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Phytochemical Composition and Antioxidant


Activity of Methanolic Extract of Ficus benjamina
(Moraceae) Leaves

Article November 2013

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Research J. Pharm. and Tech. 6(11): November 2013

ISSN 0974-3618 www.rjptonline.org

RESEARCH ARTICLE

Phytochemical Composition and Antioxidant Activity of Methanolic


Extract of Ficus benjamina (Moraceae) Leaves
Abhishek Jain, Varsha Ojha, Gaurav Kumar, Loganathan Karthik,
Kokati Venkata Bhaskara Rao*
Molecular and Microbiology Research Laboratory, Environmental Biotechnology Division,
School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu - 632 014, India
*Corresponding Author E-mail: kokatibhaskar@yahoo.co.in

ABSTRACT:
The aim of the present study was to study the phytochemical composition and antioxidant potential of the Ficus
benjamina. Antioxidant activity of the methanolic extracts of F. benjamina was screened by 2, 2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging assay, total antioxidant activity, iron chelating activity and reducing power
assay. In addition to antioxidant activity, extract was also evaluated for cytotoxic activity brine brine shrimp lethality
assay. Estimation of total phenolic content was performed by Folin-Ciocalteau reagent method and estimation of total
flavonoid content was performed by aluminium chloride method. During preliminary phytochemical analysis, F.
benjamina showed the presence of carbohydrates, phenolic compounds, oil and fats, saponins, flavonoids, alkaloids,
proteins and tannins as major phytochemical groups. Methanolic extract of F. benjamina exhibited significant
antioxidant activity in DPPH radical scavenging assay, total antioxidant activity, iron chelating activity and reducing
power assay. Phytochemical screening of methanolic extract of F. benjamina showed the presence of high levels of
phenolic (4.006 mg gallic acid equivalence/gm) and flavonoids (16.005 mg quercetin acid equivalence/gm)
compounds which could be responsible for its antioxidant potential. Extract also resulted in significant cytotoxic
activity towards brine shrimp nauplii. The obtained results emphasize the antioxidant activity of F. benjamina and
provided the scientific basis for the traditional use in prevention and therapies of diseases.

KEYWORDS: Ficus benjamina, phytochemical, antioxidant activity, cytotoxic activity.

INTRODUCTION:
Free radicals are atoms, molecules or ions with unpaired Failure of antioxidant defense system to control oxidative
electrons and are generated during normal metabolism of stress leads to several diseases in humans such as,
body. Free radicals are generally classified into reactive Alzheimer diseases, cataracts, ageing, diabetes mellitus and
oxygen species (ROS) and reactive nitrogen species (RNO). cardiovascular disease. 2
Some common examples of free radicals are superoxide
(O2), hydroxyl radical (OH) and nitric oxide radical (NO) Antioxidants are the molecules that can effectively
1 neutralize the free radicals. They safely interact with free
. Free radicals react with the cellular biomoleculs such as
DNA, lipid membranes and proteins, and damage them, radicals and terminate the chain reaction before vital
which ultimately leads to the cellular destruction 1. molecules get damaged. Phenolics, flavonoids, vitamins
Normally, these radicals are countered by antioxidant (E and C), numerous minerals (Cu, Mn, Zn, Se and Fe),
defense system of body which includes a variety of glutathione are some of the most widely recognised
enzymatic and no enzymatic antioxidants. Under the antioxidants. Antioxidants in blood, cells, and tissue fluids
influence of certain factors such as smoking, pesticides, play an important role in neutralizing the normal level of
radiations, drugs, alcohols etc. the formation of free radicals oxidative damage caused by these free radicals 3. Plants are
will increase several fold, which cannot be countered by a rich source of polyphenolic compounds which have been
antioxidant defense system of body. widely accepted antioxidant. A large number of studies are
going on worldwide directed to find natural antioxidants
from plant sources.
Received on 06.08.2013 Modified on 10.09.2013
Accepted on 15.09.2013 RJPT All right reserved
Research J. Pharm. and Tech. 6(11): November 2013; Page 1184-1189

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Research J. Pharm. and Tech. 6(11): November 2013

F. benjamina (Moraceae) is an evergreen tree, growing up protocols for the presence of carbohydrates, proteins,
to 60 feet tall and 60 to 70 feet wide. F. benjamina is native phenolic compounds, oil and fats, saponins, flavonoids,
to south and Southeast Asia and commonly known as alkaloids and tannins 15.
the Weeping Fig. F. benjamina is the official tree of
Bangkok and Thailand. Various parts of F. benjamina have Antioxidant activity:
been widely used as medicine for the treatment of certain DPPH radical scavenging activity:
skin disorders, hypotensive stomachic and anti-dysentery in The DPPH radical scavenging activity of methanolic extract
several traditionally important medicines 4. It is also used as
of F. benjamina leaves was determined by using standard
a folk medicine for the treatment of certain skin and protocols reported earlier16. Methanolic extract of F.
respiratory disorders5, 6. In recent years, various parts of F.
benjamina leaves was diluted in distilled water to make 5,
benjamina have been extensively studied for various 10, 15, 20, 25, 30 g/ml dilutions. Two millilitres of each
pharmaceutical properties by modern techniques and dilution was thoroughly mixed with one millilitre of DPPH
reported to possess hepatoprotective activity7, antimicrobial
solution (0.2 Mm/ml in methanol) and mixed thoroughly.
activity8,9, antitumor activity 9, antioxidant activity10, The mixture was incubated in dark at 20C for 40 min.
antiviral activity11, antimycobacterial activity12,
Absorbance was measured at 517 nm using UV-Vis
antinociceptive activity and antidiarrhoeal activity14.
13
spectrophotometer with methanol as blank. Experiment was
performed in triplicates at each concentration. The
The main aim of this study was to investigate the leaves of percentage scavenging of DPPH by the extract was
F. benjamina for its phytochemical composition, calculated according to the following formula:
antioxidant activity and cytotoxic activity by various in
vitro methods. % DPPH Radical scavenging = [(Ac-At)/Ac] 100

MATERIALS AND METHODS: Here,


Chemicals: Ac is the absorbance of the control (DPPH),
2, 2-diphenyl-1-picrylhydrazyl (DPPH) and Quercetin was At is the absorbance of the test sample.
purchased from Sigma-Aldrich Chemical Co. (Milwaukee,
WI, USA). Sodium carbonate (Na2CO3), Sodium phosphate Determination of total antioxidant activity:
(NaH2PO4) was purchased from Himedia Laboratories Pvt. Total antioxidant activity of methanolic extract of F.
Ltd. (Mumbai, India). Methanol, Ferric chloride (FeCl3), benjamina leaves was 17determined by using standard
Potassium Ferricyanide (K3Fe (CN)6), Trichloroacetic acid, protocols reported earlier . A volume of 1 ml of the extract
Folin- Ciocalteau reagent, Methanol, Ascorbic acid, Gallic (50, 100, 150 and 200 g/ml) was mixed with 3 ml of the
acid were purchased from SRL Pvt. Ltd. (Mumbai, India). reaction mixture (containing 10 ml of concentrated H2SO4,
Ammonium molybdate ((NH4)2MoO4) and Aluminium 1.005 g of sodium phosphate monobasic and 1.47 g of
chloride (AlCl3) were purchased from SD Fine-Chem ammonium molybdate which was dissolved in 290 ml of
Chem. Ltd. (Mumbai, India). All other chemicals used were water). The mixture was kept in water bath for one hour at
of analytical grade. 95C. The solution containing 3 ml of reaction mixture and
1 ml of distilled water was used as blank and the
Plant material: absorbance was measured using UV-Vis spectrophotometer
Fresh leaves of F. benjamina were collected from the plants at 695 nm. Experiment was performed in triplicates at each
growing in the VIT University campus, Vellore concentration.
(Lat.12.9202N, Long.79.1333E), Tamil Nadu, India,
during August 2012. The plant material was carried to the Iron chelating activity:
Molecular and Microbiology Research Laboratory, VIT Iron chelating activity of methanolic extract of F.
University. A voucher specimen was maintained in our benjamina leaves was 18determined by using standard
laboratory for future reference (FB/VIT/MMRL/ protocols reported earlier .The extract was diluted to 50,
21.08.2012-1) 100, 150 and 200g/ml in distilled water. A reaction
mixture having 1 ml of 0.05% O-Phenanthroline and 2 ml
Processing of plant: of 200 M Ferric chloride was mixed with 2 ml of the plant
Fresh and mature leaves of F. benjamina were collected and extract of the above mentioned concentrations. The mixed
washed thoroughly in distilled water and shade dried at solution was kept for incubation for 10 minutes at room
room temperature. Dried leaves were powdered uniformly temperature. Solution containing 1 ml of 0.05% O-
using a mechanical grinder. The powder was extracted in Phenanthroline, 2 ml of 200 M Ferric chloride and 2 ml of
methanol using Soxhlet apparatus. The extract was distilled water was used as blank. Absorbance was
concentrated with a rotary evaporator and dried using measured at 510 nm using UV-Visible spectrophotometer.
lyophilizer. Dried extract was collected in an air tight Experiment was performed in triplicates at each
receptacle and stored at 4C for further use. concentration.

Phytochemical screening: Reducing power assay:


Phytochemical screening of the methanolic extract of F. The reducing power of methanolic extract of the F.
benjamina leaves was carried out by using the standard benjamina was determined by ferric reducing power
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Research J. Pharm. and Tech. 6(11): November 2013

assay19. 1 ml volume of plant extracts at different of three replicates per sample. Results were analyzed using
concentrations (125, 250, 500 and 1000 g/ml) were mixed Microsoft Excel 2007 and Graph Pad Prism 5.
with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 2.5 ml of
1 % Potassium ferricyanide (K3Fe (CN)6). The mixture was RESULTS AND DISCUSSION:
incubated at 50C for 20 min. A volume of 2.5 ml of Medicinal plants are being used for the treatment of human
Trichloroacetic acid (10%) was added to the mixture, and diseases for thousands of years in several traditional and
was centrifuged at 3000 rpm for 10 min in a cooling folk medicinal systems. In developing countries medicinal
centrifuge. 2.5 ml of the supernatant was mixed with equal plants have archived a great value as to contribute to
volume of distilled water and 0.5 ml FeCl3 (0.1%). socioeconomic and health of a country. In recent years,
Absorbance was measured at 700 nm using a UVVisible World Health Organization (WHO) has stated that 80% of
spectrophotometer. Each experiment was performed in world population living in developing countries depends on
triplicates at each concentration. herbal drugs for primary healthcare 23. In last few decades,
a variety of medicinal plants have been reported to possess
Cytotoxic activity (Brine shrimp lethality assay): antitumor activity, antimicrobial activity, antidiabetic
Brine shrimp (Artemia saline Leach) eggs (Ocean Star activity, hepatoprotective activity, antioxidant activity,
International, Inc., Snowville, USA) were placed in a larvicidal activity, hemolytic activity etc 24-30. Above
hatching tank containing sea water for 48 hrs. After mentioned reports emphasized that, medicinal plants are a
hatching, 4 ml of sea water containing 20 brine shrimp valuable source of medicinal compounds and could be used
nauplii was mixed with 1 ml of methanolic extract of F. for screening new drugs. Therefore based on the traditional
benjamina leaves (50, 100, 150, 200 g/ml in sea water). uses, F. benjamina was selected for this study and screened
Survival of brine shrimp nauplii was recorded at 1, 2, 3 and for its phytochemical composition, antioxidant and
24 hrs. Experiment was performed in triplicates at each cytotoxic activity by various in vitro methods.
concentration20.
Yield of the extract:
Estimation of polyphenolic compounds: Dried leaves (10 gm) powder of the F. benjamina was
Estimation of total phenolic content: extracted in methanol to obtain the test extract. After
Total phenolic content of the methanol extract of F. drying, 10 gm of F. benjamina powder yielded 0.53 gm of
benjamina was measured using the Folin-Ciocalteau extract that is 5.3% of the initial plant powder.
reagent method as reported earlier 21. The crude methanolic
extracts were diluted to obtain different concentrations Phytochemical analysis:
(125, 250, 500 and 1000 g/ml). 100 l of each extract was Phytochemicals are non-nutritional plant chemicals that are
mixed with 2.5 ml of Folin- Ciocalteau reagent (1/10 synthesized by plant for self defense. These phytochemicals
dilution in distilled water) and 2 ml of 7.5% Na2CO3 (w/v in protect the plants from pathogens and environmental stress,
distilled water). The mixture was incubated at 45C for 15 in addition, they can also used effectively to cure several
min. The absorbance was measured at 765 nm using a UV diseases, there for it can be stated that the phytochemicals
Visible spectrophotometer. Na2CO3 solution (2 ml of 7.5% are the compound that determined the medical potential of
Na2CO3 in 2.60 ml of distilled water) was used as blank. any plant. In this study, methanolic extract of F. benjamina
The results were expressed as Gallic acid equivalence in g. was evaluated to phytochemical analysis by various
Each experiment was performed in triplicates at each biochemical methods. Preliminary phytochemical analysis
concentration. of the extract exhibited the presence of tannins,
carbohydrates, phytosterols, flavonoids, phenolics, oils and
Determination of total flavonoid content: fats, saponins. Results are summarized in Table 1. Result of
The determination of total flavonoids content of the the study are in agreement with previous finding where the
methanolic extract of F. benjamina was carried out using similar kind of observations were made by other study
Aluminium chloride method 22. A volume of 1 ml extract group, however miner variation in the results was also
(containing 125, 250, 500 and 1000 g/ml) was mixed with observed, which may be due to the use of different solvent
1 ml of AlCl3 (2% in ethanol). The mixture was incubated at used for phytochemical extraction 31
room temperature for 60 minutes. AlCl3 solution (1 ml of
2% AlCl3 + 1 ml of water) was used as blank. The Table1: Phytochemical composition of methanolic extract of F.
absorbance was measured at 420 nm using UV-Vis benjamina leaves
spectrophotometer. Total flavonoid content was expressed Phytochemicals F. benjamina
as Quercetin equivalence (QE) in g. Experiment was Saponins _
performed in triplicates at each concentration. Oil fat +
Tannins +
Alkaloid _
Statistical analysis: Carbohydrates ++
The results of DPPH radical scavenging activity, total Phytosterols ++++
antioxidant activity, iron chelating activity, reducing power Glycosides _
activity, total phenolic content, total flavonoid content and Flavonoids +++
Phenolic compounds +++
cytotoxic activity of the methanolic extract of F. benjamina Here: -= Negative, += positive
are expressed as mean standard deviation of the response
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Research J. Pharm. and Tech. 6(11): November 2013

Antioxidant activity: which shows maximum absorbance at 695 nm. In present


Free radicals have been reported to associate with more study, methanolic extract of the F. benjamina shows
than hundred different diseases in humans, therefore it is significant total antioxidant activity in a dose dependent
important to found out certain products that could manner. The results of total antioxidant activity were also
effectively protect the body from free radical mediated represent as ascorbic acid equivalence and summarized in
oxidative stress. Natural antioxidants from medicinal plants Figure 2.
are a good choice to control oxidative stress and as of
natural origin these compounds are usually non toxic. Thus
in this study antioxidant potential of methanoic extract of F.
benjamina leaves was analysed against a variety of free
radicals by DPPH radical scavenging activity, total
antioxidant activity, metal chelating activity and reducing
power activity.

DPPH radical scavenging activity:


The DPPH radical scavenging assay has been widely used
to evaluate the free radical scavenging effectiveness of
various antioxidant substances. DPPH is a stable free
radical compound with a characteristic absorption at
wavelength of 517 nm. Antioxidants upon interaction with
DPPH transfer a proton to DPPH by direct abstraction of
phenol H- atoms and electron transfer process, thus Figure2: Total antioxidant activity of varying concentrations of
neutralizing its free radical character. The colour of the methanolic extract of Ficus benjamina leaves. Data is given in
reaction mixture changes from purple to yellow with a mean SD (n = 3 test). Data was compared with ascorbic acid
equivalence (g/ml)
decrease of the 517 nm absorbance. The degree of
discolouration indicates the scavenging potential of the
Iron chelating assay:
antioxidants 32, 33. In this study, methanolic extract of F.
The antioxidant activities of phenolic compounds are also
benjamina exhibited high DPPH radical scavenging
attributed to their ability to chelate transition metal ions,
activity with an IC50 value 59.07 g/ml and the free radical
such as those of iron and copper, which have been proposed
scavenging activity was found to be increasing with
as the catalysts for the initial formation of reactive oxygen
increase in the time and in dose dependent manner. The
species. Chelating agents may stabilize pro-oxidative metal
results are expressed as percentage inhibition of DPPH
ions in living systems by complexing them 34. In iron
(n=3). The results of DPPH radical scavenging activity are
chelating assay, O-phenanthroline quantitatively forms
represented in ascorbic acid equivalence and reported in
complexes with Fe+2 which get disrupted in the presence of
Figure 1.
chelating agents 18.The methanolic extracts of the Ficus
benjamina was assessed for their ability to chelate transition
metal like iron and exhibited dose dependent activity. The
result of iron chelating activity is also represented as
ascorbic acid equivalence (g/ml) and summarized in
Figure 3. Iron chelating activity of the extract was found to
be increasing with increase in dose.

Figure1: DPPH radical scavenging activity of varying


concentrations of methanolic extract of Ficus benjamina leaves.
Data is given in mean SD (n = 3 test). Data was compared with
ascorbic acid equivalence (g/ml)

Total antioxidant activity:


The phosphomolybdate method has been routinely used to
Figure 3: Iron chelating effect of varying concentrations of
evaluate the total antioxidant capacity of extract 17. In the methanolic extract of Ficus benjamina leaves. Data is given in
presence of the extract, Mo (VI) reduced to Mo(V) and mean SD (n = 3 test). Data is represented as ascorbic acid
forms a green coloured phosphomolybdnum V complex equivalence (g/ml).
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Research J. Pharm. and Tech. 6(11): November 2013

Reducing power assay: Estimation of total phenolic content:


Reducing power assay is a good reflector of the antioxidant In the current study, total phenolic content of the
activity of the plant. The plant having higher reducing methanolic extract of F. benjamina was estimated by Folin-
power generally reported to carry high antioxidant Ciocalteau reagent method. Quantification of the phenolic
potential too. In this experiment ferric ions are reduced to compounds was carried out with respect to the standard
ferrous ions with the colour of the reaction mixture changes curve of gallic acid. Extract exhibited the presence of high
from yellow to bluish green that can be detected at 700 nm amount of phenolic compounds (4.006 mg gallic acid
using a UVVisible spectrophotometer. Higher absorbance equivalence/gm) in a dose dependent manner. The results
of the reaction mixture indicated greater reducing potential were expressed as gallic acid equivalents (GAE) in g and
of the extract. The results for ferric reducing power activity summarized in Figure 6.
of extract of F. benjamina with compared to ascorbic acid
are reported in Figure4. Extract exhibited dose dependent
reducing power potential 19.

Figure 6: Total phenolic content of varying concentrations of


methanolic extract of Ficus benjamina leaves. Data is given in
mean SD (n = 3 test) and expressed as Gallic acid equivalence
Figure 4: Reducing power activity of varying concentrations of (GAE) in g.
methanolic extract of Ficus benjamina leaves. Data is given in
mean SD (n = 3 test). Data is represented as ascorbic acid Estimation of total flavonoid content:
equivalence (g/ml).
Total flavonoids content of methanolic extract of F.
benjamina was determined using aluminium chloride
Brine shrimp lethality assay: method. Quantification of the flavonoids was carried out
The brine shrimp cytoxicity assay was considered as a with respect to the standard curve of quercetin. Extract
convenient probe for preliminary assessment of cytotoxicity exhibited the presence of high amount of flavonoids
of the plant extracts 20. Methanolic extract of F. benjamina (16.005 mg quercetin acid equivalence/gm) in a dose
exhibited significant cytotoxic activity toward the brine dependent manner. The results were expressed as quercetin
shrimp nauplii in a dose dependent and time dependent equivalents (QE) in g and summarized in Figure 7.
manner. Results of the cytotoxic activity are summarised in
graphical form Figure 5.

Figure5: Brine shrimp lethality activity of varying concentrations Figure 7: Total flavonoid content of varying concentrations of
of methanolic extract of F. benjamina leaves. Data is given in mean methanolic extract of Ficus benjamina leaves. Data is given in
SD (n = 3 test). Data is represented as mean standard deviation mean SD (n = 3 test) and expressed as Querecetin equivalence
of 3 replicates. (QE) in g.

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Research J. Pharm. and Tech. 6(11): November 2013

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