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179
Author for Correspondence:
Anju Venugopal,
JKKMMRF'S College of Pharmacy,
B-Komarapalayam, Namakkal, Tamil Nadu 638183, India.
Email: anjudimple@gmail.com
Research Article
ISSN: - 2306 –6091
Available Online at: www.ijphr.com
IN-VITRO SCREENING OF METHANOLIC LEAF EXTRACTS OF
ALBIZIA SAMAN (JACQ.)MERR, FOR THEIR ANTIOXIDANT PROPERTY
*Anju Venugopal, Suresh Arumugam, Suriya Annamalai, Senthilkumar Natesan
JKKMMRF's-Annai JKK Sampoorani Ammal College of Pharmacy,
B-Komarapalayam, Namakkal, Tamil Nadu - 638183, India.
___________________________________________________________________________
Abstract
The present study was designed to evaluate the in-vitro antioxidant potential of methanolic extract of Albizia
saman leaves. The extraction of leaves was carried out by Soxhlet apparatus using methanol. The methanolic
extract investigated for free radical scavenging activity of the 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH),
reducing power assay and nitric oxide scavenging activity. In DPPH assay the methanolic extract at a
concentration 500µg/ml was showed 79.7049±0.2158. The methanolic extracts showed maximum activity of
81.608±0.2843% at 500µg/ml in nitric oxide scavenging assay. In reducing power assay the methanolic extract
showed maximum absorbance of 0.3706±0.00158 at 500µg/ml .The phytochemical screening revealed the
presence of alkaloids and polyphenolic compounds. This suggest a potential utility of the plant as a source of
phenolic antioxidants and may provide leads in the ongoing research for natural antioxidants form Indian
medicinal plants to be used in treating diseases related to free radical reactions.
Keywords: Albizia saman, DPPH assay, Reducing power assay, Free radical scavenging, Antioxidant activity.
___________________________________________________________________________
Introduction
Antioxidants are believed to quench free radicals
free radicals are atom or molecule with singlet
unpaired electron which make them highly
reactive. Oxidative free radicals are generated by
metabolic reactions. Free radicals create a chain
reaction leading to membrane lipid per oxidation,
DNA damage etc. Free radical oxidation has been
implicated in Cancer, atherosclerosis, neuro-
degenerative diseases and inflammatory bowel
disease.1Antioxidants are added to pharmaceutical
formulations as redox systems possessing higher
oxidative potential than the drug that they are
designed to protect, or as chain inhibitors of radical
induced decomposition. In general, the effect of
oxidants is to break up the chain formed during a
hydrogen atom or an electron to free radical
receiving the excess energy possessed by the active
molecule.2Samanea saman (rain tree) is a
tropically found common plant which mitigates
multitude of diseases and ailments.3There are
several folk remedies prepared from various parts
of rain tree. The root decoction is used in hot baths
for stomach cancer.4Rain tree is a folk remedy for
colds, diarrhoea, headache, intestinal ailments, and
stomach ache.5Recent evidence suggest that free
radicals, which are generated in any bioorganic
redox processes, may induce oxidative damage in
various components of the body (e.g., lipids,
proteins and nucleic acids) and may also be
International Journal of
Pharmaceuticals and
Health care Research
180
Anju Venugopal. et al., Int. J. Pharm. & H. Care Res., Vol.–02 (03) 2014 [179 - 183]
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involved in processes leading to the formation of
mutations.6
Materials and methods
Collection of Plant
The leaves of Albizia saman were collected from
Ernakulum District in the month of march 2014
and authenticated by Dr.M.S Francis, M.Sc,
M.Phil, P.hD, Associate Professor in Botany,
Center for Post Graduate Studies and Research,
Sacred Heart College, Kochi-13. The voucher
specimen of the plant was prepared and stored in
Unibiosys Biotech Research Labs; Kochi with
reference number BIS 01/2014/1012.The plant
material was dried, powdered and stored in airtight
containers until further studies.7
Preparation of extracts
The Leaves of Albizia saman were washed with
distilled water and dried in shade. About 400g of
air dried powdered Leaves of Albizia saman was
taken in 1000ml soxhlet apparatus and extracted
with petroleum ether for 2 days. At the end of
second day the powder was taken out and it was
dried. After drying it was again packed and
extracted by using methanol as solvent, till color
disappeared. The temperature was maintained at
55ºC-65ºC. After that, the extract was concentrated
by distillation and solvent was recovered. The final
solution was evaporated to dryness and dry residue
was obtained.
In vitro anti oxidant activity
DPPH Assay
Procedure
Methanolic extract were prepared in methanolic
solution at various concentrations (100,200, 300,
400 and 500 μg/ml).To a set of test tubes, 2.9 ml of
DPPH solution (100 μM/ml in methanol) and 0.1
ml of varying concentrations of test sample were
added. The mixture was then shaken vigorously
and allowed it to stand in dark for 30 m and
absorbance was measured using a UV
spectrophotometer at 517 nm.8A control solution
was consisting of 0.1 ml of methanol and 2.9 ml of
DPPH radical solution.9Ascorbic acid was used as
standard.10 Percentage scavenging of DPPH radical
was calculated by comparing the absorbance
between the test and control. All determinations
were done in triplicate and the mean values were
determined:
Scavenging activity (%) = (Absorbance of control –Absorbance of sample) × 100
Absorbance of control
Reducing power assay
Procedure
The reducing power of extracts was determined
according to the method of different amounts of
each extracts (100,200,300,400,500μg/ ml) in water
were mixed with phosphate buffer (2.5 ml, 0.2 M,
pH 6.6) and potassium ferricyanide (2.5 ml,
1%).11The mixture was incubated at 50oC for 20
min. A portion (2.5 ml) of trichloroacetic acid
(10%) was added to the mixture to stop the
reaction, which was then centrifuged at 3000 rpm
for 10 min. The upper layer of solution (2.5 ml)
was mixed with distilled water (2.5 ml) and FeCl3
(0.5 ml, 0.1%), and the absorbance was measured
at 700 nm. Increased absorbance of the reaction
mixture indicated increased reducing power.
Ascorbic acid was used as standard. Compared the
results of test and standard. All determinations
were done in triplicate and the mean values were
determined
Nitric oxide Scavenging Assay
Procedure
The scavenging effect of the methanolic extracts of
Albizia Saman leaves on nitric oxide was measured
according to the a reaction mixture was prepared
containing: 10 mM Sodium Nitro Prusside in 0.5 M
phosphate buffer, pH 7.4, and various doses
(100,200,300,400,500/ml) of the test solution in a
final volume of 3 ml. After incubation for 60 min at
37°C, Griess reagent (1% Sulfanilamide in 5%
H3PO4 and 0.1 % Naphthyl ethylene diamine
dihydrochloride) was added.12 The pink
chromophore generated during diazotization of
nitrite ions with sulphanilamide and subsequent
coupling with a-napthyl-ethylene diamine was
measured spectrophotometrically at 540 nm.
Ascorbic acid was used as a positive control. All
determinations were done in triplicate and the mean
values were determined Nitric oxide scavenging
ability (%) was calculated by using the formula:
Scavenging activity (%) = (Absorbance of control –Absorbance of sample) × 100
Absorbance of control
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Anju Venugopal. et al., Int. J. Pharm. & H. Care Res., Vol.–02 (03) 2014 [179 - 183]
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Table No. 01: DPPH scavenger assay of methonolic extract of
Albizia samanleaves compared with standard ascorbic acid
S.No
Concentration(µg/ml)
%inhibition of
Std(Ascorbic Acid)
Ic50
(µg/ml)
%inhibition of Methanolic
Extract
Ic50
(µg/ml)
1
100
66.07±0.2121***
75.68
45.93±0.0723***
149.72
2
200
73.80±0.2121***
66.79±0.3596***
3
300
81.13±0.2157***
70.96±0.2172***
4
400
84.90±0.3597***
75.23±0.2158***
5
500
94.24±0.3597***
79.70±0.2158***
Values are mean±S.D., n=3,p<0.001=***,p<0.01=**, p<0.05=* when compared to control.
100
200
300
400
500
0
20
40
60
80
100 Std
Met.Extct
concentration(ug/ml)
%inhibition
Graph 1: DPPH scavenger assay of methonolic extract of Albizia Saman leaves compared with
standard ascorbic acid
Table No. 02: Reducing power assay of Albizia Saman leaves compared with standard ascorbic acid.
S. No
Concentration(µg/ml)
Absorbance of std
(ascorbic acid) (700nm)
Absorbance of methanolic extract.(700nm)
1
100
0.1546±0.0032***
0.1276±0.0051***
2
200
0.3776±0.0031***
0.1453±0.0051***
3
300
0.5170±0.0020***
0.2110±0.0020***
4
400
0.6390±0.0020***
0.2686±0.0022***
5
500
0.7163±0.0031***
0.3706±0.0015***
Values are mean±S.D., n=3,p<0.001=***, p<0.01=**,p<0.05=* when compared to control.
100
200
300
400
500
0.0
0.2
0.4
0.6
0.8 Std
Met.Extract
Concentration(ug/ml)
Absorbance(700nm)
Graph 2: Reducing power assay of Albizia Saman leaves compared with Standard ascorbic acid.
182
Anju Venugopal. et al., Int. J. Pharm. & H. Care Res., Vol.–02 (03) 2014 [179 - 183]
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Table No. 03: Nitric Oxide (NO) scavenging activity of Albizia Saman leaves compared
with standard ascorbic acid.
S.No
Concentration(µg/ml)
%inhibition of Std
(Ascorbic Acid)
Ic50 (µg/ml)
%inhibition of
Methanolic Extract
Ic50 (µg/ml)
1
100
57.54±0.2132***
86.89
42.81±0.2843***
173.79
2
200
66.74±0.2843***
57.54±0.2132***
3
300
79.95±0.3554***
71.35±0.3339***
4
400
84.12±0.1421***
77.89±0.1421***
5
500
92.46±0.5685***
81.61±0.2843***
Values are mean±S.D., n=3,p<0.001=***,p<0.01=**,p<0.05 when compared to control.
100
200
300
400
500
0
20
40
60
80
100 Std
Met.Extract
Concentration(ug/ml)
%Inhibition
Graph 3: Nitric Oxide (NO) scavenging activity of Albizia Saman leaves compared with
standard ascorbic acid
Results and discussion
DPPH Assay
The DPPH radical scavenging assay is an easy
rapid and sensitive method for the antioxidant
screening of plant extracts.A number of methods
are available for the determination of free radical
scavenging activity but the assay employing the
stable 2, 2-diphenyl-1-picryl-hydrazyl radical
(DPPH) has received the maximum attention owing
to its ease of use and its convenience.13
In the DPPH photometric assay method, the
methanolic extract of Albizia Saman leaves
exhibited a comparable antioxidant activity with
that of standard ascorbic acid at varying
concentration tested (100, 200, 300, 400,500
μg/ml). There was a dose dependent increase in the
percentage antioxidant activity for all
concentrations tested.
The extract at a concentration of 100μg/ml showed
a percentage inhibition of 45.9303 ± 0.07255 and
for 500μg/ml it was 79.7049 ± 0.2158. The
concentration required for 50% inhibition Ic50 was
found to be 149.72 µg/ml . Ascorbic acid was used
as the standard drug for the determination of the
antioxidant activity by DPPH method. The
concentration of ascorbic acid varied from 100 to
500μg/ml. Ascorbic acid at a concentration of
100μg/ml exhibited a percentage inhibition of
66.07±0.2121 and for500μg/ml, 94.2522±0.3597.
The Ic50 value was found to be 75.68 μg/ml (Table
1). A graded increase in percentage of inhibition
was observed for the increase in the concentration
of ascorbic acid. (Graph: 1) illustrate significant
decrease in the DPPH radical due to the scavenging
ability of extracts and ascorbic acid.
Reducing Power Assay
In this assay the yellow colour of the test solution
changes to various shades of green and blue is
depending upon the reducing power of each
compound. The presence of radicals causes the
conversion of the Fe 3+ / ferricyanide complex
used in this method to the ferrous form a higher
absorbance at 700nm indicates a higher reducing
power. The results of the reducing power assay are
given in (Table 2). Therefore by measuring the
formation of pearls Prussian blue at 700nm, the Fe
2+ concentration.
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The reducing power of the alcoholic extracts and
standards increases with the increase in amount of
sample and standard concentrations (graph: 2.Table
2). Shows increase in the absorption of methanolic
extract with increase in the concentration. The
methanolic extracts showed significant maximum
absorbance of 0.3706±0.0015at 500 μg /ml,
whereas ascorbic acid at the same concentration
exhibited 0.7163±0.0031absorbance.
Nitric Oxide Scavenging Assay
Nitric oxide (NO) is a potent pleiotropic inhibitor
of physiological processes such as smooth muscle
relaxation, neuronal signaling, inhibition of platelet
aggregation and regulation of cell mediated
toxicity. It is a diffusible free radical that plays
many roles as an effectors molecule in diverse
biological systems including neuronal messenger,
vasodilatation and antimicrobial and antitumor
activities.
Suppression of released NO may be partially
attributed to direct NO scavenging, as the extracts
of Albizia saman leaves decreased the amount of
nitrite generated from the decomposition of SNP in
vitro. The scavenging of NO by the extracts was
increased in dose dependent manner (table 4).
(Graph: 3) illustrates a significant decrease in the
NO radical due to the scavenging ability of extracts
and ascorbic acid. The methanolic extracts showed
significant activity of 81.608±0.2843 % at 500 μg
/ml, and Ic50 was 86.89 μg /ml whereas ascorbic
acid at the 500 μg /ml concentration exhibited
92.4623±0.5685% inhibition and Ic50 was 173.79
μg /ml.
Conclusion
On the basis of the above results it can be
concluded that the methanolic extract possess
significant antioxidant activities studied by in- vitro
screening methods. The phytochemical screening
revealed the presence of alkaloids and polyphenolic
compounds. Methanolic leaf extract was screened
for antioxidant activity by performing in-vitro
assay method namely DPPH radical scavenging,
reducing power method and nitric oxide
scavenging assay. In all the antioxidant assay
methods, the methanolic extract of Albizia saman
leaves exhibited a comparable antioxidant activity
with that of standard ascorbic acid at varying
concentration tested. There was a dose dependent
increase in the percentage antioxidant activity
significantly. This suggest a potential utility of the
plant as a source of phenolic antioxidants and may
provide leads in the ongoing research for natural
antioxidants form Indian medicinal plants to be
used in treating diseases related to free radical
reactions.
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