Antioxidant
Activity of the Aerial Parts of the Achyranthes aspera Var. Porphyristachya
(Wall. Ex Moq.) Hook. F.
2Dep. of Pharmacology, Pravara
Rural
3Deparrment of Pharmacognosy, M.M. College of Pharmacy, Pune.
ABSTRACT
Achyranthes aspera is common weed found in
KEYWORDS: Achyranthes aspera, DPPH assay, ABTS assay, FRAP assay.
INTRODUCTION:
A. aspera var. porphyristachya
(Wall. Ex Moq.) (Amaranthaceae)
is a weed found on wayside and at waste places throughout the
The biochemistry of oxidative stress and hydroperoxide metabolism in mammalian organs, have
been a focus of research form last few decades.2, 3 The nature of
various biological oxidants was found to cover large ranges in biological
lifetime, in concentration, and in the occurrence in cells and organs. Aerobic
metabolism entails the production of reactive oxygen species, even under basal
conditions; hence there is a continuous requirement for inactivation of these
reactive oxygen species. The steady-state of prooxidants
and antioxidants may be disrupted. A disbalance in
favor of the prooxidants and disfavoring the
antioxidants, potentially leading to damage, has been called ‘oxidative
stress’.4, 5 Such damage may afflict all types of biological
molecules, including DNA, lipids, proteins and carbohydrates. Thus, oxidative
stress may be involved in processes such as mutagenesis, carcinogenesis,
membrane damage, lipid peroxidation, protein
oxidation and fragmentation.6 In view of the variety in prooxidants, it is not surprising that nature has evolved a
battery of different types of antioxidants.7, 8 Experimental studies
revealed that cells and organisms require defense against oxidants, without
which survival under aerobic conditions would be jeopardized.
DPPH Antioxidant Assay
Table.
1. Percentage inhibition of various extracts of A. aspera at different concentrations
|
Concentration (µg/ml) |
%
Inhibition of various extracts of A. aspera at different concentrations |
|||||
|
Ascorbic
acid (ASC) |
Petroleum
ether extract (PEE) |
Chloroform
extract (CHE) |
Ethyl
acetate extract (EAE) |
Ethanol
Extract ETHE) |
Aqueous
Extract (AQE) |
|
|
25 |
95.03 |
52.02 |
42.54 |
22.66 |
33.64 |
15.34 |
|
50 |
97.34 |
53.64 |
44.05 |
36.88 |
40.69 |
20.81 |
|
75 |
98.61 |
64.05 |
53.41 |
41.39 |
51.89 |
29.83 |
|
100 |
99.19 |
63.82 |
57.46 |
37.57 |
57.34 |
36.88 |
Fig.
1. Percentage inhibition of various extracts of the aerial part of A. aspera at
different concentrations.
Fig.
2. IC50 values for various extracts of the A. aspera.
Fig.
3. Concentration response curve of Trolox for ABTS at
the absorbance at 734 nm
Fig.
4. % inhibition of various extracts of the A.
aspera.
Fig.
5 TEAC, µg/ml trolox equivalent per g dry weight of
plant
Fig.
6. Concentration response curve of Trolox for FRAP at
the absorbance at 593 nm.
Fig.
7. Antioxidant activity of various extracts to reduce the ferric ion TPTZ
complex.
Fig.
8.TEAC, µg/ml trolox equivalent per g dry weight of
plant
MATERIALS AND METHODS:
Plant material collection and
authentication:
The plant specimen for the proposed study was collected
from Ahmednagar district of Maharashtra. Care was
taken to select healthy plants and normal organs. Mr. P. G. Diwakar
Deputy Director Botanical Survey of
Table.
2. IC50 values for various extracts of the A. aspera.
|
Name of extract |
IC 50 Values. |
|
Ascorbic acid. |
12.50 |
|
Petroleum ether extract |
31.25 |
|
Chloroform extract |
71.25 |
|
Ethyl acetate extract |
96.75 |
|
Ethanol extract |
78.75 |
|
Aqueous extract |
147.50 |
ABTS Antioxidant Assay:
Table.
3 Absorbance and % inhibition of various concentrations of Trolox.
|
Concentration
of Trolox (mM) |
Absorbance |
%
Inhibition |
|
0.5 |
0.612 |
|
|
0.377 |
||
|
0.128 |
||
|
2.0 |
0.007 |
99.03 |
|
2.5 |
0.006 |
99.17 |
Table.
4. Absorbance and % inhibition of various extracts of A. aspera.
|
A. aspera
extracts (30µl of 50 µg/ml ) |
Absorbance |
%
Inhibition |
|
ABTS solution |
0.721 |
|
|
Petroleum ether extract (PEE) |
0.270 |
|
|
0.275 |
||
|
0.455 |
||
|
Ethanol extract (ETHE) |
0.517 |
28.29 |
|
Aqueous extract (AQE) |
0.604 |
16.13 |
Extraction:
Dried and
coarsely powdered aerial part of A.aspera was subjected to successive solvent
extraction in Soxhlet extractor using petroleum ether, chloroform, ethyl
acetate, and ethanol as solvent and the marc left was refluxed with water. All
the extracts were vacuum dried to produce PEE (1.28%), CLE (0.716%), EAE
(0.58%), ETE (6.94%), and AQE (7.14%), respectively.
Chemicals Used.
Ascorbic acid,
ABTS, 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), 2, 4, 6-Tri (2-pyridyl)-s-triazine (TPTZ), Trolox,
potassium per sulphate. All these chemicals necessary
for the antioxidant activity were purchased from Sigma (USA).
Evaluation of antioxidant potential:
DPPH Assay:
Free radical scavenging potential of extract was
determined by DPPH assay.9 7.886 mg of DPPH was accurately weighed
and dissolved in 100ml methanol to obtain 200 µM solution of DPPH. Different
concentrations of extracts (25-100 µg/ml) were prepared. To 2 ml methanol
solution of DPPH, 2 ml of sample solution was added. The mixture was incubated
in dark at room temp for 15 min. The degree of free radical scavenging activity
in presence of different concentration of extracts and their absorbance were
measured colorimetrically at 517 nm. The degree of
free radical scavenging activity was expressed as percentage inhibition.
% inhibition = {(A control – A sample)/ (A control)} X
100
A control – Absorbance of DPPH alone
A sample – Absorbance of DPPH along with different
concentrations of extracts.
IC50 was calculated from equation of line obtained by
plotting a graph of concentration (mcg/ml) verses % inhibition.
Table.
5. TEAC, µg/ml trolox
equivalent per g dry weight of plant
|
Name
of extract |
TEAC
µg/ml trolox equivalent per g dry weight of plant |
|
Petroleum ether extract |
1.300 |
|
Chloroform extract |
1.275 |
|
Ethyl acetate extract |
0.775 |
|
Ethanol extract |
0.575 |
|
Aqueous extract |
0.350 |
Ferric reducing Antioxidant Potential (FRAP)
Assay:
Table
6. Absorbance of various concentrations of Trolox.
|
Concentration
of Trolox (mM) |
Absorbance |
|
0.132 |
|
|
1.0 |
0.221 |
|
1.5 |
0.329 |
|
2.0 |
0.447 |
|
2.5 |
0.591 |
Table
7. Absorbance of various extracts of the aerial part of A. aspera.
|
A. aspera
Extracts (200µl of 50 µg/ml ) |
Absorbance |
|
FRAP solution |
0.160 |
|
Petroleum ether extract (PEE) |
0.479 |
|
0.360 |
|
|
0.096 |
|
|
Ethanol extract (ETHE) |
0.121 |
|
Aqueous extract (AQE) |
0.096 |
Table
8. TEAC, µg/ml trolox
equivalent per g dry weight of plant
|
Name
of extract |
TEAC
µg/ml trolox equivalent per g dry weight of plant |
|
Petroleum ether extract |
2.150 |
|
Chloroform extract |
1.600 |
|
Ethyl acetate extract |
0.275 |
|
Ethanol extract |
0.550 |
|
Aqueous extract |
0.275 |
ABTS Assay:
ABTS•+ radical cation is generated by
reacting 7 mM ABTS and 2.45 mM
potassium peroxodisulfate via incubation at room
temperature (23 0C) in the dark for 12–16 h.10 The ABTS•+
solution was diluted with 80% HPLC-grade ethanol to an absorbance of
0.700 ± 0.040 at 734 nm and equilibrated at 30 0C. Plant extracts
were diluted with distilled water or 80% methanol, such that after introduction
of a 30 µl aliquot of each dilution into the assay, it produced from 20% to 80%
inhibition of the blank absorbance. To 3 ml of diluted ABTS•+, 30 µl
of the plant extract solution was added and mixed thoroughly. The reactive
mixture was allowed to stand at room temperature for 6 min and the absorbance
was recorded immediately at 734 nm. Trolox standard
solutions (concentrations from 0 to 2.5 µg/ml) in 80% ethanol were prepared and
assayed using the same conditions. Appropriate solvent blanks were run in each
assay. The percent of inhibition of absorbance at 734 nm was calculated and
plotted as a function of concentration of Trolox for
the standard reference data.
Table
9. Phytochemical screening of the various extracts of
A. aspera.
|
Test
performed for |
Petroleum
ether extract (PEE) |
Chloroform
extract (CHE) |
Ethyl
acetate extract (EAE) |
Ethanol
extract (ETHE) |
Aqueous
extract (AQE) |
|
Carbohydrates |
- |
- |
- |
+ |
+ |
|
Proteins |
- |
+ |
+ |
+ |
- |
|
Alkaloids |
+ |
+ |
+ |
+ |
- |
|
Glycosides |
- |
- |
- |
- |
- |
|
Steroids |
+ |
+ |
- |
- |
- |
|
Flavonoids |
- |
- |
- |
- |
- |
|
Tannins and phenolic
compounds |
- |
- |
- |
- |
- |
The absorbance of the resulting oxidized solution was
compared to that of the calibrated Trolox standard.
Results were expressed in terms of Trolox equivalent
antioxidant capacity (TEAC, µM Trolox equivalents per
g dry weight of plant)
Ferric reducing antioxidant
potential assay (FRAP):
An aliquot (200
µl) of an extract (with appropriate dilution, if necessary) was added to 3 ml
of FRAP reagent (10 parts of 300 mM sodium acetate
buffer at pH 3.6, 1 part of 10 mM TPTZ solution and 1
part of 20 mM FeCl3•6H2O
solution) 11 and the reaction mixture was incubated in a water bath
at 37 0C. The increase in absorbance at 593 nm was measured at 30 min. The
antioxidant capacity based on the ability to reduce ferric ions of the extract
was expressed as µM Trolox equivalents per gram of
plant material on dry basis.
Phytochemical screening
of the crude extracts:
Various Phytochemical studies
including test for carbohydrates, proteins, alkaloids, glycosides, steroids, flavonoids, tannins and phenolic
compounds were carried out.12
RESULTS AND DISCUSSION:
Reactive oxygen species (ROS) are involved in the
pathogenesis of various diseases. Uncontrolled
oxidation is caused by free radicals. Free radicals oxidize all major classes
of biomoleculs. The products of these oxidation
reactions diffuse from the original site of attack and spread the damage all
over the body and produces serious damage to almost all the cells. Some
important biomoleculs susceptible to free radical
oxidation are Lipids, Proteins, Nucleic acids, Carbohydrates. Thus the need of
antioxidant therapy arises. As A. aspera is proved to be effective anti inflammatory agent13 and immunomodulator
14, the antioxidant property of the plant may be useful in the
treatment of these diseases.
In DPPH assay petroleum ether extract shows better
percentage inhibition (fig.1; Table 1) than other extracts. IC50 value shown by
the Petroleum ether extract was found to be lowest as compared to other
extracts (fig.2; Table 2). Results of ABTS assay depends on the percentage
inhibition shown by the different concentration of trolox,
which is used as a standard this assay. Depending upon the percentage
inhibition shown by the various extracts (Table 4), and comparison with the
percentage inhibition shown by the various concentration of trolox
(Table 3), trolox equivalent antioxidant
concentration (TEAC) of the extract was determined. It was found that in ABTS
assay, TEAC of the petroleum ether extract was highest (1.30 µg/ml trolox equivalent per g dry weight of plant) than other
extracts of the A. aspera
(fig.5; Table 5). In FRAP assay also, TEAC of the petroleum ether extract was
found to be highest (2.15 µg/ml trolox equivalent per
g dry weight of plant) than other extracts of the A. aspera (fig.8; Table 8). Highest TEAC
is related to the highest antioxidant potential. Thus petroleum ether extract
has highest antioxidant potential than the other extracts.
Phytochemical screening performed on various extracts of A. aspera
shows that petroleum ether extracts contain steroids and alkaloids (Table 9).
Thus, we can conclude that the nonpolar constituents
in the plants like steroids may be responsible for the antioxidant activity of
the plant.
Polar phytoconstituents like flavonoids, tannins and phenolic
compounds which are generally showing antioxidant property were found to be
absent in the plant, and thus we can conclude that, polar extracts like ethanol
and aqueous extracts are not showing the activity.
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Received on
08.10.2009
Accepted on
19.11.2009
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Journal of Pharmacognosy and
Phytochemistry. 1(3): Nov. – Dec. 2009, 220-223