American Journal of Chemistry 2015, 5(1): 23-27

DOI: 10.5923/j.chemistry.20150501.04

Antioxidant Activity and Total Flavonoids Content of

Aerial Parts of Ficus pyriformis Hook. & Arn. (Moraceae)
Cultivated in Egypt
Zedan Zeid Ibraheim1, Alaa Mohammed Nafady2, Mahmoud Abdullah Mostafa2,
Fahd Mohammed Amin2,*

1
Department of Pharmacognosy, Faculty of Pharmacy, Assuit University, Assuit, Egypt
2
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assuit, Egypt

Abstract The total methanolic extract and fractions of Ficus pyriformis at different concentrations were subjected to
free radical scavenging activity using 2,2-diphenyl 1-picrylhydrazyl (DPPH•) method. Different fractions of Ficus pyriformis
obtained from successive fractionation of the total extracts on vacuum liquid chromatography (VLC) with organic solvents of
different polarities. n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH); showed that, the
MeOH and EtOAc fractions have the highest activity followed by DCM and n- hexane fractions respectively. The total
flavonoids content were measured using aluminum chloride colorimetric method and quercetin [QE] as standard equivalent
for comparison, the total extract of Ficus pyriformis was also subjected to preliminary phytochemical screening tests for
different phytoconstituents present in the plant.
Keywords Antioxidant, DPPH• assay, Phytochemical screening, Ficus pyriformis

activity and many Ficus species rich in flavonoids [8, 9]. The
1. Introduction literature survey show that there is no report considering
antioxidant activity of Ficus pyriformis. In this study we
Free radicals are molecules or molecular fragments aimed to evaluate the antioxidant activity of Ficus pyriformis
containing one or more unpaired electrons in its outermost cultivated in Egypt, estimate the total flavonoids content and
atomic or molecular orbital and are capable of independent carry out preliminary phytochemical screening.
existence and are involved in the normal physiology of living
organisms [1, 2]. Under certain conditions, the excess of free
radicals and Reactive Oxygen Species (ROS) like peroxy 2. Material and Methods
radical (ROO•) have been proposed to induce cellular
damage and to be involved in several human diseases such as 2.1. Plant Material
cancer, arteriosclerosis, inflammatory disorders as well as in The aerial parts of Ficus pyriformis were collected during
ageing process [3]. Antioxidants are chemical substances the flowering and fruiting stage from El-Orman Botanical
that reduce or prevent oxidation. They have the ability to Garden, Giza, Egypt. The specimens were authenticated by
counteract the damaging effects of free radicals in tissues Ms. Trease Labib Consultant of plant taxonomy at the
and thus are believed to protect against several diseases [4]. Ministry of Agriculture.
Therefore there is great interest in finding new and safe
antioxidants from natural sources [5]. The genus Ficus have 2.2. Preparation of Plant Extracts
shown diverse biological activity, they have been The plant material was collected, dried in shade, powdered,
investigated as potential repository of natural products for sieved and kept in an amberd well closed container in dark.
treatment of various ailments including tumors, The air-dried powdered aerial parts (2 Kg) of Ficus
inflammatory disorder, diabetes and as antioxidants [6, 7]. pyriformis. Were extracted by maceration and percolation
Flavonoids considered as one of the major classes of with (70%) methanol three times and pooled together. The
phytoconstituents in plants responsible for antioxidant combined methanolic extract was concentrated under
reduced pressure (40ºC) using a rotary evaporator till
* Corresponding author:
Ph_fahd86@yahoo.com (Fahd Mohammed Amin)
constant weight to give a dark brown syrupy residue (150g).
Published online at http://journal.sapub.org/chemistry A part of the methanolic extract (100g) was subjected to
Copyright © 2015 Scientific & Academic Publishing. All Rights Reserved successive solvent fractionation on VLC with n-hexane,
24 Zedan Zeid Ibraheim et al.: Antioxidant Activity and Total Flavonoids Content of Aerial Parts
of Ficus pyriformis Hook. & Arn. (Moraceae) Cultivated in Egypt

DCM, EtOAc and finally with MeOH till complete standards and DPPH• radical scavenging activity was
exhaustion. Each fraction was filtered through filter paper calculated by using the formula [14].
Whatman no.1 and then concentrated under pressure (40ºC) 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎
using rotary evaporator yielding respectively n-hexane (13g), 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 − 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜
DCM (11g), EtOAc (21 g) and MeOH (44 g) all kept in = × 100
refrigerator till used. 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜

2.4.3. Phytochemical Screening

2.3. Apparatus
The extract was subjected to preliminary phytochemical
All spectroscopic data were acquired using the Shimadzu
tests to find out phytoconstituents present in them. The tests
1601, UV/visible Spectrophotometer. Disposable cuvettes
were carried to detect the presence of steroids, triterpenoids,
(1 cm × l cm x 4.5 cm) were used for visible absorbance
flavonoids, tannins, anthraquinones, coumarins,
measurements. Rotary Evaporator 4000 (Heidolph,
cardenolides, iridoids, carbohydrates and /or glycosides
Germany).
[15-17].
2.4. Chemicals
2, 2-Diphenyl-1-picryl hydrazyl (DPPH•), quercetin (QE), 3. Statistical Analysis
ascorbic acid and potassium acetate were obtained from
Sigma-Aldrich Chemicals Co., Germany. Aluminum Experimental results are expressed as mean ± standard
chloride obtained from El-Nasr Pharmaceutical and deviation (SD). All results are means of three replicates.
Chemical Co., Egypt (ADWIC). Other chemicals used were SPSS 16 version was used for the statistical analysis and
of high analytical grade and obtained from Merck Chemical Microsoft excel program (2010).
Co., Germany.

2.4.1. Determination of Total Flavonoids 4. Results and Discussion

Aluminum chloride colorimetric method was used for
4.1. Determination of Total Flavonoidal Content
estimation of total flavonoids [10, 11]. 0.5 ml solution of
plant extract was mixed with 1.5 ml of methanol, 0.1 ml of The basic principle in the Aluminum chloride colorimetric
10% aluminum chloride, 0.1 ml of 1 M potassium acetate method is due to formation of acid stable complexes with the
and 2.8 ml of distilled water. The mixture kept at room C-4 keto group and either the C-3 or C-5 hydroxyl group of
temperature for 30 min. The absorbance of the reaction flavones and flavonols, also Aluminum chloride forms acid
mixture measured at λ max 415 nm. Standard calibration labile complexes with the ortho- dihydroxyl groups in the A–
curve is generated by using quercetin as reference standard. or B-ring of flavonoids. This complexes measured
Stock solution of quercetin was made by dissolving 10 mg in spectrophotometrically at λmax 415nm [18]. In this work; the
methanol and transferred to volumetric flask and completes total flavonoids of the total extract obtained from the aerial
the volume to 10 ml, then makes serial dilution to make parts of were calculated from the equation of the standard
concentrations (10-100 μg/ml) in methanol. plot as follow;
Absorbance = 0.0094 × total flavonoid [μg QE/mg of dry
2.4.2. DPPH Radical Scavenging Activity (DPPH• assay) extract] + 0.039
The method of [12, 13] was adapted for testing the radical (R2 = 0.9963).
scavenging of the extracts using the stable free radical 2,
2-diphenyl-1-picrahydrazy (DPPH•) spectrophotometry. 10
× 10-5 M solution of DPPH• (394.3 g/mol.) was prepared by
dissolving 0.04 g of DPPH• in 1000 ml of methanol. In the
assay 0.2 ml of methanol solution of Ficus pyriformis aerial
parts total extract and its fraction at different concentrations
(0.0625,0.125, 0.250, 0.5, 1mg/ml) was mixed with 2 ml of
methanol solution of DPPH• (0.1mM). Similarly; 0.2 ml
methanol solution of ascorbic acid and quercetin of various
concentrations (0.0625, 0.125, 0.25, 0.5, 1 mg/ml) were
mixed with 2 ml of DPPH• solution. A mixture of 0.2 ml of
methanol and 2 ml of methanol solution of DPPH• (0.1 mM)
served as control. After mixing, all the solutions were
incubated in dark for 30 min. and then absorbance was
measured at λ max 517 nm. The experiments were carried out Figure 1. Calibration curve of standard quercetin for determination of total
in triplicate using ascorbic acid and quercetin as a reference flavonoids content in total extract of Ficus pyriformis aerial parts
 American Journal of Chemistry 2015, 5(1): 23-27 25

The total flavonoidal content of the total extract obtained obtained antioxidant activity of the total extract and fractions
from the aerial parts of Ficus pyriformis were [31.8 µg QE/ (Tab 2, Figures 2, 3, 4, 5, 6 and 7) are closely related to the
mg plant extract]. presence of poly-phenolic compound such as flavonoids.
The presence of ortho-dihydroxyl of the B-ring (3’, 4’-di OH)
Table 1. Total flavonoids content of Ficus pyriformis total extract
of the flavonoid molecule which confers high stability to the
Conc. of extract (mg/ml) Absorbance QE equivalent (µg/mg) flavonoid phenoxy radical, C2-C3 double bond in
conjugation with 4-oxo group of the ring C participates in
1 mg/ml 0.338 31.8
radical stabilization via electron delocalization over all three
ring system. The presence of both 3- and 5- hydroxyl moiety
4.2. DPPH Radical Scavenging Activity (DPPH• assay) of the rings C and A, play an important role in radical
Free radicals can be defined as any molecular species scavenging activity of the flavonoids [21, 22].
capable of independent existence that contains an unpaired
electron in an atomic orbital such as Reactive Oxygen 4.3. Phytochemical Screening
Species(ROS) like (superoxide anion (O2•–), hydroxyl (•OH ), Preliminary phytochemical screening of the total extract
peroxy (ROO•), and alkyl radical ( RO• ) which may attack showed the presence of steroids, triterpenoids, flavonoids,
biological macromolecules, giving rise to protein, lipid, and tannins, carbohydrates and or glycosides. Flavonoids and
DNA damage, cell aging, and cancer [19]. Antioxidants tannins contributed to have an antioxidant activity [8, 9].
scavenge or quench free radical. Antioxidant activity deals Value are expressed as mean ± SD; n = 3, Total ext. = total
with the kinetic of the reaction between antioxidant and the extract, n-Hexane Fr. = Hexane fraction, DCM Fr. =
free radicals that scavenges or quenches [20]. Dichloromethane fraction, EtOAc fr. = Ethyl acetate fraction,
In the present study; the antioxidant activity of Ficus MeOH fr. = Methanol fraction.
pyriformis aerial parts total extract and its fractions were
determined using DPPH• method. The DPPH• method allows
a direct investigation of the ability for the extract or
antioxidant to donate hydrogen and/or electrons to quench
the DPPH• radical.

(DPPH•) + (H-A) → DPPH-H + (A•)

(Purple) antioxidant (Yellow)

As the radical is quenched, the color of the solution

changes from a deep purple to a light yellow and the
absorbance at 517 nm decreases [13]. As a result (table 2,
figures 2, 3, 4, 5, 6 and 7) indicated good scavenging activity
of the total extract, and some fractions towards DPPH• in
comparison with reference standards (ascorbic acid and
quercetin). The MeOH fraction showed maximum activity in
comparison with total extract and other fractions, followed Figure 2. Scavenging activity of extracts with different concentrations in
by EtOAc, DCM and n-hexane fractions respectively. The comparison with standards

Table 2. Antioxidant activity of Ficus pyriformis aerial parts total extract and fractions

Concentration (mg/ml)
Fractions 0.0625 0.125 0.25 0.5 1
% inhibition
Ascorbic acid 88.7±0.28% 90.29±0.15% 91.6±1.01% 92.5±0.42% 93.8±0.19%
Quercetin 85.9±0.31% 87.3±0.32% 89.3±0.31% 90.1±0.44% 91.5±0.31%
Total ext. 34.5±0.86% 40.07±0.43% 57.4±0.99% 66.4±0.64% 84.5±0.31%
n-Hexane fr. 4.9±0.12% 10.3±0.61% 16±0.44% 20±0.44% 25±0.32%
DCM fr. 13.6±0.37% 15.8±0.37% 18.85±0.56% 23.8±0.62% 37.6±0.46%
EtOAc fr. 28.45±0.71% 39.8±0.73% 44.71±0.46% 58.45±0.61% 69.1±0.46%
MeOH fr. 36.6±0.99% 50.4±0.23% 57.96±0.39% 72.18±0.72% 86.3±0.44%
26 Zedan Zeid Ibraheim et al.: Antioxidant Activity and Total Flavonoids Content of Aerial Parts
of Ficus pyriformis Hook. & Arn. (Moraceae) Cultivated in Egypt

Figure 6. DPPH free radical scavenging activity of aerial parts total

Figure 3. DPPH free radical scavenging activity of aerial parts total extracts and fractions of Ficus pyriformis in concentration (0.5 mg/ml) in
extract and fractions of Ficus pyriformis in concentration (0.0625 mg/ml) in comparison with standards
comparison with standards

Figure 4. DPPH free radical scavenging activity of aerial parts total Figure 7. DPPH free radical scavenging activity of aerial parts total
extracts and fractions of Ficus pyriformis in concentration (0.125 mg/ml) in extracts and fractions of Ficus pyriformis in concentration (1 mg/ml) in
comparison with standards comparison with standards

5. Conclusions
For the first time the evaluation of the antioxidant activity
of the aerial parts of Ficus pyriformis was done along with its
total flavonoids content. The MeOH fraction and total
extract of Ficus pyriformis aerial parts showed highest
antioxidant activity followed by EtOAc, DCM and n-hexane
fraction respectively in comparison with reference standards
and this attributed to polyphenolic compound as flavonoids
and tannins which known to possess an antioxidant activity
[14, 22, 23].

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