Research J. Pharm. and Tech.

8(3): March 2015

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE

Phytochemical composition and antioxidant activity of Ficus elastica

Roxb. (Moraceae) leaves
Preeti1, Abhishek Jain1, Gaurav Kumar1,2, Loganathan Karthik1,
Kokati Venkata Bhaskara Rao1*
1
Molecular and Microbiology Research Laboratory, Environmental Biotechnology Division,
School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu - 632 014, India
2
School of Life Sciences, Jaipur National University, Jaipur- 302025, Rajasthan, India
*Corresponding Author E-mail: kokatibhaskar@yahoo.co.in

ABSTRACT:
The aim of this study is to analyze the phytochemical composition and antioxidant activity of Ficus elastica (F.
elastica). Antioxidant activity of methanolic extract of Ficus elastica leaves was evaluated by 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging assay, total antioxidant activity test, iron chelating activity test and reducing
power assay. Antimicrobial activity and Cytotoxicity of F. elastica was determined by agar well diffusion method and
brine shrimp lethality test respectively, while total phenolic and flavonoid contents of the extract were measured by
Folin-Ciocalteau reagent method and aluminum chloride method respectively. The extract showed significant (p<0.05)
dose dependent antioxidant activity in various in vitro antioxidant methods. F. elastica also showed broad spectrum
antimicrobial activity against Bacillus cereus, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aerugenosa.
Brine shrimp lethality activity of F. elastica unveiled significant dose dependent and time dependent cytotoxic activity
against brine shrimp nauplii. Phytochemical screening of F. elastica showed the presence of carbohydrates, phenolic,
flavonoids, proteins and tannins, which might be responsible for the above reported medicinal properties of the extract.
These findings establish F. elastica as a potential source of natural antioxidants, antimicrobial and cytotoxic
compounds and in future studies can be established to obtain the lead molecules for drug development.

KEYWORDS: Ficus elastica, Phytochemical screening, antioxidant activity, brine shrimp lethality and
antimicrobial activity.

INTRODUCTION:
Oxidation is an essential function of cells for its normal
functioning, though it leads to the production of free
radicals as byproducts which might cause several diseases 1.
Free radicals are atom or group of atoms with unpaired
electrons and these unpaired electrons cause radicals to be
highly chemically reactive and initiate a chain of reaction to
damage nucleic acid, proteins, lipid membranes etc. This
damage may lead to several diseases such as cancer,
neurodegenerative diseases, lung diseases, heart and eye
diseases in humans 2, 3. In order to prevent the body from
free radicals, antioxidants can be used which either may be
synthesized by body or obtained from the diet 4.
Received on 12.01.2015 Modified on 28.01.2015
Accepted on 06.02.2015 © RJPT All right reserved
Research J. Pharm. and Tech. 8(3): Mar., 2015; Page 259-264
DOI: 10.5958/0974-360X.2015.00043.8
259
They play an important role in neutralizing the normal level
of oxidative damage caused by these free radicals, thus
protect the body from oxidative stress 5. Some times in vivo
and dietary antioxidants are not sufficient to protect the
body form oxidative stress; in such cases antioxidant
supplements could be used. These antioxidants can either be
synthesized or obtained from natural sources such as plants,
algae and microorganisms. Plants are the most common and
widely accepted source of natural antioxidants 6, 7.
Therefore this study is designed to screen F. elastica for its
antioxidant potential.
F. elastica belongs to the family Moraceae commonly
known as the rubber fig, rubber bush, rubber tree, rubber
plant, or Indian rubber bush, native to northeast India and
southern Indonesia. The latex obtained from the bark of
branches is used for preparing rubber. F. elastica was
selected for this study because of its role as in traditional
 Research J. Pharm. and Tech. 8(3): March 2015

medicine which is used to cure various diseases such as was performed in triplicates at each concentration 11. The
skin infections, allergies, anemia, neurodegenerative percentage scavenging of DPPH by the extract was
disorders and hepatic problems; in addition it is used as calculated according to the following formula:
diuretic agent 8. The limitation of scientific literature to
validate the traditional knowledge has limited its %DPPH Radical scavenging = [(Ac-As)/Ac] × 100
application in modern medicine; in this regard the aim of
this study was to study F. elastica for its phytochemical Here,
composition and antioxidant activity, in addition Ac is the absorbance of the control (DPPH)
antimicrobial and cytotoxic activity of the extract was also As is the absorbance of the test sample/standard
evaluated.
Determination of total antioxidant activity
MATERIALS AND METHODS: One milliliter leaf extract (50, 100, 150, 200, 250 and 300
Chemicals µg/ml) was mixed with 3 ml of the reaction mixture
2, 2-diphenyl-1-picrylhydrazyl (DPPH) and Quercetin was (containing 10 ml of concentrated H2SO4, 1.005 g of
purchased from Sigma-Aldrich Chemical Co. (Milwaukee, sodium phosphate monobasic and 1.47g of ammonium
WI, USA). Sodium carbonate (Na2CO3), Sodium phosphate molybdate which was dissolved in 290 ml of water). The
(NaH2PO4) was purchased from Himedia Laboratories Pvt. mixture was kept in water bath for one hour at 95ºC. The
Ltd. (Mumbai, India). Methanol, Ferric chloride(FeCl3), solution containing 3 ml of reaction mixture and 1 ml of
Potassium Ferricyanide (K3Fe (CN)6), Trichloroacetic acid, distilled water was used as blank and the absorbance was
Folin- Ciocalteau reagent, Methanol, Ascorbic acid, Gallic measured using UV-Vis spectrophotometer at 695 nm.
acid were purchased from SRL Pvt. Ltd. (Mumbai, India). Experiment was performed in triplicates at each
Ammonium molybdate ((NH4)2MoO4) and Aluminium concentration 12.
chloride (AlCl3) were purchased from SD Fine-Chem
Chem. Ltd. (Mumbai, India). All other chemicals used were Iron chelating activity
of analytical grade. Two milliliter of extract (50, 100, 150 and 200µg/ml) was
mixed with one milliliter of reaction mixture (Containing 1
Plant material ml of 0.05% O-Phenanthroline and 2 ml of 200 µM Ferric
The plant sample was collected from the nursery of VIT chloride). Mixtures were kept for incubation for 10 minutes
University, Vellore (Lat.12.9202˚N, Long.79.1333˚E), at room temperature. Solution containing 1 ml of reaction
Tamil Nadu, India, during August 2012. The plant material mixture and 2 ml of distilled water was used as blank. The
was carried to the Molecular and Microbiology Research absorbance was measured at 510 nm using UV- Visible
Laboratory, VIT University. Herbarium was prepared and spectrophotometer. Experiment was performed in triplicates
maintained in our laboratory for future reference. at each concentration 13.

Plant processing Reducing power assay

Fresh plant leaves were collected and washed thoroughly One milliliter of plant extract at different concentrations
with distilled water and then dried at room temperature in (125, 250, 500 and 1000 μg/ml) was mixed with phosphate
shade. Dried leaves were uniformly powdered using a buffer (2.5 ml, 0.2 M, pH 6.6) and 2.5 ml of 1 % Potassium
mechanical grinder. The powder was extracted in methanol Ferricyanide. The mixture was then incubated at 50˚C for
using a Soxhlet extractor. The extract was concentrated at 20 min. A volume of 2.5 ml of Trichloroacetic acid (10%)
40°C under reduced pressure (72 mbar) with a rotary was added to the mixture, and was centrifuged at 3000 rpm
evaporator and dried using lyophilizer. Dried extract was for 10 min in a cooling centrifuge. Two milliliter of the
collected in air tight container and stored at 4°C for further supernatant was mixed with equal volume of distilled water
use. The extract was then stored in an air tied container at and 0.5 ml ferric chloride (0.1%). Absorbance was
4˚C until used. measured at 700 nm using a UV–Visible
spectrophotometer. Each experiment was performed in
Phytochemical screening triplicates at each concentration 14.
Phytochemical screening of methanolic extract of F.
elastica was carried out by using the standard protocols for Estimation of polyphenolic compounds
carbohydrate, saponin, oil and fat, flavonoids, alkaloids, Estimation of total phenolic content
phytosterols, tannins and phenolic compounds 9, 10. Total phenolic content of the methanol extract of the F.
elastica leaves was determined using the Folin-Ciocalteau
In vitro antioxidant activity reagent method [12]. The crude methanolic extract was
DPPH radical scavenging activity diluted to obtain different concentrations (125, 250, 500 and
Extract was diluted in methanol to prepare dilutions of 5, 1000 μg/ml). 100 μl of each extract was mixed with 2.5 ml
10, 15, 20, 25, 30µg/ml. One milliliter of DPPH solution of Folin- Ciocalteau reagent (1/10 dilution in distilled
(0.2 mM/ml in methanol) then was mixed with 2 ml of each water) and 2 ml of 7.5% sodium carbonate (w/v in distilled
dilution. The mixed solution was incubated in dark at 20˚C water). The mixture was incubated at 45˚C for 15 min. The
for 40 minutes. Absorbance was measured at 517 nm using absorbance was measured at 765 nm using a UV–Visible
UV spectrophotometer with methanol as blank. Experiment spectrophotometer. Sodium carbonate solution (2 ml of
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 Research J. Pharm. and Tech. 8(3): March 2015

7.5% Na2CO3 in 2.60 ml of distilled water) was used as drying, F. elastica yielded 0.65 gm of extract that is 6.5%
blank. The results were expressed as Gallic acid of the initial plant powder (10 gm).
equivalence in μg. Each experiment was performed in
triplicates at each concentration 15. Phytochemical analysis
The results of the phytochemical analysis of methanolic
Determination of total flavonoid content extract of F. elastica showed the presence of tannins,
The determination of total flavonoid content of the carbohydrates, phytosterols, phenolics and flavonoids and
methanolic extract of F. elastica leaves was carried out absence of oil fat and saponin. Result of phytochemical
using aluminum chloride reagent method. A volume of 1 ml screening is summarized in Table.1
extract (containing 125, 250, 500 and 1000 µg/ml) was
mixed with 1 ml of aluminum chloride (2% in ethanol). The Table.1. Phytochemical composition of the Ficus elastica
mixture was incubated at room temperature for 60 minutes. Phytochemical F. elastic
Aluminum chloride solution (1 ml of 2% AlCl3 + 1 ml of Saponins _
Oil fat _
water) was used as blank. The absorbance was measured at Tannins ++
420 nm using UV-Vis spectrophotometer. Total flavonoid Alkaloid _
content was expressed as Quercetin equivalence (QE) in μg. Carbohydrates +
Experiment was performed in triplicates at each Phytosterols ++++
concentration 16. Flavonoids ++++
Phenolics ++++
Here: -= Negative, += positive
Cytotoxic activity (Brine shrimp lethality assay)
Brine shrimp (Artemia saline, Leach) eggs (Ocean Star Antioxidant activity
International, Inc., Snowville, USA) were placed in a DPPH radical scavenging activity
hatching tank containing sea water for 48 hours [16]. After DPPH is a relatively stable free radical compound with
hatching, 4 ml of sea water containing 20 brine shrimp maximum absorption at wavelength of 517nm. Antioxidant
nauplii was mixed with 1 ml of methanolic extract of F. on reacting with DPPH neutralizes it and the reaction
elastica (50,100,150 and 200 µg/ml in sea water). Survival mixture turn purple to yellow. The degree of discoloration
of brine shrimp nauplii was recorded at 1, 2, 3 and 4 hours. is directly correlates with the antioxidant potential of the
Experiment was performed in triplicates at each sample. During this study, F. elastica was found to exhibit
concentration 17. significant (p < 0.05) dose dependent DPPH radical
scavenging activity with an IC50 value 20.17 µg/ml. The
Antimicrobial activity results were compared with ascorbic acid standard and
The following isolates of bacteria were used for the study: expressed as percentage DPPH scavenging activity in
Escherichia coli, Bacillus cereus, Pseudomonas Figure 1.
aerugenosa, Klebsiella pneumoniae and Staphylococcus
aureus. Antimicrobial activity of the methanolic extract of
plant was determined by the agar well diffusion method 18.
The test organisms were inoculated on Muller Hinton agar
plates using sterilized cotton swabs. Agar surface was bored
using sterilized gel borer to make wells. 100µl of the test
extract and 100µl of distilled water were poured in two
separate wells and the plates were kept for incubation at
37˚C for 24 hrs and plates were observed for zone of
inhibition.

Statistical analysis
The results of DPPH radical scavenging activity, total
antioxidant activity, iron chelating activity, reducing power
activity, cytotoxic activity, antimicrobial activity, total
phenolic content and total flavonoid content of the
methanolic extract of F. elastica is expressed as mean ± Figure 1: DPPH radical scavenging activity of varying
standard deviation in the response of three replicates per concentrations of methanolic extract of F. elastica leaves. Data is
sample. Statistical significance between the groups was given in mean ± SD (n = 3 test; p<0.05). Data was compared with
ascorbic acid standard.
determined by one-way ANOVA coupled with Tukey’s
post hoc test at p < 0.05. Statistical analysis was performed
Total antioxidant activity
with Microsoft Excel 2007 and GraphPad Prism 5.
The phosphomolybdate method has been routinely used to
evaluate the total antioxidant capacity of extract 12. In the
RESULTS AND DISCUSSION presence of the fractions, the Mo(VI) is reduced to Mo(V)
Yield of the extract and forms a green coloured phosphomolybdnum(V)
Ten grams of dried leaf powder of the F. elastica was complex which shows maximum absorbance at 695 nm. In
extracted in methanol to obtain the test extract. After
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 Research J. Pharm. and Tech. 8(3): March 2015

this study, F. elastica exhibited significant (p < 0.05) dose
dependent increase in total antioxidant activity with an
optical density of1.03 ± 0.26 at 1 mg/mL. The result was
compared with the ascorbic acid standard in Figure 2.
Reducing power potential
Reducing power assay is a good reflector of antioxidant
activity of the sample. The samples having high reducing
power generally reported to carry high antioxidant potential
too. In this experiment, Ferric ions are reduced to ferrous
ions with the color of the reaction mixture changing from
yellow to bluish green. In this study, F. elastica exhibited
significant (p < 0.05) dose dependent increase in reducing
power potential with an optical density of 1.25 ± 0.047 at
1mg/ml. The results were compared with ascorbic acid
standard in Figure 4.

Figure 2: Total antioxidant activity of varying concentrations of

methanolic extract of F. elastica leaves. Data is given in mean ± SD
(n = 3 test; p<0.05). Data was compared with ascorbic acid
standard.

Iron chelating assay

The antioxidant activity of phenolic compounds was
attributed by their ability to chelate transition metal ions,
such as those of iron and copper. These chelating agents
may stabilize pro-oxidative metal ions in living systems by
complexing them 19. In iron chelating activity, O-
phenanthroline quantitatively forms complexes with Fe+2
which get disrupted in the presence of chelating agents 20.
The alcoholic extract interfered with the formation of a
ferrous-o-phenanthroline complex, thereby suggesting that
the extract has metal chelating activity. In this study, F.
elastica exhibited significant (p < 0.05) dose dependent
increase in iron chelating activity with an optical density of
0.22 ± 0.1 at 300 µg/ml. The results were compared with
ascorbic acid standard in Figure 3.
Figure 4: Reducing power activity of varying concentrations of
methanolic extract of F. elastica leaves. Data is given in mean ± SD
(n = 3 test; p<0.05). Data was compared with ascorbic acid
standard.
Estimation of total phenolic content and total flavonoid
content
Total phenolic and flavonoid content of the extract were
measured by Folin-Ciocalteau reagent method and
aluminum chloride method respectively. The quantification
experiments unveiled the presence of high quantity of
phenolics and flavonoids in extract. The total phenolic and
flavonoid content of F. elastica was found to be 64.005 mg
gallic acid equivalence/gm dry weight of the extract and
43.003 mg quercetin equivalence/gm dry weight of the
extract, respectively. The results of total phenolic content
and total flavonoid content are reported in Figure 5 and 6.

Brine shrimp lethality assay

The brine shrimp cytotoxicity assay was considered as a
convenient probe for preliminary assessment of cytotoxicity
of the plant extracts 17. This method can be extrapolated for
cell-line toxicity and antitumor activity 21. Dose and time
dependent increase in cytotoxic activity of F. elastica was
Figure 3: Iron chelating activity of varying concentrations of seen. Result of brine shrimp lethality has been summarized
methanolic extract of F. elastica leaves. Data is given in mean ± SD as percentage cytotoxicity in graphical form in Figure 7.
(n = 3 test; p<0.05). Data was compared with ascorbic acid
standard.

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Antimicrobial activity
The methanolic extract of F. elastica showed high
antimicrobial activity against B. cereus followed by P.
aerugenosa, E. coli and K. pneumoniae whereas F. elastica
showed no antimicrobial activity against S. aureus. The
results of antimicrobial activity have been summarized in
Table 2.

Table 2: Antimicrobial activity of methanolic extract of F. elastica

Organisms Zone of clearance (mm)
F. elastic PC NC
Escherichia coli 6.66±0.57 32.0±2.0 No zone
Bacillus cereus 14.0±1.0 33.66±1.52 No zone
Pseudomonas 8.0±1.0 14.0±1.0 No zone
aerugenosa
Klebsiella 6.33±0.57 15.66±3.78 No zone
pneumoniae
Staphylococcus No zone 10.6±1.52 No zone
aureus
Figure 5: Total Phenolic content in varying concentrations of Here, PC: positive control, NC: negative control [Vancomycin (10
methanolic extract of F. elastica leaves. Data is given in mean ± SD μg/disc) was used as positive control for S. aureus, Chloroamphenicol
(n = 3 test; p<0.05) and expressed as Gallic acid equivalence (GAE) (30 μg/ disc) for K. pneumoniae; Erythromycin (10 μg/disc) for E.
in μg. coli]; Values are expressed as mean ± standard deviation of the three
replicates,

CONCLUSION:
On the basis of the results obtained in the present study, it is
concluded that F. elastica is showing good antioxidant
activity and thus can be used as a source of safe and natural
antioxidant compound. It has also showed a good
antimicrobial activity against B. cereus and thus can be
used as a potential drug in future against food poisoning
and other diseases caused by B. cereus. Also, in future we
can isolate the antioxidant compound for their potential use
in the field of therapeutics.

ACKNOWLEDGEMENT:
Authors are thankful to department of applied
microbiology, Schools of Biosciences and Technology, VIT
Figure 6: Total flavonoid content in varying concentrations of University, Vellore, for providing necessary facilities and
methanolic extract of F. elastica leaves. Data is given in mean ± SD support for the completion of this work.
(n = 3 test; p<0.05) and expressed as Quercetin equivalence in μg.

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