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Journal of Pharmacognosy and Phytochemistry 2018; 7(6): 1146-1151
E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2018; 7(6): 1146-1151
Received: 01-09-2018
Accepted: 05-10-2018
R Arockia Badhsheeba
Research Scholar PG and
Research Department of Botany
VO Chidambaram College,
Tuticorin, Tamil Nadu, India
V Vadivel
PG and Research Department of
Botany VO Chidambaram
College, Tuticorin, Tamil Nadu,
India
Correspondence
V Vadivel
PG and Research Department of
Botany VO Chidambaram
College, Tuticorin, Tamil Nadu,
India
Evaluation of in vitro antioxidant activity of
Acrostichum aureum Linn. Rachis
R Arockia Badhsheeba and V Vadivel
Abstract
Acrostichum aureum Linn is a medicinal fern collected from Puthalam, Kanyakumari District, Tamil
Nadu, India. This plant is used by the local people for curing pharyngitis, chest pain and diabetics.
Hence, in the present study, petroleum ether, benzene, ethyl acetate, methanol and ethanol extracts of the
rachis of A. aureum have been tested for antioxidant activity by using 1, 1-diphenyl-2-picryl-hydrazyl
(DPPH) radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging
activity, antioxidant activity by radial cation (ABTS•+) and reducing power assays. The antioxidant
potential is dose dependent in all the assays carried out. It is concluded that the A. aureum can be used as
a medicine against free radical associated oxidative damage.
Keywords: medicinal plant, Acrostichum aureum, antioxidant, free radicals, DPPH, ABTS
Introduction
Antioxidants may be defined as compounds that inhibit or delay the oxidation of other
molecules by inhibiting the initiation or propagation of oxidizing chain reactions [1].
Antioxidants can also protect the human body from free radicals and reactive oxygen species
(ROS) effects. Antioxidants retard the progress of many chronic diseases as well as lipid
peroxidation [2]. Also, antioxidants have been widely used as food additives to provide
protection against oxidative degradation of foods [3].
At the present time, the most commonly used antioxidants are butylatedhydroxyanisole
(BHA), butylatedhydroxytoluene (BHT), propyl gallate (PG) and tert butyl hydroquinone
(TBHQ). The safety of these antioxidants has recently been questioned due to toxicity [4].
Therefore, there is a growing interest on natural and safer antioxidants [5]. Development of
safer natural antioxidants from extracts of oilseeds, spices and other plant materials that can
replace synthetic antioxidants has been of interest [6].
Natural antioxidants are known to exhibit a wide range of biological effects including
antibacterial, antiviral, antiinflammatory, ant allergic, antithrombotic and vasodilatory
activities. Thus, there is a need of antioxidants of natural origin because they can protect the
human body from the diseases caused by free radicals [7].
Acrostichum aureum Linn (Family- Pteridaceae), common name: Swamp Fern, Mangrove
Fern, occurs Worldwide in mangrove swamps, salt marshes, canal margins, and low
hammocks. It is widely distributed throughout South Florida [8], Brazil, South and West
Mexico, Guyanas, Central America, Colombia, Venezuela, Ecuador, Paraguay, Barbados,
Trinidad, South China, Taiwan, Japan, North Australia, India, Sri Lanka and Bangladesh [9]. It
is an evergreen shrub, can be grown as annual which is locally used as choice of medicinal
plant in the treatment of major and minor complaints. Several studies have reported the
traditional use of A. aureum’s rhizome for curing wounds, non-healing ulcers, boils, syphilitic
ulcers, sore throat, chest pains, elephantiasis, purgative, febrifuge, cloudy urine in women, and
rheumatism in Malaysia [10], Bangladesh [11], India [12], and Yap islands and Micronesia [13].
In the present study, the mode of antioxidant action in petroleum ether, benzene, ethyl acetate,
methanol and ethanol extracts of A. aureum was investigated. In vitro methods of assessment
were used to determine the scavenging activity of the extract on 1, 1-diphenyl-2-picryl
hydrazyl (DPPH), hydroxyl radical, superoxide scavenging, 2, 2-azinobis 3-
ethylbenzothiazoline 6-sulfonate (ABTS) and reducing power.
Materials and Methods
Plant material
Rachis of Acrostichum aureum Linn were collected from Puthalam, Kanyakumari District,
Tamil Nadu and was authenticated at Botanical Survey of India, Southern circle, Coimbatore,
Tamil Nadu. The gathered rachis were cut into small pieces and shade dried at room
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Journal of Pharmacognosy and Phytochemistry
temperature. The shade dried rachis were ground into coarse
powder using mechanic grinder and stored in air tight
container for further use.
Extract preparation
The coarsely powdered rachis (20g) was extracted separately
with petroleum ether, benzene, ethyl acetate, methanol and
ethanol in a Soxhlet apparatus for 24h. The extracts obtained
were filtered through Whatman No. 41 filter paper. Then the
filtrates were concentrated under vacuum using rotary
evaporator (Heidolph, Schwabach, Germany). The
concentrated extracts were stored at 4ºC for further
investigation of potential in vitro free radical scavenging
activity.
Determination of DPPH radical scavenging activity
DPPH free radical scavenging activity of the extracts were
measured in vitro using 1, 1-diphenyl-2-picryl-hydrazyl
(DPPH) assay described by Blois [14]. 50mg of the extract and
ascorbic acid (standard) were taken and dissolve in methanol
and final volume make up to 50ml which was used as a stock
solution with the concentration 1000µg/ml. Then different
concentrations like 50, 100, 200, 400 and 800μg/ml were
prepared by diluting with the methanol from stock solution.
3ml of different concentration (50, 100, 200, 400 and
800μg/ml) of test solution and standard was taken in different
test tubes. To this add 1ml DPPH working solution (0.1mM
DPPH in methanol) and the mixtures were shaken vigorously
and allowed to stand at room temperature for 30min. The
absorbance was measured against methanol as blank at 517nm
using a UV-VIS spectrophotometer (Genesys 10S UV:
Thermo electron corporation). The percentage inhibition was
calculated by comparing the absorbance values of the test
sample with those of the controls (not treated with extract).
The capability of scavenging the DPPH radical was calculated
by using the following formula.
DPPH scavenging effect (% inhibition) =
Where, A0 refers to the absorbance of the DPPH control, and
A1 refers to the absorbance of DPPH in the presence of
extract/ascorbic acid. The inhibitory concentration (IC50)
value was calculated. IC50 values denotes the concentration
of extract/standard required to scavenge 50% of free radicals
Determination of hydroxyl radical scavenging activity
The effect of extracts on hydroxyl radical scavenging activity
was assayed by using the deoxyribose method [15] with some
modification. Stock solutions of EDTA (1mM), FeCl3
(10mM), ascorbic acid (1mM), H2O2 (10mM) and
deoxyribose (10mM), were prepared in deionized distilled
water. 1ml of different concentration (50, 100, 200, 400 and
800μg/ml prepared with deionized distilled water) of test
solution and control (ascorbic acid) was taken in different test
tubes. To this 0.1ml EDTA, 0.01ml of FeCl3, 0.1ml H2O2,
0.36ml of deoxyribose and 0.33ml of phosphate buffer
(50mM, pH 7.4) were added and the mixture was then
incubated at 37ºC for 1h. 1ml of the incubated mixture was
taken out and was mixed with 1ml of (10%) trichloroacetic
acid and 1ml of (0.5%) thiobarbituric acid (TBA) [in 0.025M
NaOH containing 0.025% butylated hydroxyl anisole (BHA)]
to develop the pink chromogen. The absorbance of the test
solution and standard were measured at 532nm. Deionized
distilled water was used as blank. The percentage inhibition
was calculated by comparing the absorbance values of the test
sample with those of the controls (not treated with extract).
The hydroxyl radical scavenging activity of the extract was
reported as percentage inhibition of deoxyribose. The
degradation is calculated by using the following equation.
Hydroxyl radical scavenging activity =
Where, A0 is the absorbance of the control, and A1 is the
absorbance test samples and reference.
Determination of superoxide radical scavenging activity
The superoxide anion scavenging activity was measured as
described by Srinivasan et al [16]. 1ml of different
concentration (50, 100, 200, 400 and 800μg/ml prepared with
methanol) of test solution and control (ascorbic acid) was
taken in different test tubes. To this 0.5ml of 16mM Tris-HCl
buffer pH 8, 0.5ml of 0.3mM nitroblue tetrazolium (NBT),
0.5ml of 0.936mM of Nicotinamide Adinie Dinucleotide
Reduced (NADH) and 0.5ml of 0.12mM of phenazine
methosulphate (PMS) were added and incubated at dark for 5
min. The absorbance was measured at 560nm. Distilled water
used as blank. The percentage inhibition was calculated by
comparing the absorbance values of the test sample with those
of the controls (not treated with extract). The inhibition
percentage was calculated as superoxide radical scavenging
activity as follows.
Superoxide radical scavenging activity =
Where, A0 is the absorbance of the control, and A1 is the
absorbance test samples and reference.
Determination of ABTS scavenging activity
2, 2-Azinobis 3-Ethylbenzothiazoline 6-Sulfonate (ABTS)
radical scavenging activity of A. aureum extracts was
measured by Huang et al [17] method with some modifications.
Unlike DPPH assay, the assay that involves scavenging of
ABTS radicals required generation of the radicals. The ABTS
radical cation (ABTS•+) was generated by mixing ABTS stock
solution (7mM) with potassium persulfate (2.45mM). The
reaction mixture left in the dark for 12h at room temperature
and the resulting dark coloured solution was diluted using
ethanol to an absorbance of 0.70 ± 0.02 at 734nm. 0.lml of
different concentrations (50, 100, 200, 400 and 800µg/ml
prepared with methanol) of extracts and trolox (reference
standard) was mixed with 3.9ml of radical solution in clean
and labeled test tubes. The tubes were incubated in dark for
6min at room temperature followed by measuring the
absorbance of the reaction mixture in spectrophotometer at
734nm. Methanol replacing the extract / trolox served as
control (i.e., 0.1ml methanol + 3.9ml ABTS radical solution).
The ABTS radical scavenging activity of the extracts was
calculated using the following formula and the results were
expressed as trolox equivalent antioxidant capacity (TEAC)
values.
ABTS radical scavenging activity =
Where, A0 is the absorbance of the ABTS solution without
extract/trolox and A1 is the absorbance the ABTS solution in
the presence of extract/trolox. The IC50 value was calculated.
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Journal of Pharmacognosy and Phytochemistry
IC50 denotes the concentration of extract required to scavenge
50% of the radicals.
Determination of reducing power
The reducing power of the extract was determined by the
method of Kumar and Hemalatha [18]. 1ml of each extract of
different concentration (50, 100, 200, 400 and 800µg/ml
prepared with methanol) and standard (ascorbic acid) were
mixed with 5ml of sodium phosphate buffer (0.2M, pH 6.6)
and 5ml of 1% potassium ferricyanide and the mixture was
incubated at 50ºC for 20min. After incubation, 5ml of 10%
trichloroacetic acid (TCA) was added to the mixture and
centrifuged at 980g for 10min in a refrigerator centrifuge.
About 5ml of supernatant of solution was taken and diluted
with 5ml of distilled water and shaken with 1ml of freshly
prepared 0.1% ferric chloride and the absorbance was
measured at 700nm in UV-VIS spectrophotometer. A blank
was prepared without adding extract. This result indicates that
increase in absorbance of reaction mixture indicates increase
in reducing power.
Statistical analysis
Grap Pad PRISM software (version 4.03) was used for
calculating IC50 values for DPPH radical scavenging activity,
hydroxyl radical scavenging activity, superoxide radical
scavenging activity and antioxidant activity by radical cation
(ABTS•+). The results were expressed as mean and all the
experiments were done in triplicate.
Result and Discussion
DPPH radical scavenging activity
Free radicals are molecules, usually of oxygen, which have
lost an electron and are continuously generated during human
body metabolism. DPPH is a stable free radical and accepts
an electron or hydrogen radical to become a stable
diamagnetic molecule which is widely used to investigate to
investigate radical scavenging activity. In DPPH radical
scavenging assay, antioxidants react with DPPH (deep violet
colour) and convert it to yellow coloured α, α-diphenyl-β-
picryl hydrazine. The degree of discoloration indicate the
radical-scavenging potential of the antioxidant [14].
The effect of different solvent extracts of rachis of A. aureum
and standard ascorbic acid on DPPH radical scavenging
activity were shown in figure 1.
Fig 1: DPPH radical scavenging activity of different extracts of Acrostic hum aureum rachis
The DPPH radical scavenging activity of all the samples was
highly depend on concentration, namely, antioxidant activity
increased with increase in extract concentration. Among the
tested solvents, benzene extract of rachis of A. aureum
exhibited high DPPH radical scavenging activity. Results
shows that 800μg/ml concentration of benzene extract from
the rachis of A. aureum exhibited the highest DPPH
(118.56%) scavenging activity compared to other extracts.
The IC50 value of ascorbic acid (Table 1) was 32.84µg/ml;
whereas, methanol extract was found to contain 36.54µg/ml
IC50 value.
Table 1: IC50 values of different solvent extracts of rachis of A. aureum
Solvent
DPPH
Hydroxyl radicals
ABTS
Superoxide
Petroleum ether
31.56
25.16
26.12
26.18
Benzene
34.13
30.18
22.46
24.16
Ethyl acetate
30.36
31.48
27.16
28.16
Methanol
36.54
32.16
30.11
30.96
Ethanol
32.16
30.84
28.36
34.84
Ascorbic acid
32.84
29.93
33.06
30.15
Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity was measured by
studying the competition between deoxyribose and the
extracts for hydroxyl radicals generated from
Fe2+/EDTA/H2O2 system (Fenton reaction). The hydroxyl
radicals attack deoxyribose, which eventually results in the
formation of thiobarbituric acid reacting substance (TBARS)
[19].
Among the reactive oxygen species, the hydroxyl radicals are
the most reactive and predominant radicals generated
endogenous during aerobic metabolism. A single hydroxyl
radical results in the formation of many molecules of lipid
hydroperoxides in the cell membrane which may severely,
disrupts its function and leads to cell death [20].
The petroleum ether, benzene, ethyl acetate, methanol and
ethanol extracts of rachis of A. aureum were found to possess
concentration dependent scavenging activity on hydroxyl
radicals and the results were given in figure 2.
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Journal of Pharmacognosy and Phytochemistry
Fig 2: Hydroxyl radical scavenging activity of different extracts of Acrostichum aureum rachis
Among all the tested extracts, ethyl acetate and methanol
extracts (800µg/ml) showed high levels of hydroxyl radical
scavenging activity (126.92% and 126.32% respectively).The
IC50 value of ascorbic acid was 31.48%; whereas, IC50 value
of methanol extract was found to be 32.16%.
ABTS radical cation scavenging activity
The effect of A. aureum rachis extracts and standard ascorbic
acid on ABTS radical cation were compared and shown in
figure 3. At 800μg/ml concentration of methanol, ethanol,
ethyl acetate extracts of A. aureum rachis possessed 119.22%,
108.16%, 106.32% scavenging activity on ABTS. All the
concentration of A. aureum rachis extract showed lower
activity than the standard ascorbic acid 121.36%. The IC50
value of ascorbic acid was 33.06% whereas methanol extract
was found to be 30.11%. This scavenging activity of ABTS
radical by the plant extracts were found to be appreciable; this
implies that the plant extract useful for treating radical related
pathological damage especially at higher concentration [21].
Fig 3: ABTS radical cation scavenging activity of different extracts of Acrostichum aureum rachis
Superoxide radical scavenging activity
Superoxide anion plays an important role in the formation of
more reactive species such as hydrogen peroxide, hydroxyl
radical and singlet oxygen, which induce oxidative damage in
lipids, proteins and DNA [22]. Therefore, estimating the
scavenging activity of plant extracts on superoxide radical is
one of the most important ways of clarifying the mechanism
of antioxidant activity.
In the present investigation, different solvent extracts of
rachis of A. aureum were found to possess concentration
dependent scavenging activity on superoxide and the results
were given in figure 4. Among all the solvent extracts, ethanol
extracts (800µg/ml) of the plant material exhibited the highest
superoxide radical scavenging activity; whereas, the standard
ascorbic acid had 109.54% of superoxide radical scavenging
activity. The mean IC50 value of ascorbic acid was 30.15%;
whereas, ethanol extract was found to be 34.84%.
Fig 4: Superoxide radical scavenging activity of different extracts of Acrostichum aureum rachis.
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Journal of Pharmacognosy and Phytochemistry
Reducing power
Reducing power reflects the electron donating capacity of its
bioactive compounds, which serves as a significant indicator
of its antioxidant activity. Reduced Fe3+ / ferricyanide
complex to the ferrous form, which indicated existence of
reductants in the sample solution. The reductants have been
exhibiting antioxidative potential by breaking the free radical
chain and donating a hydrogen atom [23]. The reducing power
of the extracts of A. aureum rachis was shown in figure 5. The
reducing ability of the extract increased with increasing
concentration, our results were in accordance to studies done
by Vijaya et al [24]. In the present study, 800μg/ml
concentration of methanol extract showed the highest
reducing power than the ascorbic acid.
Fig 5: Reducing power ability of different extracts of Acrostichum aureum rachis
Conclusion
In the present study, it can be concluded that different solvent
extracts of rachis of A. aureum have wide range of antioxidant
and/or free radicals scavenging activity. The antioxidant
activity of plant may vary depending upon the geographical
area, seasonal variation and also the method and solvent used
for extraction. Literature survey reveals that flavonoids [25]
and phenolic compounds [26] are responsible for antioxidant
activity. However, we do not know what components in the
plant extracts show these activity. More detailed studies on
chemical composition of the plant extracts, as well as in vivo
assays are essential to characterize them as biological
antioxidant. It should be kept in mind that antioxidant activity
measured by in vitro methods may not reflect in vivo effects
of antioxidant [27]. Many other factors such as
absorption/metabolism are also important.
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