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Journal Name, Year, Volume 1
Assessment of Total Phenolic Content and Antioxidant Activity of Ficus
carica and Olea europaea L. Leaves Extracts
Abderrahim BENSLAMA*a, Amirouche DEGHIMAb and Nadjat RIGHIc
a
Department of Biochemistry and Microbiology , Faculty of Sciences, University of Msila, Msila, Algeria.
b
Department of Nature and Life Sciences, Faculty of Exact, Nature and Life Sciences, University of Biskra, Algeria.
c
Laboratory of Characterization and Valorization of the Natural resources, University of Bordj Bou Arerridj, Algeria.

*Correspondence: benslama_abderrahim@hotmail.fr; Tel.: +213-664-486-6391

Abstract:
Background: Ficus carica and Olea europaea are two medicinal plants are widely distributed in Algeria, and are used
as food and in Algerian folk medicine
Objective: The aim of this work is the valorization of the Algerian flora; by the evaluation of the antioxidant capacity of
differents extracts of F. and O. europaea.
Methods: The dried leaves of plants of Ficus carica and Olea europaea were submitted to sequential extraction with
solvents of increasing polarity to give hexane, chloroform, ethyl acetate and methanolic extracts. The total phenolic and
flavonoids was determined spectrophoto-metrically. The antioxidant activity of extracts was evaluated using 1,1’-
diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging and ferric reducing power test.
Results: The results of the quantitative analysis showed that the methanolic extract (ME) give the highest yield with
16.43% and 19.98% for F. carica and O. europaea respectively. The analysis showed that the highest amounts of phenolic
compounds was recorded in the ME of O. europaea (171.40±6.79 µg GAE/ mg E), when the the highest amounts of
flavonoids was recorded in the CHE of the F. carica.( 34.06± 0.05 µg QE/ mg E). The results showed that the EAE
exhibited the highest antiradical activity against DPPH free radical with an EC 50=45.21±1.12 and 8.20±0.37 µg/ml for F.
carica and O. europaea, respectively. Moreover, the EAE of the two plants present the highest reducing effect compared
to authors extracts at concentration of 200 µg/ml.
Conclusion: Our results revealed the presence a height correlation between the antioxidant activity of extract and the
total phenolic content of this extract was reported. Moreover, the result reported that the EAE had a considerable antioxidant
capacity, may be an alternative antioxidants used as additives in food and pharmaceutical preparation.
Keywords: Ficus carica, Olea europaea, phenolic compounds, antioxidant activity, free radical, reducing power.

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Journal Name, Year, Volume
1. INTRODUCTION
The plants have been used for generations by the people
as medicines for health care and treatment of diseases.
Natural products contribute largely to the today drug
development industry and constitute an affordable and
available system of health care for people in the developing
countries [1]. Most of the therapeutic effects of the medicinal
plants are due to their content in secondary metabolites that
include aromatic substances like polyphenols and their
oxygen-substituted derivatives. Many of these compounds
have antioxidant properties [2]. Oxidative stress is the
common point of many types of disease, such as
hypertension, atherosclerosis, acute renal failure, diabetes
mellitus, and neurodegenerative disorders. This alteration
occurs when there is an imbalance between the production of
reactive oxygen species (ROS) and the ability of the
biological system to neutralize these reactive intermediates
or repair the resulting damage [3]. Plants naturally produce
antioxidants for their defense and to counter the oxidative
stress caused by the production of ROS during
photosynthesis and thus represent a good source of new
antioxidant compounds. Lately, there has been an increase in
the scientific interest for the natural antioxidants present in
traditional medicinal plants, not only for their presumed
safety but also for their nutritional and therapeutic value and
for the will to replace the synthetic antioxidants that are
commonly used in processed foods, such as butylated
hydroxytoluene (BHT) and butylated hydroxyanisole (BHA),
which have side effects and have been reported to be
carcinogenic [4,5]. The aim of this work is the valorization
of two Mediterranean medicinal plants, Ficus carica and
Olea europaea that are widely distributed in Algeria, and are
used as food and in Algerian folk medicine, by evaluating
the antioxidant capacity of their leaves extracts.
2. MATERIALS AND METHOD
2.1. Materials
The plant materials were collected in November 2016,
from Biskra (south of Algeria), authenticated by Dr.
Salemkour Noura from the center of scientific and technical
research on arid regions (C.S.T.R.A.R), Biskra, Algeria. The
leaves were dried at room temperature in the dark then
crushed up and stored until use. For chemicals and reagents:
Aluminum chloride (AlCl3), Potassium ferricyanide
[K3Fe(CN)6], 1,1’-diphenyl-1-picrylhydrazyl (DPPH),
Sodium carbonate (Na2CO3), Folin-Ciocalteau reagent,
Ascorbic acid; Quercetin; andBbutylated hydroxyanisole
(BHA), Trichloroacetic acid (TCA) and Iron chloride
(FeCl3).
2.2. Methods
2.2.1. Extraction
The dried powdered leaves of both plants have been used
for the extraction following the method of [6]. Briefly, 100 g
of the plant materiel was submitted to sequential extraction
using solvents of increasing polarity to afford, hexane,
chloroform, ethyl acetate and methanolic extracts
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2
respectively. The extracts were dried under reduced pressure
using a rotatory evaporator and stored at room temperature
until use.
2.2.2. Total phenolic content
The total phenolic content (TPC) of the extracts were
measured using the Folin-Ciocalteau colorimetric method
described by [7], with gallic acid as standard. Basically, 0.2
ml of extract suitably diluted was mixed with 1 ml of Folin-
Ciocalteu’s phenol reagent (1:10 fold diluted). After 4 min,
0.8 ml of Na2CO3 (7.5 %) solution was added and the
mixture was allowed to stand for 2 h. Absorbance was
measured at 765 nm. The amount of total polyphenols in
different extracts were expressed as µg of gallic acid
equivalent (GAE)/ mg extract.
2.2.3. Total flavonoids content
The total flavonoids content of extracts (TFC) was
quantified using aluminum chloride reagent (AlCl 3) method
described by [8]. Briefly, 1 ml of extract, dissolved in
corresponding solvent was added to 1 ml of AlCl3 (2%). The
absorbance was measured at 430 nm, after incubation at
room temperature for 10 min. The flavonoids content was
expressed as µg quercetin equivalents (QE)/ mg extract.
2.2.4. Free radical scavenging using DPPH assay
The antioxidant activity of the different extracts was
evaluated using the DPPH free radical scavenging assay
according to [9]. 1ml of 0.1 mM DPPH methanolic solution
was mixed with 3 ml of varying concentrations of the
extract, the reaction mixture was shaken and incubated in the
dark for 30 min, the absorbance was taken at 517 nm. For the
control, the extract was replaced with methanol. BHA and
BHT; two synthetic antioxidants were used as standards.
Percent inhibition was calculated using the following
formula: Radical-scavenging activity (%)=(AC−AS/AC)
×100
Where AC and AS are respectively the control and the
sample absorbance.
2.2.5. Reducing power
The ability of extracts to reduce ferric ions (Fe +3) was
estimated by the method described by Bneslama [10]. 800 µl
of extract was mixed with 400 µl phosphate buffer (0.2 M,
pH=6.6) and 800 µl of a 1% potassium ferricyanide [K 3Fe
(CN)6], then the mixture was incubated at 50°C for 20 min.
800 µl (10%) of trichloroacetic acid (TCA) was added to the
mixture and centrifuged for 10 min. Finally, 400 µl of the
supernatant solution was mixed with 400 µl of distilled water
and 80 µl FeCl3 (0.1%) and the absorbance was recorded at
700 nm. Increased absorbance of the reaction mixture
indicated increased reducing power.
2.3. Statistical analysis
Experimental results were expressed as mean±SD of
triplicate. The data were analysed by Student’s t-test to
determine statistical significance. p-values and p<0.05 was
considered as indicative of significance. The EC50 values was
© 2014 Bentham Science Publishers
Short Running Title of the Article
calculated from linear regression analysis. All statistical
analysis and graphing of data were performed using Graph
pad prism 6 software.
3. RESULTS AND DISCUSSIONS
3.1. Extraction and quantitative analysis
The extraction was carried out following the method of
[6] by sequential extraction using solvents of increasing
polarity. The extraction resulted in four extracts for each
plant, hexane extract (HE), chloroform extract (CHE), ethyl
acetate extract (EAE) and methanolic extract (ME) giving
different yields (Table 1). The TPC and the TFC in the
extracts were determined spectrophotometrically using
Folin-Ciocalteu and aluminum chloride reagents,
respectively. The content of total phenolic was expressed in
terms of µg GAE/ mg dry extract, while the content of
flavonoids was expressed in terms of µg QE/mg dry extract.
The results showed that the highest amounts of phenolic
compounds was recorded in the ME of O. europaea
(171.40±6.79 µg GAE/ mg E), when the the highest amounts
of flavonoids was recorded in the CHE of the F. carica
(34.06± 0.05 µg QE/mg E).
The study of medicinal plants starts with the pre-
extraction and the extraction procedures, which is an
important step in the processing of the bioactive constituents
from plant materials. There are several extraction methods
and using different solvents to extract the active compounds
from plants [6]. The yield of extraction varies according to
the plant species, the organ used in the extraction, the drying
conditions and the richness of each species with secondary
metabolites [11]. The region and harvest period are also
determinants for the yield [12]. Moreover, recovery of
phenolic compounds also depends on the type of solvent
used, its polarity and the solubility of the phenolic
compounds in extraction solvents [13]. Solvents could
significantly affect total phenolics due to differences in
solvent polarities, which could influence the solubility of
various constituents present in plants parts [14].
The total phenolic contents in plant extracts depend on
the type of extract, i.e. the polarity of solvent used in
extraction. High solubility of phenols in polar solvents
provides high concentration of these compounds in the
extracts obtained using polar solvents for the extraction [15].
3.2. Antioxidant activity
3.2.1. Free radical scavenging
DPPH radicals are widely used as model system to
investigate the scavenging activities of several natural
antioxidant compounds [9]. DPPH radical scavenging
activity of the extracts is shown in Fig. 1. The results showed
that the ME and the EAE of O. europaea exhibited the
highest scavenging activity against DPPH radical with EC 50=
17.18±1.36 and 8.20±0.37 µg/ml respectively, the two
extracts have very good scavenger effects, better than that of
the Vit. C used as standard. In addition, the ME of F. carica
have a higher scanvengig activity compared to other extracts
of the same plant (EC50=45.21±1.12 µg/ml). Moreover, the
ability of extracts of O. europaea to scavenge the DPPH free
Journal Name, 2014, Vol. 0, No. 0 3
radical is higher than the ability of F. carica extracts,
exception the HE. In fact, the scavenger effects of extracts
were very far from the standards used BHA and Vit. C
(Exception for the EAE of O. europaea).
The bioactive secondary metabolites have been shown to
reduce the risk and progression of diseases such as cancer,
cardiovascular, neurodegenerative diseases, etc. by
scavenging free radicals through various biological
mechanisms [16]. The anti-radical capacity is very important
to determine the antioxidant activity of compounds and
molecule, this importance due to the deleterious role of free
radicals in biological systems. Diverse assays are used to
evaluate the antioxidant capacity of the natural product from
plant. Chemical methods are based on the capacity of
compounds to scavenge the synthetic free radicals [17]. As
shown in this study, there is a considerable correlation
between total polyphenol contents and the antiradical activity
of extract. Many studies indicate a linear relationship
between total phenolics and antioxidant activity [18-20].
Indeed, it has been reported that the compounds extracted by
the highly polar solvents exert a greater scavenging effect
than those extracted by low-polar solvents [21]. The
antiradical activity of natural antioxidants such as phenolic
acid and flavonoids may be due to their richness by
hydroxyl-ring. The spatial configuration and the number of
OH group of flavonoid structures can influence the different
antioxidant mechanisms [22].
3.2.2. Reducing power
The capacity of plant extracts to reduce Fe +3 was assessed
by the method described by Benslama [10]. The potassium
ferricyanide reduction method is a widely-used method for
evaluating the reducing power of plant extracts. In this assay,
the extract reduced the iron+3/ferricyanide complex to the
ferrous form by donating an electron, and it was compared to
that of ascorbic acid, which is known to be a strong reducing
agent. The reducing power of a compound serves as a
significant indicator of its potential antioxidant [23]. As
shown in Fig. 2, the reducing powers of the of O. europaea
extracts was higher than of the Ficus carica extracts
(exception CHE) at concentration of 200 µg/ml. Moreover,
the EAE of O. europaea showed the best reducing power
with an absorbance (A=1.017±0.06), followed by the ME of
the same plant (A=0.405±0.05). The Vit. C present a high
reducing power with A=1.622±0.08 at concentration of 200
µg/ml. of In addition, the result revealed a high correlation
between reducing effect of extracts and their content of
polyphenols, this shows that the phenolic compounds are the
effective element in this extracts. Another reaction pathway
in electron donation is the reduction of an oxidized
antioxidant molecule to regenerate the active reduced
antioxidant [24].
Reducing power is a very important aspect for the
estimation of the antioxidant activity. Moreover, higher ferric
antioxidant reducing activity at a lower concentration of
litchi extract indicates its potential as an antioxidant for food
application at the commercial level [25]. Previous studies
have also reported that the higher reducing power is
associated with more antioxidant activity. They have
demonstrated the reducing power of polyphenols, its appear
4 Journal Name, 2014, Vol. 0, No. 0
to function as good electron and hydrogen-atom donors and
therefore should be able to terminate radical chain reactions
by converting free radicals to more stable products [26].
CONCLUSION
In conclusion, the results has demonstrated that the ME
EAE of O. europaea have a high antioxidant capacity this
activity due to their high content of phenolic compounds.
Therefore, they can be used to treat several diseases in which
there is an increase in free radical production. For the
perspectives, further studies are needed to identify which
phenolic compounds are responsible for the antioxidant activity
of the extracts, and assess the way in which the phenolic
substances contribute to this activity. Additional in vitro and in
vivo antioxidant assays are needed to confirm the potential use
of these extracts in disease treatment.
LIST OF ABBREVIATIONS
ME: Methanolic extract
CHE: Chloroform extract
EAE: Ethyl acetate extract
AE: Aqueous extract
GAE: Gallic acid equivalent
QE: Quercetin equivalent
ACKNOWLEDGEMENTS
We would like to thank by Dr. Salemkour Noura, the
center of scientific and technical research on arid regions
(C.S.T.R.A.R), Biskra, Algeria, for the identification of the
plant material.
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Journal Name, Year, Volume 6

Received: March 20, 2014 Revised: April 16, 2014 Accepted: April 20, 2014

Table 1. Total polyphenol and flavonoids contents of the different extracts.

F. carica O. europaea
ME HE CHE EAE ME HE CHE EAE
Yield (%) 16.43 0.88 1.81 1.05 19.98 0.71 2.30 1.95
TPC
16.41±3.92 8.26±0.05 67.06±0.118 58.18±0.094 171.40±6.79 4.29±0.33 27.85±2.04 68.96±8,76
(µg GAE/mg E)
TFC
5.7 ± 0.25 5.28 ± 0.07 34.06± 0.05 4.12 ± 0.017 9.85±0.54 1.69 ±0.31 11.75 ±1.36 15.47±1.11
(µg QE/mg E)

Fig. 1: The scavenging activity of extracts using DPPH free radical test (Values were
expressed as means ± SD of triplicate, ns: p> 0.05; * p: ≤ 0.05; **:p ≤ 0.01; ***:p ≤ 0.001).

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Fig. 2. The reducing power of extracts expressed as absorbance at 700 nm at concentration of

200 µg/ml of extract. (Values were expressed as means ± SD of triplicate; ns: p> 0.05; * p: ≤
0.05; **:p ≤ 0.01; ***:p ≤ 0.001).