INTRODUCTION:
Every cell releases free
radicals when it utilises oxygen for energy production i.e. All the biochemical
process results in the production of free radicals1. Also several
metabolic process, smoke and UV radiations results in free radical generation2.Various
activated forms of oxygen together is termed as reactive oxygen species (ROS)
which includes free radicals like nitric oxide radical (NO), superoxide radical
(O2), hydroxyl radical (OH), and lipid peroxide radical (LOO) and
non free radicals like hydrogen peroxide (H2O2), singled
oxygen (O2) 3. Naturally the production of these free
radicals is balanced by the antioxidative defence mechanism4. When
there is an imbalance in the free radical generation, oxidative stress develops
which brings oxidative damage to the cells. Oxidation of biological molecules
such as lipids, DNA, proteins and carbohydrates by toxic ROS causes mutation of
DNA, cell senescence and even death of the cell5.
The antioxidants behaves as free radical scavengers by
protecting and reprogramming the ROS damaged cells thus enhancing the immune
defence mechanism and lowers the probability of cancer and degenerative
disorders1.
The concentration of
pro-oxidant balance and the antioxidants determines the diseased condition of
the body. Increase in the amount of free radicals or higher oxidative stress
due to insufficient dietary antioxidant can develop pro-oxidant dominance.
Even though natural
antioxidant protect our system from the detrimental effect of free radical
there is always a need for supply of antioxidant from external natural source6.
Those natural antioxidant act by interfering in the oxidation reaction of
scavenging oxygen species, free radical in the body7. Thus this
external supplied antioxidant can check various diseases caused due to effect
of free radical accumulation in the biological system8. In this
scenario attention is given to natural antioxidant that plays a vital role in
human health as whole9.
Studies till date explain
that Medicinal plants delivers several therapeutic effects since it contains
antioxidant phytochemicals which are non-toxic in nature and possess various
mechanisms of action like stimulation of immune system, scavenging of oxidative
agents, antiviral and antibacterial effects and hormone metabolism10.
Therefore to control and prevent the oxidative damages caused to the cells,
screening of medicinal plant antioxidants becomes necessary. Extraction and
characterization of antioxidant properties and various phytochemical elements
of natural origins are done all over the world on medicinal plants11,12.
Aegle marmelos is locally known as bael, golden apple, bili and
belongs to the family Rutaceae. The tree is perennial, grows 6-7.5 m tall and
90-120 cm of width13. It is a native Indian species known for its
religious significance. It is cultivated all over India and in few other
sub-continental countries. This plant is used in traditional medicinal practise
which uses dried roots to treat dyspepsia, dysentery, chronic diarrhoea,
vomiting, rheumatism, nervous disorders. Seeds, barks and fruits of the plant
exhibit antioxidant activity. Thereby plant on whole has anti-inflammatory,
antipyretic and hypoglycaemic activity14.
To get an insight
into the knowledge of traditional medicinal plant of India, present study was
carried out to study antioxidant activity of aqueous extract of Aegle marmelos leaves extract using hydrogen peroxide and DPPH radical
scavenging activity.
MATERIALS AND METHODS:
Extraction of plant:
Leaves were washed and dried
at room temperature and finely grounded before extraction. The Aqueous extract
was prepared by dissolving 25 grams of dried leaf powder in 50 ml of distilled
water and kept in rotary overnight. It was air dried in laminar air flow
chamber to remove water and a greenish-black powder was obtained.
DPPH radical scavenging activity:
To determine the
free radical-scavenging activity of the extracts stable 1, 1-diphenyl- 2-picryl hydroxyl (DPPH) was
used1. 1 ml of plant extract at various concentrations (0.1- 0.5 mg)
was taken along with 1 ml of freshly prepared 0.1 mM methanolic DPPH. After 15
minutes at the room temperature the absorbance was measured at 517 nm. For the
assay ascorbic acid was used as a standard. Lower the absorbance at 517 nm,
higher the scavenging activity2. Percentage of DPPH scavenging
activity of Aegle marmelos was
calculated using the following equation.
Where,X0 is the
absorbance of the control and X1 is the absorbance of aqueous extract of Aegle marmelos.
Hydrogen peroxide scavenging activity:
The determination of the
extracts to scavenge hydrogen peroxide was done by the following method. 40 mM
of hydrogen peroxide was prepared in phosphate buffer (pH 7.4). 1 ml of aqueous
plant extract was taken in different concentrations (0.1- 0.5 mg) and added to
0.6 ml of 40 mM of hydrogen peroxide solution. After 10 minutes absorbance of
the hydrogen peroxide was noted down at 230 nm. Ascorbic acid is considered as
standard and blank solution contains only phosphate buffer without hydrogen
peroxide solution1. The percentage of hydrogen peroxide scavenging
exhibited by aqueous plant extract and standard solutions was calculated as
follows.
|
Hydrogen
peroxide scavenging effect (%) = |
|
Where, X0
represents the absorbance of the control and X1 represents the
absorbance of aqueous extract of Aegle
marmelos.
RESULTS AND DISCUSSION:
It is evident that venomous
consequences due to the oxidative stress can be checked with the help of native
antioxidants. Medicinal plants, herbs and spices known to contain natural
antioxidant compounds which possess protective activity against free radicals
that are of much interest15,16. In the present study antioxidant
activity of aqueous extracts is calculated by DPPH scavenging assay and H2O2
assay. Generally, antioxidant properties of the extract were found to be
concentration dependent. The aqueous extract of Aegle marmelos was showing good antioxidant activity in all assays.
Table 1: Shows the radical
scavenging activity estimated by DPPH scavenging assay and H2O2
assay in aqueous extract of Aegle
marmelos.
|
Sample Name |
Concentration (µg/ml) |
% Inhibition in
DPPH scavenging |
% Inhibition in H2O2
scavenging |
|
Aegle mar-melos |
100 |
91.38485 |
74.88568 |
|
200 |
92.88849 |
85.31889 |
|
|
300 |
95.84846 |
86.8231 |
|
|
400 |
97.37101 |
90.73406 |
|
|
500 |
98.31669 |
94.03129 |
DPPH free radical scavenging activity:
The DPPH scavenging activity
is major method used for screening the antioxidant activity of plant
extract. The stable nitrogen-centred
free radical changes the colour upon reduction from violet to yellow by either
hydrogen or electron donation. A substance which undergoes the above reaction
is termed as antioxidants1. An odd electron present in DPPH radical
results in the absorbance at 515-517 nm3. The free radical
scavenging activity of the aqueous extract of Aegle marmelos increase along with the increase in concentration.
The results were recorded in the form of % inhibition as given in Table 1 and
Figure(1) gives the graphical representation of the inhibition. The aqueous
extract of Aegle marmelos considering
in various concentrations (100-500 µg/ml) produce moderate level to high level
DPPH scavenging activity. The highest level of inhibition was recorded for 500
µg/ml as 98.31669%.
Hydrogen peroxide scavenging activity:
The phenolic compounds donate
its electrons to H2O2 and neutralise it to water1.
The ability to scavenge effectively the aqueous extract by hydrogen peroxide is
done by the method of Ruch1. The scavenging activity of extract was
seen in a concentration-dependent manner. The results were noted down in the
form of % inhibition as given in Table 1 and Figure(2) gives the graphical
representation of the inhibition. The
aqueous leaf extract of Aegle
marmelos taking in different concentrations (100-500 µg/ml) produce
moderate level to high level hydrogen peroxide scavenging activity. The highest
level of inhibition was recorded for 500 µg/ml as 94.03129%.
Anyhow hydrogen peroxide is not very much reactive, sometimes it causes
cytotoxicity by releasing hydroxyl radicals in the cells. Therefore removing H2O2
radical throughout the food system is very important1.
Figure(1): DPPH scavenging
activity of aqueous extract of Aegle
marmelos leaves.
Figure(2): H2O2
scavenging activity of aqueous extract of Aegle
marmelos leaves.
CONCLUSION:
The
leaves of Aegle marmelos was used to study the antioxidant properties. The
result explains that it has a high potential for antioxidant activity. Aqueous extract of Aegle marmelos showed high DPPH free radical scavenging and H2O2
scavenging activity.This medicinal plant exhibit the antioxidant properties since it contain
the hydroxyl group.
The
present investigation suggests that medicinal plants such as Aegle
marmelos which possess good antioxidant potential are the better supplements for
the diseases associated with oxidative stress. For further research, the effect
of temperature, the extraction method is to be considered and the components of
the extracts should be identified.
ACKNOWLEDGMENT:
The authors thank Sathyabama
University for providing an
opportunity for this particular research and the head of the department for allowing to utilise the laboratory
facilities to carry out this study.
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