml
of 90 μM DPPH solution (18 mg in 50 ml 95% methanol prepared daily). The mixture
was vortexed thoroughly for 1 min and left at room temperature for 10 min, than the ab-
sorbance was measured against a control at 517 nm. The blank probe contained all com-
ponents except for the radicals. The range of investigated concentrations was 0.0033-
0.167 mg/ml. The capability to scavenge the DPPH radicals was calculated using the
following equation:

AADPPH• (%) = (AControl – ASample)/AControl × 100

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Original scientific paper

where AControl is the absorbance of the control reaction (containing all reagents except for
the extract) and ASample is the absorbance in the presence of the berry extract. The values
of antioxidant activity were investigated for the various concentrations of the berry
extracts.
Reducing power. The reducing power of the extracts was determined by the method
of Oyaizu (21). The capacity of the extracts to reduce the ferric-ferricyanide complex to
the ferrous-ferricyanide complex of Prussian blue was determined by recording the absor-
bance at 700 nm after incubation. For this puropse, suspension of extracts (0.05–1 mg) in
1 ml of distilled water or 1 ml of distilled water (control) was mixed with 1 ml of phos-
phate buffer (pH 6.6) and 1 ml of 1% potassium ferricyanide K3[Fe(CN)6]. The mixture
was incubated at 50°C for 20 min. Following this, 1 ml of trichloroacetic acid (10%) was
added and the mixture was then centrifuged at 3000 rpm for 10 min. A 2 ml aliquot of the
upper layer was mixed with 2 ml of distilled water and 0.4 ml of 0.1% FeCl3 and the
absorbance of the mixture was measured at 700 nm. A higher absorbance of the reaction
mixture indicates a higher reduction capability.

RESULTS AND DISCUSSION

Quantitative analysis of antioxidative components

The Folin–Cioacalteu method is a rapid and widely used assay to investigate the total
polyphenolics content. This method is based on the reducing power of phenolic hydroxyl
groups, but it is known that different polyphenolic compounds have different responces
to the Folin–Cioacalteu reagent. The total flavonoids content of extracts were measured
according to the method of Zhinsen et al. (18). The pH differential method is a simple, ra-
pid, and economical means for determining the amount of anthocyanins, and this method
has been verified by AOAC‟s strict validation guidelines.
Results of the spectrophotometric determination of antioxidative components are
shown in Table 1. The highest contents of total polyphenols, total flavonoids and total
anthocyanins were found in the bilberry pomace extract (1116.24, 1047.39 and 1279.49
mg/100g fresh pomace, respectively).

Table 1. Total polyphenolics, flavonoids and anthocyanins content in BPE

Berry fruits Antioxidant compounds content (mg/100 g fresh pomace)

pomace extracts Total polyphenolics Total flavonoids Total anthocyanins
Bilberry 1116.24 1047.39 1279.49
Blackberry 804.50 245.48 149.12
Strawberry 488.12 296.11 19.48
Raspberry 637.77 591.65 65.21

Wang et al. reported that total polyphenolics contents of blackberry, raspberry and
strawberry were 226 ± 4.1, 234 ± 5.1 and 103 ± 2.0 mg gallic acid per 100 g of fresh
fruit, respectively (22), whereas Šavikin et al. reported that bilberry total polyphenolics
content was 890 ± 9.0 mg gallic acid per 100 g f fresh fruit (23). If compared with these

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results, the polyphenolics contents in our study were higher. Also, the reported total
anthocyanins contents (22, 23) of bilberry, blackberry, raspberry and strawberry (610.0,
152.8, 68.0 and 31.9 mg/100g fresh fruit, respectively) show that the investigated pomace
extracts had higher values.

Antioxidant assays

The antioxidant activity of the four BPEs was determined using DPPH free radical
scavenging assay and reducing power. The model of scavenging the stable DPPH radicals
is a widely used method to evaluate antioxidant activity in a relatively short time com-
pared with other methods (24). The dose-response curve for the DPPH radical scavenging
capacity (RSCDPPH) of investigated BPEs is shown at Figure 1. The RSCDPPH value of
BPE extracts increased with increasing concentration. Based on the RSC DPPH, the IC50 va-
lues were determined. The IC50 value, defined as the concentration of the extract required
for 50% scavenging of radicals under experimental condition employed, is a parameter
widely used to measure the free radical scavenging activity (25). A smaller IC50 value
corresponds to a higher antioxidant activity. The IC50 of the obtained bilberry, blackberry,
strawberry and raspberry pomace extracts were 0.040, 0.017, 0.038 and 0.040 mg/ml,
respectively. Šavikin et al. reported that the IC50 value of bilberry fruit was 0.82 mg/ml
(23). The bilberry pomace in our test had a higher antioxidant activity than bilberry fruit
reported by these researchers.
In order to determine the relative importance of the various classes of phenolic com-
pounds in free radicals scavenging activity, correlation analyses were made between the
measures of IC50 and the polyphenolics, flavonoids and anthocyanins contents. There was
a high linear correlation between the IC50 and the content of anthocyanins (r2=0.98) and
polyphenols (r2=0.85). Also, a good correlation was found between the IC50 and the con-
tent of flavonoids. The analysis of the correlation between the IC50 values and the results
of the DPPH assay indicated the great importance of phenolic compounds in the radical
scavenging activities.
100

80

60
RSCDPPH(%)

40

Bilberry
20
Blackberry
Strawberry
Raspberry
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18
Concentration (mg/ml)

Figure 1. DPPH free radical scavenging activity of the four BPEs

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Original scientific paper

For the measurments of the reducing power, the Fe3+ - Fe2+ transformation was
investigated in the presence of the four BPEs using the method of Oyaizu (21). Figure 2
shows the reducing power of the four BPEs. The reducing power of all BPEs increased
with increasing concentration. The strawberry pomace extract showed the highest
(RP0.5=0.35 mg/ml), while the blackberry pomace extract showed the lowest (RP0.5=0.57
mg/ml) reducing power.

1.4

1.2

0.8
A

0.6
Bilberry
0.4
Blackberry
Strawberry
0.2
Raspberry
0
0 0.2 0.4 0.6 0.8 1
Concentration (mg/ml)

Figure 2. Reducing power of the four BPE

The obtained results show that the berry fruits pomaces are a good source of antioxi-
dant compounds and can be used as a potential value-added ingredient in the food, cos-
metic and pharmaceutical industry.

CONCLUSION

A quantitative analysis of antioxidative components of the four berry (blueberry,

blackberry, strawberry and raspberry) fruits pomaces showed high content of these com-
ponents. The highest contents of total polyphenols, flavonoids and anthocyanins were
found in the bilberry pomace extract (1116.24, 1047.39 and 1279.49 mg/100g fresh po-
mace, respectively).
The DPPH radical scavenging capacity (RSCDPPH) of investigated BPEs increased
with increasing concentration. The highest DPPH radical scavenging activity was obser-
ved in the case of blackberry pomace extract (IC50 = 0.017 mg/ml).
The correlation analyses were made between the measures of IC50 and the polyphe-
nolics, flavonoids and anthocyanins contents. There was a high linear correlation between
the IC50 and the content of anthocyanins (r2=0.98) and polyphenols (r2=0.85). Also, a
good correlation was found between the IC50 and the content of flavonoids.
The strawberry pomace extract showed the highest (RP0.5=0.35 mg/ml), while the
blackberry pomace extract showed the lowest (RP0.5=0.57 mg/ml) reducing power.

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The obtained results show that berry pomace has a high antioxidant activity and iso-
lated nutrients should be integrated into food products with health-promoting advantages
to consumers.

Acknowledgement

This research is a part of the Project of Serbia-Slovenia technology co-operation for

2010-2011, project No. 69-00-02/2009-01/10.

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Original scientific paper

САДРЖАЈ ПОЛИФЕНОЛА И АНТИОКСИДАТИВНА АКТИВНОСТ

ЕКСТРАКАТА ТРОПА БОБИЧАСТОГ ВОЋА

Јелена Ј. Вулић, Весна Т. Тумбас, Слађана М. Саватовић, Соња М. Ђилас,

Гордана С. Ћетковић и Јасна М. Чанадановић Брунет

Универзитет у Новом Саду, Tехнолошки факултет, Булевар цара Лазара 1, 21000 Нови Сад, Србија

Tроп четири врсте бобичастог воћа (боровница, купина, јагода и малина), спо-
редни производ производње сока, есктрахован је 80% метанолом са додатком
0.05% сирћетне киселине. Највећи садржај антиоксидативних компоненти, укупних
полифенола, флавоноида и антоцијана, био је у екстракту тропа боровнице
(1116.24, 1047.39 и 1279.49 mg/100g свежег тропа). Антиоксидативна активност
екстраката тропа четири врсте бобичастог воћа одређена је DPPH спектрофотомет-
ријским тестом и редукционом способношћу. Утврђена је висока линеарна корела-
ција између IC50 и садржаја антоцијана (r2=0.98) и полифенола (r2=0.85). Такође,
добра корелација је нађена између IC50 и садржаја флавоноида. Екстракт тропа ја-
годе показао је највећу (RP0.5=0.35 mg/ml), а екстракт купине најнижу редукциону
способност (RP0.5=0.57 mg/ml). Резултати указују на могућност коришћења екстра-
ката тропа бобичастог воћа као доброг функционалног додатка у прехрамбеној,
козметичкој и фармацеутској индустрији.

КЉУЧНЕ РЕЧИ: троп бобичастог воћа, полифеноли, антиоксидативна активност

на DPPH радикале, редукциона способност

Received 22 August 2011

Accepted 15 November 2011

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