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Int J Adv Pharm Biol Sci Vol.2, Issue 1, 99-106 Rajkumar et al.
99
INTERNATIONAL JOURNAL OF ADVANCE PHARMACEUTICAL AND BIOLOGICAL SCIENCES
Vol. 2, Issue. 1, JANUARY-MARCH 2012 ISSN 2249 –8966
Research Article Available online http://www.ijapbs.in
EVALUATION OF ANTIOXIDANT PROPERTIES OF ALBIZIA AMARA LEAVES
T.Rajkumar* 1, E.Satheesh Kumar1and B.N.Sinha2
1Kottam Institute of Pharmacy, Eravally ‘X’Roads, Mahaboob nagar District, AP. 509125
2Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi,India.
.
Received: 10th December 2011, Revised and Accepted: 30th December 2011
*Corresponding author: E-mail: rajkutty1983@gmail.com. Phone: 7207504238
ABSTRACT
The aim of the study is to evaluate the anti oxidant and free radical scavenging property and
to determine the total phenolic content of the petroleum ether and methanolic extracts of
Albizia amara. The anti oxidant activity of the extracts were investigated by three different
methods, 2,2-diphenyl-1-picrylhydrazyl radical assay, nitric oxide free radical scavenging
assay and reducing power assay. While the Folin–Ciocalteu method was used to determine
the total phenolic content. The Methanolic extract exhibited the strongest anti oxidant activity
when compared to petroleum ether extract. Total Phenol Content in terms of Gallic acid
equivalent was found to be 243.37 for Methanolic extract and 161.87 for petroleum ether
extract. There was a positive correlation between total phenolic content and antioxidant
activity, R2= 0.9919 & 0.9953 for Methanolic extract and petroleum ether extract respectively
in the plant samples. The results indicate the presence of phenolic compounds as well as
significant antioxidant activity.
Keywords: Anti oxidant; Total phenolic content; Flavonoids; Albizia amara
INTRODUCTION
Recently there has been an upsurge of
interest in the therapeutic potential of
medicinal plants as antioxidants in
reducing such free rad ical-induce d
tissue injury Besides, well known and
traditionally used natura l antioxidants
from teas, wines, fruits, vegetables and
spices, some natural antioxidants (e.g.
ros e m a ry an d sage ) are already
exploited commerci al l y e i t h e r a s
antioxidant additives or as nutritional
supplements .The major constituents of
biolog ica l me mbranes ar e lipid s and
proteins. The number of functions of
membranes increases as the protein
amount increases. Reactive oxygen species
can easily initiate the lipids causing
damage of the cell membrane
constituent i.e. phospholipids, lipoproteins
by propagating a reaction cycle It has
been mentioned that antioxidant activity
of plants might be due to their phenolic
compounds Flavonoids are a group of
polyphenolic compounds with known
properties which include free radical
scavenging, inhibition of hydrolytic
oxidative enzymes and anti-
inflammatory action . Antioxidants are
Int J Adv Pharm Biol Sci Vol.2, Issue 1, 99-106 Rajkumar et al.
100
compounds that inhibit or delay the
oxidation process by blocking the
initiation or propagation of oxidizing chain
reactions. There are two basic categories
of antioxidant namely synthetic and
natural ones. Restriction on the use of
synthetic anti-oxidants is being imposed
because of their carcinogenicity. Thus the
interest in natural antioxidants has been
increased considerably. As resources of
natural antioxidants much attention has
been paid to plants. Especially, the
antioxidants present in edible plants have
recently been considered as food additives
[1].
The antioxidant activity of several Indian
plants has been reported. In this paper the
antioxidant activity of Albizia amara
(Fabaceae) known as “Oil Cake tree”
which is an endemic plant in dry areas of
Tamilnadu, Andhra and Karnataka in India
is reported. The seeds of Albizia amara
(Fabaceae) used as an astringent, treating
piles, diarrhoea, gonorrhoea, leprosy,
leucoderma, erysipelas and abscesses. The
leaves and flowers have been applied to
boils, eruptions, and swellings, also
regarded as an emetic and as a remedy for
coughs, ulcer, dandruff and malaria [2, 3].
MATERIALS AND METHODS:
Chemicals
All chemicals used were of analytical
grade. DPPH was obtained from Sigma
Chemicals, USA. sulphanilamide,
phosphoric acid, napthylenediamine
dihydrochloride, vitamin- C, Folin-
Ciocalteu reagent, ferric chloride,
potassium ferricyanide, trichloro acetic
acid, sodium phosphate, sodium
carbonate, sodium nitrite, ammonium
molybdate, gallic acid and methanol
were obtained from CDH , Mumbai,
India.
Materials
The authenticated leaves of Albizia amara
were collected from medicinal garden of
Medicinal plants Revitalisation and
Rehabilitation Centre, Sevaiyur,
Tamilnadu on the month of july 2009 and
authenticated furtherly by Dr.S.Jha,
Professor, Birla Institute of Technology,
Mesra, Ranchi, India and a voucher
specimen (PHARM/HS/14/09-10) was
retained in our laboratory for future
reference.
Sample preparation and extraction
The leaves were dried under shade for 4-6
days. Then the dried materials were milled
to powder. This powdered material was
again dried in the oven at 40 0 C for 4 h
and used for extraction followed by
concentration, screening. The coarsely
dried powdered leaves were extracted with
Petroleum Ether (b.p.600-800) cold
maceration for 72 h, and hot percolation by
90% methanol about 72 h. Both extracts
were recovered and concentrated to
dryness.
The dried extracts were dissolved in
respective solvents to a final concentration
of 500 µg/ml (Sample stock solution).
Preliminary phytochemical screening
The various extracts of Albizia amara were
subjected to qualitative tests for
preliminary phytochemical screening. This
was carried out by the method described
by Harborne and Kokate [4, 5] to show the
presence of various compounds
flavonoids, alkaloids, tannins, saponins
and phenolic compounds etc.
DPPH Radical scavenging assay
The free radical scavenging activity of the
Petroleum ether and Methanolic extracts of
Albizia Amara leaf was measured in vitro
by 1, 1-diphenyl-2-picrylhydrazyl (DPPH)
assay. 0.3 mM solution of DPPH in
methanol was prepared and 1 ml of this
solution was added to 3 ml of the extract
solutions at different concentrations. The
mixture was shaken and allowed to stand
at room temperature for 30 min and the
absorbance was measured at 517 nm using
a spectrophotometer. Lower absorbance of
the reaction mixture indicated higher free
Int J Adv Pharm Biol Sci Vol.2, Issue 1, 99-106 Rajkumar et al.
101
radical scavenging activity. The
percentage scavenging activity at different
concentrations was determined and the
IC50 value of the fractions was compared
with that of ascorbic acid (vitamin C),
which was used as the standard [6].
The IC50 value was defined as the
concentration in (µg/ml) of extract that
inhibits the formation of DPPH radicals by
50%.
Percentage of inhibition (I %) was
calculated in the following formula:
I % = 100 x (AC – AS)/AC
Where,
AC is the absorbance of the control and
AS is the absorbance of the sample.
The extract methanolic solution without
DPPH was used as a blank.
AC is the absorbance of the control
(containing all reagents except the
sample), and AS is the absorbance of the
sample.
Nitric oxide radical scavenging activity
The interaction of petroleum ether and
methanolic extract of sample with nitric
oxide was assessed by the nitrite detection
method. Sodium nitroprusside (5mM) in
phosphate buffered saline was mixed with
3.0 ml of different concentrations (50 –
250 µg/ml) of extracts dissolved in the
suitable solvent system and incubated at
25°C for 150 minutes. The samples from
the above were reacted with Greiss reagent
(1% sulphanilamide, 2% H3PO4and 0.1%
napthylenediamine dihydrochloride). The
absorbance of the chromophore formed
during the diazotization of nitrite with
sulphanilamide and subsequent coupling
with napthylethylenediamine was read at
546 nm. The same reaction mixture
without the extract but with equivalent
quantity of distilled water served as
control. Ascorbic acid was used as
reference standard [7].
Reducing power assay
The reducing power of the extracts
was assessed by the method of Oyaizu (8).
Different concentrations (50 – 250 µg/ml)
of the extracts (2.5 ml) were mixed
with 2.5 ml of 200 mM sodium
phosphate buffer (pH 6.6) and 2.5 ml
of 1% potassium ferricyanide. The
mixture was incubated at 50°C for 30 min.
After 2.5 ml of 10% trichloroacetic acid
(w/v) was added, the mixture was
centrifuged at 1000 rpm for 10 min. The
upper layer (5 ml) was mixed with 5 ml of
deionised water and 1 ml of 0.1% of ferric
chloride, and the absorbance was measured
spectrophotometrically at 700 nm. Blank
sample was prepared using distilled water
instead of extract. The values are
presented as the means of triplicate
analyses. The extract concentration
providing 0.5 of absorbance (EC50) was
calculated from the graph of absorbance at
700 nm against extract concentration.
Ascorbic acid was used as standard [9].
Total antioxidant capacity
(Phosphomolybdenum reduction assay)
Phosphomolybdenum (PMo) assay was
used to estimate the capability of the
samples to reduce transition metal ions.
The reagent solution contained ammonium
molybdate (4 mM), sodium phosphate (28
mM), and sulfuric acid (600 mM) mixed
with the samples diluted in methanol. The
samples were incubated at 90°C for 90
min, cooled down to room temperature,
and the absorbance of the green
phosphomolybdenum complex was
measured at 695 nm. The reducing
capacity of extracts was calculated using
the following equation [10, 11].
Abs Final = Abs Sample - Abs Blank -
Abs Extract
Where:
Abs extract = absorbance of sample where
molybdate solution was replaced by water;
Abs blank = absorbance of blank
containing methanol instead of extract
sample.
For reference, the appropriate solutions of
ascorbic acid have been used, and the
reducing capacity of the analyzed extract
was expressed as the ascorbic acid
Int J Adv Pharm Biol Sci Vol.2, Issu
e 1,
equivalent (AAE)
per gram of sample dry
weight.
Determination of total Phenolic content
(TPC) of leaves in terms of Gallic acid
Content
The concentration of the total soluble
phenolic compounds in petroleum ether
and methanol extracts of
Albizia amara
leaf was determined
with the Folin
Ciocalteu reagent. Gallic acid was used a
stand
ard phenolic compound. 1 ml of
p
et.ether and methanol extracts
(1000µg/ml) were separately pipetted into
a volumetric flask and diluted with
distilled water (46 ml). One millilitre of
Folin-Cioc
alteu reagent was added and
the contents of the flask were mixed
thoroughly. After 3 min, 3 ml of sodium
carbonate (2%) was added and the mixture
was allowed to stand for 2
intermittent shaking. The absorbance was
measured at 760 nm in a
spectropho
tometer. The concentration of
total phenolic compounds in the leaf
extracts was determined as µg of gallic
acid equivalents using an equation that was
obtained from
the standard gallic acid
graph [12].
Absorbance = 0.0053 x Total Phenols
[Gallic Acid Equivalent (µg)] -
Statistical analysis
All the experiments were carried out in
triplicates. The IC 50 values were
presented by their respective 95%
confidence limits. The TPC
(µg
shown as mean ±SEM. One way analysis
of variance (ANOVA) follo
Dunnett’s test was used
to assess
signifcant
differences (p<0.05) between
extracts. All the
statistical analy
accomplished using the
computer software
GraphPad Prism 3.02 for
(GraphPad Software, San Diego, CA
USA).
RESULTS
TABLE 1: Ic 50 Values o
f DPPH And
Nitric Oxide Free Radical Scavenging
Assay (Nrsa), Ec 50
Value of Reducing
e 1,
99-106
Rajkumar
per gram of sample dry
Determination of total Phenolic content
(TPC) of leaves in terms of Gallic acid
The concentration of the total soluble
phenolic compounds in petroleum ether
Albizia amara
with the Folin
-
Ciocalteu reagent. Gallic acid was used a
ard phenolic compound. 1 ml of
et.ether and methanol extracts
(1000µg/ml) were separately pipetted into
a volumetric flask and diluted with
distilled water (46 ml). One millilitre of
alteu reagent was added and
the contents of the flask were mixed
thoroughly. After 3 min, 3 ml of sodium
carbonate (2%) was added and the mixture
was allowed to stand for 2
h with
intermittent shaking. The absorbance was
measured at 760 nm in a
tometer. The concentration of
total phenolic compounds in the leaf
extracts was determined as µg of gallic
acid equivalents using an equation that was
the standard gallic acid
Absorbance = 0.0053 x Total Phenols
0.0059.
All the experiments were carried out in
triplicates. The IC 50 values were
presented by their respective 95%
(µg
/ml) were
shown as mean ±SEM. One way analysis
of variance (ANOVA) follo
wed by
to assess
differences (p<0.05) between
statistical analy
sis was
computer software
GraphPad Prism 3.02 for
Windows
(GraphPad Software, San Diego, CA
,
f DPPH And
Nitric Oxide Free Radical Scavenging
Value of Reducing
Power Assay(Rpa), Total Phenolic
Content Tpc) In Terms of Gallic Acid
And Total Anti Oxidant Activity (Tac)
In Ascorbic Acid Equivalents
Note: The IC
50
values are presented with
their respective 95% confi
dence limits.
The TPC values are means ± SEM
of three determinations.
DPPH: 1, 1-diphenyl-
2
picrylhydrazyl
NRSA: Nitric oxide free
scavenging assay
RPA: Reducing power assay
TPC: Total phenolic content
TAC: Total anti oxidant activity
Fig. 1:
Anti oxidant activity by using
DPPH
Plant
extract IC
50(DPPH)
(µg/ml)
IC
50(NRSA)
( µg/ml )
EC
50(RPA)
(µg/ml)
Ascorbic
acid
73.8 103
0.126
Methanolic
extract 164 205
0.087
Pet. ether
extract 213 148
0.1513
Rajkumar
et al.
102
Power Assay(Rpa), Total Phenolic
Content Tpc) In Terms of Gallic Acid
And Total Anti Oxidant Activity (Tac)
In Ascorbic Acid Equivalents
.
values are presented with
dence limits.
The TPC values are means ± SEM
of three determinations.
2
-
NRSA: Nitric oxide free
radical
RPA: Reducing power assay
TPC: Total phenolic content
TAC: Total anti oxidant activity
Anti oxidant activity by using
EC
50(RPA)
(µg/ml)
TPC
( µg/ml)
in terms
of
Gallic
acid
TAC
( µg/ml )
Ascorbic
acid
equivalent
0.126
- -
0.087
243.37 0.541
0.1513
161.86 0.388
Int J Adv Pharm Biol Sci Vol.2, Issu
e 1,
Fig. 2:
Nitric oxide radical scavenging
activity using Griess reagent
Fig. 3: Reducing power assay
e 1,
99-106
Rajkumar
Nitric oxide radical scavenging
Fig. 4:
Percentage inhibition by DPPH
Fig. 5:
Percentage inhibition by nitric
oxide radical scavenging assay
Rajkumar
et al.
103
Percentage inhibition by DPPH
Percentage inhibition by nitric
oxide radical scavenging assay
Int J Adv Pharm Biol Sci Vol.2, Issu
e 1,
Fig. 6:
Curve of Reducing Power ability
Fig. 7: Curve of
Total Anti oxidant
Capacity
DISCUSSION
Figures 1-7
showed the antioxidant
activity of Albizia amara
leaves, extracts
examined as a fraction of their
concentration. Several biochemical assays
were used to screen the antioxidant
properties: scavenging activity on DPPH
radicals (measuring th
e decrease in
DPPH radical absorption after exposure
to radical scavengers), nitric oxide
scavenging activity and reducing power
(measuring the conversion of a
Fe3+/ferricyanide complex to the
ferrous form). The assays were performed
e 1,
99-106
Rajkumar
Curve of Reducing Power ability
Total Anti oxidant
showed the antioxidant
leaves, extracts
examined as a fraction of their
concentration. Several biochemical assays
were used to screen the antioxidant
properties: scavenging activity on DPPH
e decrease in
DPPH radical absorption after exposure
to radical scavengers), nitric oxide
scavenging activity and reducing power
(measuring the conversion of a
Fe3+/ferricyanide complex to the
ferrous form). The assays were performed
for
each extract separately. Additive and
synergistic effects of phytochemicals in
fruits and vegetables are responsible for
their potent bioactive properties and the
benefit of a diet rich in fruits and
vegetables is attributed to the com
mixture of phytochemicals present in
whole foods . This explains why no single
antioxidant can replace the combination
of natural phytochemicals to achieve
the health
benefits. Analysis of (Figures
1-5
) revealed that antioxidant activity
increased with the concentration, good
results being obtained, eve
n at low extract
concentrations
[6]
.
DPPH
, a stable free radical with a
characteristic absorption at 517nm, was
used to study the radical scavenging
effects
of petroleum ether and methanol
extracts.
The decrease in absorption is
taken as a measure of the extent of radical
scavenging. The radical
activity (RSA) values were expressed
as the ratio percentage of sample
absorbance decrease and the absorbance
of DPPH˙ solut
ion in the absence of
extract at 517 nm. F
rom the analysis of
Figure 1 & 4
, we can conclude that the
scavenging effects of all extr
increased the DPPH radicals
to
concentration and were good, especially
in the case
Methanolic extract.
was found to be 164
µg/ml for methanolic
extract and 213 µg/ml for petroleum ether
extract.
Nitric oxide was generated from sodium
nitroprusside and measured by the Greiss
reduction. Sodium nitroprusside in
aqueous solution
at physiological pH
spontaneously generates nitric oxide,
which interacts with oxygen to produce
nitrate ions that can be estimated by use of
Greiss reagent. Scavengers of nitric oxide
compete with the oxygen, leading to
reduced production of nitric oxide.
plant or plant products may have the
property to counteract the formation of
nitric oxide generation in the human body.
From figure 2 & 5
significant scavenging
activity was observed for the extracts of of
Rajkumar
et al.
104
each extract separately. Additive and
synergistic effects of phytochemicals in
fruits and vegetables are responsible for
their potent bioactive properties and the
benefit of a diet rich in fruits and
vegetables is attributed to the com
plex
mixture of phytochemicals present in
whole foods . This explains why no single
antioxidant can replace the combination
of natural phytochemicals to achieve
benefits. Analysis of (Figures
) revealed that antioxidant activity
increased with the concentration, good
n at low extract
, a stable free radical with a
characteristic absorption at 517nm, was
used to study the radical scavenging
of petroleum ether and methanol
The decrease in absorption is
taken as a measure of the extent of radical
scavenging. The radical
-scavenging
activity (RSA) values were expressed
as the ratio percentage of sample
absorbance decrease and the absorbance
ion in the absence of
rom the analysis of
, we can conclude that the
scavenging effects of all extr
acts
increased the DPPH radicals
with respect
concentration and were good, especially
Methanolic extract.
IC 50 value
µg/ml for methanolic
extract and 213 µg/ml for petroleum ether
Nitric oxide was generated from sodium
nitroprusside and measured by the Greiss
reduction. Sodium nitroprusside in
at physiological pH
spontaneously generates nitric oxide,
which interacts with oxygen to produce
nitrate ions that can be estimated by use of
Greiss reagent. Scavengers of nitric oxide
compete with the oxygen, leading to
reduced production of nitric oxide.
The
plant or plant products may have the
property to counteract the formation of
nitric oxide generation in the human body.
significant scavenging
activity was observed for the extracts of of
Int J Adv Pharm Biol Sci Vol.2, Issue 1, 99-106 Rajkumar et al.
105
Albizia amara leaf. IC 50 value was found
to be 205 µg/ml for methanolic extract and
148 µg/ml for petroleum ether extract [7].
The reducing capacity of a compound may
serve as a significant indicator of its
potential antioxidant activity. A higher
absorbance indicates a higher ferric
reducing power. Figure 3 & 6 showed the
reducing powers of the methanol extracts
as a function of their concentration. The
reducing power also increased with
concentration, and the values obtained for
all the extracts were good. Methanolic
extract had the better reducing power.
EC50 values of reducing power ability
assay were found to be 0.0870 & 0.1513
µg/ml for methanolic and petroleum ether
extract respectively. With regard to
reducing power, higher reducing activities
can be attributed to higher amounts of
polyphenolics, and the reducing capacity
of a compound may reflect its antioxidant
potential. It has been reported that the
reducing properties are generally
associated with the presence of reductones,
which have been shown to exert
antioxidant action by breaking the free
radical chain by donating a hydrogen atom
Hence, Methanolic extract may have the
higher amounts of reductones and
polyphenol in compare to the petroleum
ether extract of Albizia amara leaf [8,9].
The phosphomolybdenum assay was based
on the reduction of Mo (VI) to Mo (V) by
antioxidant and subsequent formation of a
green phosphate/Mo (V) complex at acid
pH. The high absorbance values indicated
that the sample possessed significant
antioxidant activity. Herein, the total
antioxidant activities of solvent extracts
were measured and compared with that of
ascorbic acid and the control, which
contained no antioxidant component. The
assay is successfully used to quantify
vitamin E in plant (seed or leaf or other
parts) and, being simple and independent
of other antioxidant measurements
commonly employed, it was decided to
extend its application to plant extracts.
Moreover, it is a quantitative one, since the
antioxidant activity is expressed as the
number of equivalents of ascorbic acid.
The antioxidant activity of Methanolic
extract is equivalent of 0.541 mg ascorbic
acid while petroleum ether extract is
equivalent of 0.388 mg ascorbic acid
(Figure 7). The study reveals that the
antioxidant activity of the extract exhibited
an increasing trend with increasing
concentration of the plant extract [11].
Phenolic compounds may contribute
directly to antioxidative action. It has been
suggested that polyphenolic compounds
have an inhibitory effect on mutagenesis
and carcinogenesis in humans, when up to
1.0 g is ingested daily from a diet rich in
fruits and vegetables. In addition, it has
been reported that phenolic compounds are
associated with antioxidant activity and
play an important role in stabilizing lipid
peroxidation. Phenols are very important
plant constituents because of their radical
scavenging ability due to their hydroxyl
groups [12]. A positive relationship between
total phenols and antioxidant activity has
also been found in Albizia amara. It was
observed that methanol & petroleum ether
extracts were equivalent of 243.37
and1161.86 µg gallic acid respectively.
The difference between the phenolic
contents of the Methanolic extract and
petroleum ether extract was significant.
ACKNOWLEDGEMENT
The corresponding author wish to extend
his profound gratitude to the Birla Institute
of Technology, Mesra, Ranchi for
providing the necessary fund for the
research.
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