Industrial Crops and Products 80 (2016) 165–176

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Extracts of wild apple fruit (Malus sylvestris (L.) Mill., Rosaceae) as a

source of antioxidant substances for use in production of
nutraceuticals and cosmeceuticals
Dragana Stojiljković a,∗ , Ivana Arsić b , Vanja Tadić c
a
Farmakop Public Pharmacy, 92 Nemanjić Blvd., 18000 Niš, Serbia
b
Department of Pharmacy, Faculty of Medicine, University of Niš, 81 Dr Zoran Ðind̄ić Blvd., 18000 Niš, Serbia
c
Department for Pharmaceutical Research and Development, Institute for Medicinal Plant Research “Dr Josif Pančić”, 1 Tadeuša Košćuška St., 11000
Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: Wild apple fruit (Malus sylvestris (L.) Mill., Rosaceae) is a good source of polyphenolic compounds which
Received 1 June 2015 have a therapeutic effects on many diseases caused by reactive oxygen species and oxidative stress
Received in revised form 29 October 2015 (e.g. cardiovascular or degenerative diseases, atherosclerosis, diabetes, osteoporosis, cancer, dermatitis,
Accepted 8 November 2015
phototoxity). The aim of our study was in vitro characterization of extracts of wild apple fruit originated
Available online 6 December 2015
from Serbia, obtained by different extraction methods and solvents, in order to examine their potential
use as the source of substances with antioxidant properties in the production of nutraceuticals and
Keywords:
cosmeceuticals. Extracts were made by maceration, percolation, Soxhlet and ultrasonic extraction using
Wild apple fruit
Variation of extraction methods and
polar solvents: 70% ethanol, 45% and 80% propylene–glycol, and distilled water, and non-polar solvents:
solvents sunflower and olive oil. The extracts were characterized by determination of total content of phenols (TPC)
Polyphenolic compounds by Folin–Ciocalteu assay and expressed as gallic acid equivalents (GAE), flavonoids (TFC) by Markham’s
Antioxidant activity method with rutin as standard (RE), tannins (TT) by vanillin assay and expressed as catechin equivalents
(CE) and percentage content of anthocyanins (TA) by Eur.Ph.6.0. Besides, the antioxidant activity of the
investigated extracts was estimated by three assays: DPPH (1,1-diphenyl-2-picryl-hydrazyl) test, FRAP
(Ferric Reducing Antioxidant Power) test and test with linoleic acid, and expressed as %RSC (Radical
Scavening Capacity), FRAP value (mM Fe2+ ) and %AOA (AntiOxidant Activity), respectively. The evidence
of correlation between antioxidant properties and polyphenolic compounds content has been shown
to be very good. The content of polyphenolic compounds and antioxidant activity depended on used
extraction method and solvents. Namely, TPC in extracts ranged from 172.91–1556.99 mg GAE/100 g dry
weight (d.w.) (dry plant material), TFC from 3.97 to 182.22 mg RE/100 g d.w., TT from 72.73 to 8872.73 mg
CE/100 g d.w., and TA from 0.70 to 14.63%. %RSC ranged from 8.12 to 78.43 %RSC, FRAP value from 2.13
to 7.65 mM Fe2+ and %AOA from 57.73% to 95.32 %AOA. The study provided the data that the extracts
obtained by ultrasonic extraction using ethanol and distilled water as solvents contained the highest
amount of polyphenolic compounds and demonstrated the best antioxidant activity, as well. The results
of our study indicated that wild apple fruit might be taken into consideration as a source of antioxidant
substances for food, dermocosmetic and cosmetic industry.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
Oxidative stress (OS) is an imbalance between cellular produc-
tion of reactive oxygen species (ROS) and cell’s ability in their
“scavenging”. ROS may lead to lipid peroxidation, damage of mem-
∗ Corresponding author.
E-mail addresses: s.dragana83@yahoo.com (D. Stojiljković),
ivana.arsic@medfak.ni.ac.rs (I. Arsić), vtadic@mocbilja.rsb (V. Tadić).
brane proteins and DNA mutations, which can lead to various
structural, functional and aesthetic changes in the organism, as well
as to initiation of the development of many diseases such as car-
diovascular disease, atherosclerosis, diabetes, pulmonary disease,
cancer (Almeida et al., 2008). All biological systems possess antiox-
idant defence system, that protects from oxidative stress, as well as
enzymes for repair and removal of damaged molecules, and if this
defence system failures, damages in biological systems are larger,
so the application of natural antioxidant compounds is very impor-

http://dx.doi.org/10.1016/j.indcrop.2015.11.023
0926-6690/© 2015 Elsevier B.V. All rights reserved.
166 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176
tant in the protection from ROS and OS influences (Almeida et al.,
2008; Khan et al., 2013; Stojiljković et al., 2014).
Increased consumer’s demands for safe and secure application
of oral and topical products rich in antioxidants lead to a greater
need on search for new sources of natural antioxidants to replace
synthetic ones. Therefore, there is a great scientific interest in the
investigation of plants rich in bioactive antioxidant compounds that
could be used in the food and cosmetic industry, with the goal to
discover a new opportunities in the prevention and/or treatment
of aging and in the prevention and/or treatment of many diseases
caused by ROS (Royer et al., 2013; Žugić et al., 2014).
The use of plant extracts and consumption of fruits and
vegetables rich in natural antioxidant compounds, primarily in
polyphenolic compounds, are very important for strengthening of
protective endogenous system, as well as in the prevention and/or
treatment of damages and diseases caused by oxidative stress.
Polyphenols are the most common antioxidant compounds in the
diet, because, depending on the geographical area, it’s found that
people consume about 1 g of polyphenols per day (Amzad Hossain
et al., 2009; Royer et al., 2013). Also, there is a growing interest
in investigation of polyphenols benefits on human health, espe-
cially those of plant origin (Alberti et al., 2014; Kchaou et al.,
2013; Kunradi Vieira et al., 2011). Several studies have shown that
polyphenols inhibit the ROS formation as their “scavengers”, reduce
the penetration of UVB rays to sensitive tissues and neutralize free
radicals (Kohen, 1999). Hence, possessing the antioxidant and anti-
inflammatory properties, they might participate in the prevention
and treatment of many diseases, including disorders caused by
oxidative stress and cell aging, cardiovascular or degenerative dis-
eases, hypertension, atherosclerosis, osteoporosis, cancer, arthritis,
type II diabetes, and others (Halliwell, 1996; Kchaou et al., 2013;
Shahidi, 2012). The use of nutraceuticals and cosmeceuticals in food
and cosmetic industry, rich in polyphenolic compounds, represents
a good basis for health improving and prevention of age-related-
chronic diseases (Alberti et al., 2014; Royer et al., 2013; Žugić et al.,
2014). Beside antioxidant properties, these active compounds pos-
sess antimicrobial, antimutagenic and anticarcinogenic properties
(Amzad Hossain et al., 2009).
Apples are the most often produced and consumed fruits around
the world. It is an important part of human diet and it is an excellent
source of nutrients because of its biochemical composition (Maria
John et al., 2014; Šavikin et al., 2014). Epidemiological studies have
shown an inverse correlation between the consumption of apples
and/or similar products and the risk of development of cardiovas-
cular diseases, respiratory system dysfunction, cancer of prostate,
liver and colon (Tsao et al., 2003). Polyphenols are the major group
of compounds responsible for preventive and antioxidant proper-
ties of apples (Hagena et al., 2007; Šavikin et al., 2014). Apples are
not important only for its nutritional properties, but also for its
traditional use in folk medicine (Sivaci, 2006).
Indigenous species of fruit have proven to be much better,
because they show good adaptability to the local environment
and represent a valuable source of genetic variability for the crop
resistance to various changes, as well as for quality properties. Tra-
ditional varieties of apples are generally not suitable for large-scale
production, because they don’t meet the standards for appearance,
taste, smell and preservation, which are economically less signif-
icant (Goland and Bauer, 2004), even many studies have shown
that, in order to increase intake of antioxidant compounds as well
as positive effects, it is better to consume local indigenous apple
varieties than cultivated purchasing cultures from market (Šavikin
et al., 2014). Some researchers have shown that the majority of wild
varieties showed better antioxidant potential (Cekic and Ozgen,
2010; Kubola et al., 2011). The standardization and globalization
in the markets in Serbia and all over the world have led to a sig-
nificant reduction in the number of local varieties of apples, and
some of them are disappeared (Mratinić and Fotirić-Akšić, 2011).
Many studies were done with a cultivated apple species (Panzella
et al., 2013; Lata et al., 2009; Mikulič Petkovšek et al., 2009), much
less studies exist about polyphenolic composition and antioxidant
capacity of local indigenous varieties (Begić-Akagić et al., 2011;
Jakobek et al., 2013; Wojdylo et al., 2008) especially those of wild
origin, and even less about the varieties from the territory of the
Republic of Serbia (Lesjak et al., 2011; Žugić et al., 2014; Šavikin
et al., 2014). Serbia has a good geographical position, good natu-
ral conditions and areas for the cultivation of a large number of
fruit species (Bulatović et al., 2013), and the production of indige-
nous apples has great significance for the Serbia agriculture for
industrial, food and cosmetic production (Šavikin et al., 2014).
For this study, wild apple (Malus sylvestris (L.), Mill., Rosaceae)
from the territory of Serbia was selected. Investigated wild apple
represents a biomarker of territory of Serbia, and a plant used in
Serbian folk medicine for the treatment of various diseases such as
hypertension, atherosclerosis, diabetes, rheumatism, urinary infec-
tions (Sarić, 1989; Žugić et al., 2014; Šavikin et al., 2014), as well
as a row material which can be potentially used for a production
of nutraceuticals and cosmeceuticals. The extraction of bioactive
compounds from the wild apple fruits, studying of polypheno-
lic composition and the antioxidant potential are important for
understanding of their potential use (Luthria, 2008; Šavikin et al.,
2014). The extraction of bioactive substances from plant mate-
rial depends on many factors, such as solvents, extraction method,
extraction time, the nature of the drug used for extraction, the ratio
of drug/extract, the nature of compounds to be extracted (Alberti
et al., 2014; Haminiuk et al., 2012; Luthria, 2008; Vongsak et al.,
2013). Theoretically, the optimal extraction method for obtaining
good and suitable extracts for application in food and cosmetic
industry, should be simple, fast, economical, with skin-compatible
solvents and suitable for production in large industry (Vongsak
et al., 2013).
In this context, the aim of our study was to select a suitable
extraction method and extraction agents which will provide the
wild apple fruits extracts with the highest content of polyphe-
nolic compounds and the most pronounced antioxidant activity.
The achieved results might enable a better understanding of the
potential of local indigenous species from the territory of Serbia.
Therefore, the extraction of active substances from the wild apple
fruit (Malus sylvestris fructus) was performed using different extrac-
tion methods and solvents, as well as the characterization of the
extracts in vitro: the content of bioactive polyphenolic compounds
(phenols, flavonoids, tannins and anthocyanins), as active antiox-
idant substances of these extracts was examined. The antioxidant
potential of the investigated extracts was evaluated, as well, in
order to examine the possibility of their potential application in the
food, dermocosmetic and cosmetic industry, as a possible source of
antioxidant substances.
2. Materials and methods
2.1. Materials
2.1.1. Chemicals
Solvents 96% ethanol (v:v—volume:volume) were purchased
from Hemos (Belgrade, Serbia), 45% propylene-glycol—PG and 80%
propylene-glycol (w:w—weight:weight – PG:H2O – 45:55 and
80:20) were purchased from Centrohem doo (Stara Pazova, Serbia),
distilled water was from Medical faculty of Niš (Niš, Serbia), olive
oil was from supplier Medsol (Molfetta (BA), Italy) and sunflower
oil from Sunce (Sombor, Serbia).
The reagents Folin–Ciocalteus radical (FCR), DPPH (1,1-
diphenyl-2-picryl-hydrazyl) radical and linoleic acid were
 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176
purchased from Sigma–Aldrich (USA). Vanillin reagent and
TPTZ (2,4,6-tripyridyl-s-triazine) reagent were purchased from
Merck-chemicals (Darmstadt, Germany). Sodium carbonate
(Na2CO3), aluminum chloride (AlCl3), potassium acetate (K-
acetate), hydrochloric acid (HCl), acetate buffer, iron(III) chloride
(FeCl3), phosphate buffer, ammonium thiocyanate were purchased
from Centrohem doo (Stara Pazova, Serbia). Absolute ethanol was
from supplier Hemos (Belgrade, Serbia), absolute methanol from
Analar Normapur, VWR International SAS (France) and distilled
water from Medical faculty (Niš, Serbia).
The standards gallic acid, rutin, catechin and BHT- (butylated
hydroxytoluene) were purchased from Sigma–Aldrich (USA) and
FeSO4 × 7H2 O from Merck-chemicals (Darmstadt, Germany).
All chemicals used in the experimental work were analytical
purity (p.a.).
2.1.2. Plant material
The wild apple fruit (M. sylvestris (L.) Mill., Rosaceae) was col-
lected in September 2012 on the slopes of Kopaonik Mount (village
Perunika)—southern Serbia. Seeds were removed from the fruits,
fruits than were cut into thin slices (the degree of fragmentation
was 5 mm) and air-dried at temperature of 22 ± 2 ◦ C, for three
weeks. Before extraction, the drug was pulverized for 2 min using a
mill (IKA – Werke, Staufen, Germany) and drug degree of fragmen-
tation was reduced to 1 mm.
2.2. Extraction methods
Liquid extracts of wild apple fruit with polar solvents were pre-
pared by following extraction methods: maceration, percolation,
Soxhlet extraction and ultrasonic extraction, and with non-polar
solvents extraction methods were modified maceration and diges-
tion with oil, according to the procedure of Pharmacopoeia of
Jugoslavica (Ph Jug IV, 1984). As solvents 70% ethanol (v:v), 45%
propylene–glycol (w:w), 80% propylene-glycol (w:w), distilled
water, olive oil and sunflower oil, and 96% ethanol (v:v) in sol-
vent system ethanol:oil were used. Liquid extracts were made in
the drug:extract (D:E) ratio 1:5 (w:v—weight:volume). All extracts
were made, stored and used for further investigation as liquid
extracts.
2.2.1. Maceration as extraction method
The dried powdered fruit (20 g) was macerated with 5 parts of
solvent for 5 days in a tightly closed erlenmeyer flask, protected
from direct sunlight, at temperature of 22 ± 2 ◦ C, with occasional
shaking. Macerate was separated from drug by straining and sub-
sequent pressing and left for two days in a cold place protected
from light and than filtered.
2.2.2. Percolation as extraction method
The dried powdered fruit (20 g) was separately moistened with
solvent and allowed to swell for at least two hours. Then the drug
was transferred into a percolator, percolator was filled with solvent
(final proportion 1:5—w:v), and drug was macerated for 12 h, at
temperature of 22 ± 2 ◦ C. The percolation was done at temperature
of 22 ± 2 ◦ C, with a flow rate of 1 mL/min until prescribed volume
of percolat (100 mL) was obtained.
2.2.3. Soxhlet extraction
The dried powdered fruit (20 g) was placed into a paper tube,
then in a Soxhlet apparatus, poured with a 2.5 capacity of Soxh-
let apparatus with solvent and left overnight at temperature of
22 ± 2 ◦ C. It was then heated to boiling and extracted in a Soxh-
let apparatus to discoloration for 4 cycles (one cycle take one hour;
total extraction period take 4 h).
167
2.2.4. Ultrasonic extraction
The dried powdered fruit (20 g) was poured with a solvent
(D:E = 1:5 (w:v)), left in an ultrasonic bath under maximum oper-
ating conditions and then filtered. The parameters of ultrasonic
extraction were: time—30 min, the temperature 22 ± 2 ◦ C, power
10 × 10.
2.2.5. Maceration with oil
The dried powdered fruit (20 g) was macerated with 5 parts of
olive or sunflower oil on a water bath for 4 h, in a tightly closed
erlenmeyer flask, protected from direct sunlight, at temperature of
45 ± 2 ◦ C, with occasional shaking. Oil extract was separated from
drug by straining and subsequent pressing and than filtered.
2.2.6. Digestion with oil
The dried powdered fruit (20 g) was macerated with 3 parts of
96% ethanol for 24 h, in a tightly closed erlenmeyer flask, at tem-
perature of 22 ± 2 ◦ C, followed by maceration with 5 parts of olive
or sunflower oil extraction, in a water bath for 4 h, in a tightly
closed erlenmeyer flask, protected from direct sunlight, at temper-
ature of 45 ± 2 ◦ C, with occasional shaking, with removing ethanol
throughly at same time. Oil extract was separated from drug by
straining and subsequent pressing and than filtered.
2.3. Determination of pH values of investigated extracts
pH values of investigated liquid extracts, made using polar
solvents, were measured potentiometrically by pH 211 Micro-
processor pH Meter (Hanna Instruments, USA) (Stojiljković et al.,
2013). The method was based on direct potentiometric determi-
nation of hydrogen ions in the extracts. Results were presented as
mean of three measurements ± standard deviation (SD).
2.4. Methods for determination of active polyphenolic compounds
2.4.1. Determination of total phenolic content
Total phenolic content (TPC) in the extracts was determined
by colorimetric method using Folin–Ciocalteu reagent (FCR), with
minor modifications (Singleton and Rossi, 1965). Briefly, the reac-
tion mixture was made by mixing 0.1 mL of investigated liquid
extract, 7.9 mL of distilled water, 0.5 mL of FCR and 1.5 mL of
20% solution of Na2CO3. After 2 h of standing at temperature of
22 ± 2 ◦ C, the absorbance was measured at 765 nm using Evolu-
tion 60 UV/VIS spectrophotometer (Thermo Fisher scientific, USA).
Gallic acid (GA) was used as a standard (0.01–0.1 mg/mL), and the
results were expressed as milligrams of gallic acid equivalents per
100 g of dry plant material (mg GAE/100 g d.w.—dry weight) (equa-
tion of the calibration curve: y = 1.281x + 0.020). The measurement
was performed in triplicate, and the results were presented as
mean ± standard deviation (SD).
2.4.2. Determination of total flavonoids content
Determination of total flavonoids content (TFC) in the extracts
was performed by Markham’s method, with minor modifications
(Ordon-ez et al., 2006). This colorimetric method is based on
the complexation of flavonoids with Al3+ -ions. Briefly, 0.5 mL of
investigated liquid extract was measured and mixed with 1.5 mL
of methanol, 100 ␮L of 10% AlCl3, 100 ␮L of 1 M K-acetate and
2.8 mL of distilled water. After 30 min incubation at temperature of
22 ± 2 ◦ C, the absorbance was measured at 420 nm. Rutin was used
as a standard (0.05–0.35 mg/mL) and the results were expressed as
rutin equivalents (RE), i.e. as mg RE/100 g d.w. (calibration curve:
y = 3.397x + 0.039). Measurements were made in triplicate, and the
results were presented as mean ± SD.
168 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176
2.4.3. Determination of condensed tannins content by vanillin
assay
Condensed tannins (TT—eng. Total Tannins) were determined by
vanillin assay (Price et al., 1978) using vanillin reagent, with minor
modifications. Briefly, 1 mL of each made liquid extract was trans-
ferred into two tubes, so that each sample has a blind test. 0.5 mL of
vanillin reagent (1% vanillin in methanol: 8% HCl in methanol = 1:1
(v:v)) was added into the first set of tubes, a solution was prepared
immediately before determination. 0.5 mL of 4% HCl in methanol
was added into the second set of tubes. Between the addition of
these reagents should not take more than 1 min. The tubes were
incubated for 20 min in a water bath (30 ± 2 ◦ C). The values of
absorbance were also measured at intervals of 1 min at 500 nm,
against blank control. The absorbance of the blank (liquid extract
without vanillin reagent and with 4% HCl in methanol) was sub-
stracted from the absorbance of the sample containing the vanillin
reagent. Catechin was used as a standard (0.05–0.3 mg/mL), and the
results were shown as catechin equivalents, i.e. mg CE/100 g d.w.
(calibration curve: y = 0.055x + 0.004). Measurements were per-
formed three times, and the results were presented as mean ± SD.
2.4.4. Determination of total anthocyanins content
The percentage content of total anthocyanins (TA) in the inves-
tigated liquid extracts was determined according to the European
Pharmacopoeia (Ph Eur 6.0, 2008). Briefly, 750 ␮L of methanol
was added to 250 ␮L of investigated extract. Mixture was stirred
mechanically for 30 min and then made 50-times dilution (0.1 mL
of made solution was taken and quantitatively transferred to a
flask of 5 mL) and the flask was then supplemented with 0.1%
(v/v) solution of HCl in methanol. The absorbance was measured at
528 nm with a compensating solution of 0.1% (v/v) HCl in methanol.
The percentage content of anthocyanins was expressed through
cyanidin-3-glucoside chloride and calculated using the equation:
(A × 5000)
%TA =
(718 × m)
A—absorbance of analysis at 528 nm, m—mass of investigated drug
in grams.
2.5. Methods for determination of antioxidant activity
2.5.1. Radical-scavenging activity
To determine Radical Scavenging Capacity (RSC) of investigated
extracts, DPPH (1,1-diphenyl-2-picryl-hydrazyl) assay was used
(Brand-Williams et al., 1995), with minor modifications. Briefly,
100 ␮L of investigated liquid extract (before made in D:E ratio = 1:5)
was mixed with 900 ␮L of fresh 0.04 mg/mL methanol solution of
DPPH. Work and blind solution (solution without extract) were
incubated for 20 min in the dark at temperature of 22 ± 2 ◦ C, until
stabilization. The absorbance was read at 517 nm using a spec-
trophotometer, with methanol as the reference solution. Results
were expressed as percentage inhibition of DPPH reduction, i.e.
as %RSC. Results were presented as mean ± SD of three measure-
ments. The synthetic antioxidant butylated hydroxytoluene (BHT)
was used as a positive control. The percentage of inhibition, i.e.
%RSC was calculated using the following equation:
 
Asp − As
%RSC = × 100
Asp
%RSC—percentage of RSC, i.e. percentage of inhibition,
Asp —absorbance of the blank (containing 100 ␮L of methanol
instead sample), As —the absorbance of sample.
2.5.2. Ferric-reducing antioxidant power
To determine the total antioxidant activity, FRAP (Ferric Reduc-
ing Antioxidant Power) assay was used as a direct method to
measure the total antioxidant power of the extracts (Benzie and
Strain, 1996). Briefly, the FRAP reagent was prepared by mixing
10 mL of 300 mmoL/L acetate buffer, pH 3.6, with 1 mL of 10 mmoL/L
TPTZ in 40 mmoL/L HCI and 1 mL of 20 mmL/L FeCl3. 20 ␮L of
the investigated liquid extract (D:E = 1:5) were mixed with 1.5 mL
of a freshly prepared FRAP reagent and incubated for 10 min at
22 ± 2 ◦ C. Absorbances then were read at 593 nm. Aqueous solu-
tions of FeSO4 × 7H2 O (100–1000 ␮M) were used for calibration
and the results were expressed as FRAP value (mM Fe2+ ) of sam-
ples (calibration curve: y = 0.377x + 0.006). Results were presented
as mean ± SD of three measurements.
2.5.3. Inhibition of linoleic acid oxidation
To determine the extract ability to inhibit lipid peroxidation,
linoleic acid and thiocyanate method was used, with minor modi-
fication (Gordon and Maisuthisakul, 2009). Briefly, 0.5 mL of linoleic
acid solution was mixed with 0.5 mL of 0.05 M phosphate buffer (pH
7), then added 100 ␮L of the investigated liquid extract (D:E = 1:5)
and 150 ␮L of distilled water to obtain a mixture which was
incubated. After incubation (24 h, 40 ± 2 ◦ C), 100 ␮L of incubated
mixture was mixed with 9.7 mL of 75% methanol, 100 ␮L of 30%
ammonium thiocyanate, and 100 mL of 0.02 M in 3.5 mL of HCl. The
same procedure was performed without incubation. Absorbance
was read at 500 nm. Results were presented as mean ± SD of three
measurements. Antioxidant activity, i.e. the level of linoleic acid
peroxidation was calculated using the following equation:
 
(Ak − Au )
%AOA = × 100
Ak
%AOA—percentage of antioxidant activity, Ak —the difference in
absorbance of control (mixture without sample) with or without
incubation, Au —the difference in absorbance of sample with or
without incubation.
2.6. Statistical analysis
All results were expressed as the arithmetic mean of three
independent measurements ± standard deviation (SD). Linear
regression analyses and correlation coefficients to determine the
relationship between two variables (polyphenolic compounds and
antioxidant activity) were performed using the MS-Windows soft-
ware (Excel, 2007), with statistical significance of *P < 0.05. The
content of polyphenolic compounds in the investigated liquid
extracts and antioxidant activity of these extracts in correlation
to the applied solvents and extraction methods, were analysed by
analysis of variance (ANOVA), followed by Tukey’s t-test, with sta-
tistical significance of *P < 0.05 and **P < 0.01.
3. Results and discussion
3.1. Analysis of pH values of investigated extracts
Solvents compatible with the skin were used to prepare liquid
extracts, in order to determine their potential applications, except
in food, also in cosmetic industry, for making preparations intended
for use on the skin. The pH values of these extracts were in the rec-
ommended pH range for preparations intended for skin application
and were in the range of 3.17 in aqueous macerate to 5.48 in 80% PG
extract made by Soxhlet extraction. Results of pH values are shown
in Fig. 1.
 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176
Fig. 1. pH values of investigated extracts of wild apple fruit made using polar solvents.
3.2. Analysis of content of polyphenolic compounds in the
investigated extracts
Polyphenolic compounds have a therapeutic effect on var-
ious diseases such as cancer, cardiovascular diseases, diabetes
and atherosclerosis. Their antioxidant properties might enable the
usage of theses compounds in prevention of the mention dis-
eases. They act as “scavengers” of intermediated ROS, which are
essential for initiation of radical reactions. Also, these compounds
show anti-inflammatory activity. One of the major sources of these
polyphenolic compounds is food and supplements of plant ori-
gin (Cekic and Ozgen, 2010). It is known that among other edible
fruit, apple has the highest content of free polyphenolic compounds
with the highest bioavailability for eventual absorbtion into the
bloodstream. The content of polyphenolic compounds (phenols,
flavonoids, tannins, anthocyanins) varied between different fruit
cultures. Even, Belviso et al. (2013) have shown that the polyphe-
nolic content of the same plant was changed over a three-years of
study period. Antioxidant potential and the content of polypheno-
lic compounds in fruits are very difficult to evaluate and compare,
because of using different extraction methods, solvents, different
varieties of apples, parts of plants, fruit, sun exposure (Carbone
et al., 2011; Kalinowska et al., 2014; Rössle et al., 2010).
Extraction of bioactive polyphenolic compounds from the
medical plant material is widely used in the production of phyto-
preparations, for oral use and for use on the skin. The proper choice
of solvent and extraction method is very important for good extrac-
tion of active principles, as well as cost-extraction process (Savić
et al., 2013). Extraction is the most important phase of polyphenolic
isolation (Milutinović et al., 2013). There are some polypheno-
lic compounds that are bound, so they can not be identified and
extracted using only one method or solvent, or in order to prevent
oxidation of polyphenols it is necessary to apply different solvents
and extraction methods (Kalinowska et al., 2014).
In this context, in our stady, different solvents and extraction
methods were used for extraction of polyphenolic compounds in
order to determine their influence on the polyphenolic content and
antioxidant activity of investigated liquid extracts of wild apple
fruit. Results of the total content of phenols, flavonoids, tannins
and anthocyanins in investigated samples are shown in Fig. 2.
Depending on used solvents and applied extraction method,
obtained liquid extracts of wild apple fruit showed different con-
tent of polyphenolic compounds, as well as a different biological
activities. Solubility of polyphenolic compounds as well as the
efficiency of the extraction depends on the polarity, pH and selec-
tivity of the solvent (Milutinović et al., 2013), so we used polar
and non-polar solvents for extraction. Also, the efficiency of the
solvent depends on the physical and chemical characteristics of
tested polyphenolic compounds (polarity, interaction with other
compounds in the investigated plant material, stability) (Franquin-
Trinquir et al., 2014). Extraction by using several solvents (one
169
after another or a mixture of solvents) provides better separa-
tion and isolation of polyphenolic compounds (Kalinowska et al.,
2014), so we extracted polyphenols from wild apple fruit using
mixture of solvents (water–ethanol and water–PG system), and two
solvents, one after another (in case of ethanol-oil system). Polyphe-
nols are often more soluble in solvents which are less polar than
water. Franco et al. (2008) investigated the effect of solvents on
the polyphenolic content and antioxidant activity of extracts from
seeds of different plants and found that ethanol, as less polar sol-
vent than water, gave a better yield of extraction expressed through
DPPH radical inhibition, which is in correlation with our results,
where 70% ethanol has proven to be a very good extraction agent.
Water as a solvent has many advantages versus organic solvents,
primarily because of safety and economy, but it is less selective
solvent, and the aqueous extracts, among the polyphenols, usu-
ally contain a large amount of other components. Beside that, the
pH value of water as a solvent can significantly affect on extrac-
tion of some polyphenolic components (Milutinović et al., 2013),
as in our case, with tannins and anthocyanins whose content was
generally higher in the water extracts. In our work, the extrac-
tion with distilled water gave average, but a satisfactory content
of total polyphenolic compounds and a relatively good antioxi-
dant capacity. Water-cosolvent system has been quite investigated
for extraction of a large number of biologically active compounds,
and the most water–ethanol system (Milutinović et al., 2013). The
water–propylene glycol system, on the other hand, has not been
so investigated in increasing the efficiency of extraction of natural
compounds, despite it’s well-known pharmaceutical application,
including solubility increase of lipophilic drugs in water, as well
as the use as preservative in medicine and food industry. Further,
propylene–glycol has been generally recognized as safe and secure
substance by the Food and Drug Administration (FDA), the aque-
ous system with up to 40% of propylene–glycol is considered to
be non-toxic for human oral administration, and in the cosmetic
industry, the propylene-glycol is considered to be very good emol-
lient (Tubtimdee and Shotipruk, 2011). Oils such as sunflower and
olive oil are regularly used in food industry, and have very good
cosmetic properties (Arsić et al., 2011). The use of 96% ethanol in
the extraction with oil was additionally increased content of active
principles (almost twice) as well as the antioxidant capacity of oil
extracts with ethanol, compared to the extraction with oil solvents
only, and maceration and digestion with oil generally showed the
best content of polyphenolic compounds.
Determination of total phenolic content by Folin–Ciocalteu
(FeC) method is often used test, because it enables very quickly
and easily determination of the level of total phenols in dietary
supplements and cosmetic products of plant origin (Kalinowska
et al., 2014; Kubola et al., 2011). But, it is very difficult to compare
results obtained by different authors, mainly due to the application
of different methodologies (different extraction procedure, solvent,
temperature . . .) as well as the expression of the obtained results
170 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176

Fig. 2. Content of (a) total phenols (TPC), (b) total flavonoids (TFC), (c) total tannins (TT) and (d) total anthocyanins (TA) in the investigated liquid extracts of wild apple fruit.

for total phenol content as different equivalents of gallic, caffeic teins, thiols, vitamin derivatives, inorganic anions) (Carbone et al.,
and chlorogenic acid. Also, FeC reagent, except with phenols, can 2011; Kalinowska et al., 2014). Content of TP in our investigated liq-
react with many other active compounds in plant extracts (pro- uid extracts ranged from 172.91 mg GAE/100 g d.w. (solvent-pure
 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176
sunflower oil) to 1556.99 mg GAE/100 g d.w. (80% propylene-glycol
extract made by Soxhlet extraction). Better solubility of phenols
was showen in polar solvents than in non-polar solvents, and
ultrasonic extraction was generally good extraction method that
showed good TPC for all polar solvents.
Flavonoids, as one of the most diverse and the largest group
of natural compounds are very important natural polyphenols
(Prasad et al., 2009). In addition to their antioxidant activity,
they have a wide spectrum of biochemical and pharmacologi-
cal capabilities, such as anti-inflammatory, anti-viral, anti-allergic,
anti-carcinogen, anti-oxidative and “anti-age” activity (Kubola
et al., 2011). The highest values of TFC showed the extract with
pure olive oil as solvent, 182.22 mg RE/100 g d.w., and the low-
est percentage content of anthocyanins (0.70%), while the lowest
TFC showed aqueous extract made by percolation, 3.97 mg RE/100 g
d.w. Extraction with solvent system 96% ethanol-sunflower oil gave
the best percentage content of anthocyanins (14.63%). Jakobek et al.
(2013) also showed a good content of polyphenolic compounds of
local varieties of apples from southeast European region, and these
results are similar to ours, especially anthocyanins content (1–7%).
The content of anthocyanins, as the most abundant compounds in
the apple fruit, is a very important qualitative parameter of wild
apple fruit, because of the color that antocyanins give to fruits, as
well as of the antioxidant effect. The content of anthocyanins in the
wild apple fruit varied, depending on the type, climatic conditions,
work methodology, which additionally complicate the comparison
of the results with the literature (Jakobek et al., 2013; Kunradi Vieira
et al., 2009).
Tannin content ranged from 72.73 mg CE/100 g d.w. in 80% PG
extract made by ultrasonic extraction to 8872.73 mg CE/100 g d.w.
in the extract obtained by extraction with 96% ethanol and olive
oil. In general, the content of tannins and anthocyanins was signifi-
cantly better in aqueous extracts compared to other extracts made
using other polar solvents, but the great solubility of flavonoids,
tannins and anthocyanins was in non-polar solvents, especially in
ethanol-oil system, where extraction was first carried out with 96%
ethanol (as very good solvents for extraction from plant materials),
and then oil was incorporated in extract, with removing ethanol at
same time.
Extracts obtained by 80% PG as solvent showed statistically
higher content of phenols compared to extracts with water as sol-
vent (*P < 0.05), while the extracts with 70% ethanol as solvent had
higher content of flavonoids compared to water extracts (**P < 0.01)
(Fig. 2). There was no statistically significant difference in the con-
tent of phenols and flavonoids among the other extracts. Also, there
was no statistically significant difference in the content of tannins
and anthocyanins in the investigated extracts in relation to the
using solvents, but non-polar solvents generally showed very good
content of polyphenolic compounds. Analysis also showed no sta-
tistically significant difference in the content of active principles in
relation to the applied extraction method (*P > 0.05), but ultrasonic
extraction for polar solvents generally gave almost the best content
of active principles, and digestion for non-polar solvents (Fig. 2).
Extracts in our study that showed very good antioxidant
activity as well as TPC (ethanol extracts) had similar values as
the TPC from the literature data (7.36–10.20 mg/g d.w.) (Amzad
Hossain et al., 2009). One study showed that the content of
TP (73.59–74.6 ␮g/g—whereby our results were higher) and TF
(40.56–52.30 mg/100 g—similar to our results) of wild apple fruit
was better compared to cultivated varieties of apples (Maria John
et al., 2014). Imeh and Khokhar (2002) also showed good TPC
(302.3–534 mg GAE/100 g) and TFC of wild apple fruit compared
to the cultivated species, and they were in the range of our results.
Also, our research showed slightly better content of TP and similar
TFC of the wild apple fruit versus published results of cultivated
local varieties of apples from our region (TPC 9.37–1440 mg/100 g
171
f.w.—fresh weight) (Šavikin et al., 2014). One study showed the
influence of different solvents and the methodology on the TPC,
where the results were similar to ours (Franquin-Trinquir et al.,
2014). Kalinowska et al. (2014) also showed increased level of
polyphenolic compounds in organically cultivated and wild apple
than in cultivated cultures, which can be a result of the action
of plant defense mechanisms. They also showed the influence
of different solvents and extraction methods on the content of
polyphenolic compounds in apple fruits (extracts of two apple
species obtained at 60 ◦ C with solvent system ethanol–water had
TPC 3.49 and 6.80 mg GAE/g, whereby we showed better content
of polyphenolic compounds). The application of classical method
(maceration), Soxhlet extraction or extraction in ultrasonic bath
gave a different structure and content of polyphenolic compounds
in the tested varieties of apples (Kalinowska et al., 2014). Our results
for the TPC were higher compared to some literature data for TPC in
apple fruits (160.3–4727.8 mg GAE/kg f.w.—fresh weight), TFC was
lower in our study (114.2–3045.3 mg RE/kg f.w.), while content of
anthocyanins was between the 12.1 and 1758.4 mg C3GE/kg f.w.
(cyanidin-3-glucoside equivalents/kg fresh weight) (Xiaoqian et al.,
2014).
3.3. Analysis of antioxidant activity of investigated extracts
Apples are a good source of bioactive polyphenolic compounds
(Altisent et al., 2014). The investigation of the antioxidant potential
of wild apple fruit was performed to estimate the possible applica-
tion of wild apple fruit from Serbia as a rich source of antioxidant
substances, due to the fact that antioxidant supplements in nutri-
tion and antioxidant cosmetic products can be used in prevention
and/or treatment of many diseases caused by the action of free
radicals (Lesjak et al., 2011). There is no consensus regarding the
standard methodology for determining antioxidant activity, so it
is the best to use different methods to estimate the antioxidant
activity of fruits (Kunradi Vieira et al., 2011). Antioxidant activ-
ity can be manifested in different ways, as inhibition of oxidative
enzymes, donation of hydrogen ions to the free radicals, free radical
“scavenging”, lipid peroxidation prevention or metal ions chela-
tion. Therefore, the antioxidant activity should be measured in vitro
by using several different antioxidant methods in order, as much
precisely as in vitro methods allow, to determine the antioxidant
potential of compounds or extracts. Namely, it is known that use
of only one assay is not applicable (Lesjak et al., 2011; Žugić et al.,
2014). In complex systems, such as natural extracts, with different
polyphenolic components it is useful to make a comparative exam-
ination of antioxidant activity by different methods (Milutinović
et al., 2013). Therefore, in our research we applied three spec-
trophotometric assays, different in their working principles.
The antioxidant activity of tested extracts of wild apple fruit
was investigated by radical scavenging activity determination,
using the DPPH method, by determination of total antioxidant
capacity, using the FRAP method and ability to prevent lipid
peroxidation was determined using linoleic acid. DPPH method
determines the capacity of bioactive molecules for donating hydro-
gen and it is based on monitoring the transformation of purple
colored stable DPPH radicals into the reduced yellow form of
DPPH-H. The degree of discoloration indicates the antioxidant
potential of the extracts. Antioxidants from the wild apple fruit
react with free DDPH radical and thus interrupt the oxidation
chain reaction by donating hydrogen from the hydroxyl group of
polyphenols. They also provide a stable final product and pre-
vent the lipid oxidation (Carbone et al., 2011; Milutinović et al.,
2013). FRAP method belongs to the methods which determine
the antioxidant properties, which are based on electron transfer,
whereby the capacity of antioxidants to reduce present oxidative
172 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176

component is monitored by color change. At low pH, the reduc- Table 1

Correlations between antioxidant capacities and main polyphenolic compounds in
tion of ferric-2,4,6-tripyridyl-s-triazine [Fe3+ –TPTZ] complex into
the investigated extracts of wild apple fruit.
a ferrous-2,4,6-tripyridyl-s-triazine [Fe2+ –TPTZ] complex, which
*
has an intense blue colour, can be monitored by measuring the Compound Method R Regression formula P
absorbance at 593 nm. The degree of colour change is proportional TPC DPPH 0.0914 a d
y = 0.0051x + 44.2222 0.70156
to the concentration of antioxidants. In contrast to the inhibition FRAP 0.2703 yb = 0.0012xd + 4.9895 0.24901
of the DPPH radical, which is suitable for antioxidants soluble in Lipid perox. 0.4073 yc = 0.0101xd + 65.3061 0.07469

organic solvents (ethanol), FRAP method is applicable for estima- TFC DPPH 0.4394 ya = −0.1834xe + 58.1576 0.05257
tion of the antioxidant properties of water-soluble antioxidants. FRAP 0.5433 yb = −0.0185xe + 7.0492 0.01330
Lipid perox. 0.4527 yc = −0.0840xe + 78.8109 0.04502
The test with linoleic acid might be suitable for assessment of pre-
vention of lipid peroxidation, and it is usually combined with DPPH TT DPPH 0.6659 ya = −0.0062xf + 58.7554 0.00135
method; this method should be the best suited for oil extracts. FRAP 0.4512 yb = 0.0003xf + 6.6607 0.04584
Lipid perox. 0.1819 yc = −0.007xf + 75.7669 0.44280
Results of DPPH assay were presented as %RSC (Radical Scaven-
ing Capacity), of FRAP assay as mM Fe2+ , and of assay with linoleic TA DPPH 0.4903 ya = −3.2316xg + 62.6796 0.02817
acid as %AOA (Anti Oxidant Activity). In all assays, there is a direct FRAP 0.1221 yb = −0.0656xg + 6.3962 0.60799
Lipid perox. 0.1046 yc = −0.3066xg + 75.8775 0.66060
correlation, i.e. higher values indicate higher antioxidant activity.
*
%RSC values of investigated liquid extracts (D:E = 1:5) of wild P: statistical significance (*P < 0.05).
a
y: Radical Scavenging Capacity (%RSC).
apple fruit ranged from 8.12 %RSC (80% PG extract made by Soxh- b
y: FRAP value (mM Fe2+ ).
let extraction) to 78.43 %RSC (ethanol extract made by ultrasonic c
y: AntiOxidant activity (%AOA).
extraction). The lowest FRAP value showed the extract with pure d
x: total phenols content (mg GAE/100 g d.w.).
sunflower oil (2.13 mM Fe2+ ), and the highest ethanol percolate e
x: total flavonoids content (mg RE/100 g d.w.).
f
(7.65 mM Fe2+ ). Ethanol extract made by ultrasonic extraction x: total tannins content (mg CE/100 g d.w.).
g
x: total anthocyanins content (%).
showed the best prevention of lipid peroxidation (95.32 %AOA),
which also showed the best free radical scavenging, while the low-
est ability to prevent lipid peroxidation showed 45% PG extract
made by Soxhlet extraction (57.73 %AOA). Oil extracts showed the a good content of polyphenolic compounds and a very good antiox-
best antioxidant activity using the test with linoleic acid versus idant activity in relation to the content of polyphenolic compounds
other two antioxidant tests, as we expected. The results are shown (45% PG extracts showed the best antioxidant activity using DPPH
in Fig. 3. and FRAP test, and 80% PG extracts using the test with linoleic
Maria John et al. (2014) showed that the methanol extract of acid). Since the water is considered to be the safest and solvent
wild apple fruit had better antioxidant activity (89.27 % RSC), which for both, oral and topical administration, as well as the most eco-
was slightly higher than our results, compared to cultivated vari- nomical, our water extracts showed good content of polyphenolic
eties of apple. Cekic and Ozgen (2010) and Kubola et al. (2011) also compounds, as well as antioxidant activity (much better through
showed better antioxidant activity of wild cultures than cultivated. the test with linoleic acid compared to other two tests). Ultra-
They showed, too, that the maceration is the best method for the sonic extraction improved the antioxidant properties of aqueous
extraction of active principles and to obtain extracts with the high- extracts, too (%RSC = 62.37%, mMFe2+ = 7.37, %AOA = 80.14%). Oil
est antioxidant potential. Amzad Hossain et al. (2009) showed a extracts, as non-polar solvents, gave a good content of active princi-
good antioxidant activity of investigated wild apple fruit extracts ples, but the lowest antioxidant activity compared to polar solvents
using linoleic acid (62.82–92.34 %AOA), which was in correlation (the best activity was shown using the test with linoleic acid, %AOA
with our results. ranged from 62.21% to 66.88%). Also, the use of 96% ethanol in the
Statistically significant difference was not found in the antiox- digestion as extraction method, improved antioxidant activity of oil
idant activity of investigated extracts in relation to applied extracts in all three test, twice more using DPPH and three times
extraction methods (*P > 0.05), but in our study, ultrasonic extrac- using FRAP assay (sunflower oil), but antioxidant activity of these
tion was the best method, because the extracts prepared by this extracts was still lower compared to extracts made using polar
method had generally almost the highest antioxidant activity com- solvents, despite to high content of total flavonoids, tannins and
pared to extracts made by other methods and using the same anthocyanins. It might be because of lower content of total phenols,
solvent. Also, the adventages of this extraction method are better bearing in mind that they make their biggest part of. Some another
penetration of solvent into plant material, low extraction tempera- explanation might be that oil can good extracted polyphenolic
ture, reduced extraction time, but the maceration as a conventional compounds, but it is possible that after extraction, oil emulsified
method is simpler and more economical. Maceration method also antioxidant compounds, so they readily release them later, and
gave good results (Fig. 3). polyphenols can not demonstrate their antioxidant activity. Diges-
Comparing the antioxidant activity of investigated extracts in tion was better method for extraction of antioxidant compounds,
relation to using solvents, the analysis showed that the extracts and for antioxidant activity, compared to maceration with oil, prob-
obtained using 80% PG as solvent showed statistically lower values ably because of ethanol, as co-solvent, but these oil extracts showed
of %RSC (DPPH assay) compared to extracts obtained using 45% PG lower antioxidant activity compared to extracts made using polar
and 70% ethanol as solvents (**P < 0.01) and compared to water solvents, probably because of the same reasons as extracts made
extracts (*P < 0.05) (Fig. 3). There was no statistically significant by maceration with oil.
difference in the antioxidant activity of these extracts, using the The antioxidant activity of investigated extracts of wild apple
FRAP assay and assay with linoleic acid (**P > 0.05). Polar solvents fruit was dependet on used solvent and content of polypheno-
generally showed better antioxidant activity then non-polar. lic compounds. The difference in the antioxidant capacity that
70% ethanol has proven to be a very good solvent for extraction investigated extracts demonstrated, could be attributed to method-
of polyphenolic compounds (these extracts showed good content of ologically different antioxidant tests. DPPH test is based on free
polyphenolic compounds) and these extracts generally showed the radicals elimination, the FRAP test demonstrate the capability of
best antioxidant activity through all three assays (as the best—70% the investigated extracts to provide the ions reduction, and the test
ethanol extract made by ultrasonic extraction—%RSC = 78.43%, with linoleic acid might be suitable for assessment of prevention of
%AOA = 95.32% and mM Fe2+ = 7.65). Propylene–glycol extracts gave peroxidation. The correlation between the antioxidant capacities
 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176 173

Fig. 3. Antioxidant activity of investigated liquid extracts of wild apple fruit. (a) DPPH assay—(%RSC), (b) FRAP assay—(mM Fe2+ ), (c) Assay with linoleic acid (%AOA).
174 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176

Fig. 4. Correlation of (a) TPC and DPPH free radical scavenging activity (%RSC), (b) TPC and FRAP (mM Fe2+ ), (c) TPC and AOA (%), (d) TFC and DPPH free radical scavenging
activity (%RSC), (e) TFC and FRAP (mM Fe2+ ), (f) TFC and AOA (%), (g) TT and DPPH free radical scavenging activity (%RSC), (h) TPC and FRAP (mM Fe2+ ), (i) TPC and AOA (%),
(j) TA and DPPH free radical scavenging activity (%RSC), (k) TPC and FRAP (mM Fe2+ ), (l) TPC and AOA (%).

and the major polyphenolic compounds are shown in Table 1 and
Fig. 4.
Antioxidant activity, generally, increased proportionally with
increasing of content of polyphenolic compounds. An impor-
tant correlation between antioxidant activity and the content of
polyphenolic compounds existed in most extracts (R > 0.4), with
statistical significance of *P < 0.05. The best linearity was shown
between a tannins content and %RSC (R = 0.6659), as well as a
flavonoids content (R = 0.4394) in the DPPH assay, and (R = 0.5433)
in the FRAP assay (Table 1, Fig. 4). However, slight and low linear
correlation (R < 0.4) was detected between phenols and antioxi-
dant activity, but without statistical significance (*P > 0.05) (Table 1,
Fig. 4). One explanation for high antioxidant activity can be the
presence of other reducing compounds such as sugars, amino
acids and ascorbic acid, which can react with FC reagent (Almeida
et al., 2008; Georgé et al., 2005). Because of this, linear correlation
 D. Stojiljković et al. / Industrial Crops and Products 80 (2016) 165–176
between the TPC and antioxidant capacity only for the 70% ethanol
extracts were done, which generally showed the highest antiox-
idant activity, and very high correlation was demonstrated, even
(R = 0.9495), with *P < 0.05. The content of phenols and tannins in
the previously published results from our region (Žugić et al., 2014)
very well correlated with antioxidant activity, as we showed, too,
with a satisfactory results for the TT, while there was no correla-
tion for the content of TP. Low values of correlation between TPC
(R = 0.2076) and %RSC, and using FRAP assay (R = 0.0245) showed
the same authors (Iqbal et al., 2012; Žugić et al., 2014), while our
results showed a good correlation between flavonoids content and
antioxidant activity. Also, our results were in contrast with the
results published by Kunradi Vieira et al. (2011) for TPC (they found
significant correlation R = 0.8468), but it might be partly explained
by the climate influencing the polyphenolic content (McGhie et al.,
2005). Since we used the same wild apple fruit, the difference in the
content of polyphenolic compounds and antioxidant activity was
dependet on the used solvents and extraction methods, whereby,
extracts made by ultrasonic extraction gave the best results of
antioxidant activity using 70% ethanol as solvent and very good
results using distilled water as solvent.
4. Conclusions
Wild apple fruit represents a good source of bioactive polyphe-
nolic compounds. Depending on used solvents, polar or non-polar
(70% ethanol, 45% propylene-glycole, 80% propylene–glycole, dis-
tilled water, olive oil and sunflower oil, and 96% ethanol in solvent
system ethanol:oil) and used extraction methods (maceration, per-
colation, Sohxlet and ultrasonic extraction) we obtained extracts
with content of total phenols ranged from 172.91 to 1556.99 mg
GAE/100 g d.w., total flavonoids from 3.97 to 182.22 mg RE/100 g
d.w., total tannins from 72.73 to 8872.73 mg CE/100 g d.w. and
total anthocyanins from 0.70 to 14.63%. Content of these active
substances caused different antioxidant activity of extracts, which
was determinated using three different tests (DPPH test, FRAP test
and test with linoleic acid). Extracts made using 70% ethanol and
distilled water as solvents and ultrasonic extraction as extraction
method showed a good polyphenolic content and the best antioxi-
dant activity. Interesting results were obtained for the investigated
samples proving to be rich in phenolic constituents and demon-
strating satisfying antioxidant activity, while giving the evidence
of good correlation between antioxidant properties and phenol
compounds content, as well (especially high in ethanol and water
extracts). Due to these results, wild apple fruit extracts might rep-
resent a valuable source of natural antioxidants, and thus may be
considered as a great potential for application in food (or dietary
supplements production) and cosmetic industry representing fea-
sible alternatives to synthetic additives.
Acknowledgements
Serbian Ministry of education, science and technological devel-
opment supported this study through the Research Project
“Functional and physiologically active plant materials with added
value for usage in pharmaceutical and food industries” (Project No.
III 45017).
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