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