Indian Journal of Natural Products and Resources

Vol. 3(2), June 2012, pp. 228-231

FRAP (Ferric reducing ability of plasma) assay and effect of Diplazium esculentum
(Retz) Sw. (a green vegetable of North India) on central nervous system
Atul Kaushik*2, Chanderesh Jijta1, Jeevan J Kaushik3, Robel Zeray2, Anghesom Ambesajir2 and Lwam Beyene2
1

Department of Pharmaceutical Chemistry, GRD (PG) IMT, 214-Rajpur, Dehra Dun, Uttarakhand, India
2
School of Pharmacy, Asmara College of Health Sciences, Asmara, Eritrea, Africa.
3
Department of Basic and Behavioral Sciences, Asmara College of Health Sciences, Asmara, Eritrea, Africa.
Received 28 July 2011; Accepted 23 April 2012
The FRAP assay (Ferric Reducing Ability of Plasma), a simple test to determine the total antioxidant power; has
been chosen to assess the free radical scavenging effects of Diplazium esculentum (Retz) Sw. FRAP assay
depends upon the ferric tripyridyltriazine [ Fe (III)-TPTZ] complex to the ferrous tripyridyltriazine [Fe (II)-TPTZ] by
a reductant at low pH. Fe (II)-TPTZ has an intensive blue color and can be monitored at 593 nm. Antioxidant activity of
petroleum ether, chloroform, acetone, methanol and aqueous extracts was evaluated. D. esculentum is a well known green
leafy vegetable of North Indian food, therefore, stimulant effect of its aqueous extract on central nervous system (CNS) was
also determined on mice using the actophotometer. Results suggested that water extract (7.6 mM/dry wt) possesses
highest antioxidant activity. CNS stimulant effect of the extract at all doses level is much higher when compared to the
control and standard caffeine (a known stimulant). Stimulant effect was significant (P<0.0001 at all doses tested) and this
was also found to be dose dependent. Thus it can be concluded that D. esculentum is very rich to scavenge the free
radicals and stimulate the CNS system as compared to the standard.
Keywords: Antioxidants, Caffeine, TPTZ, Aqueous extract, Fern, Diplazium esculentum, FRAP.
IPC code; Int. cl. (2011.01)A61K 36/00, A61P 25/00

Introduction
Free radicals released during oxidative stress pose
the major endogenous damage in the biological
system. This type of damage is often associated
with various degenerative diseases and disorders
such
as
cancer,
cardiovascular
disease,
immunofunction decline and aging. Free radicals
are highly reactive molecules having unpaired
electrons and produced by radiation or as
by-products of the metabolic processes1,2 . To gain
stability, free radicals capture the electrons quickly
from other compounds and the attacked compound
becomes a free radical itself, which continues to
attack other compounds and leads to a chain
reaction. These results in the disintegration of cell
membranes and cell compounds, including lipid,
protein and nucleic acids. Besides damage to living
cells, free radicals are the major cause of food
deterioration through lipid oxidation, which
_________
*Correspondent author:
E- mail: atul_kaushik29@rediffmail.com

ultimately affects the organoleptic properties and

edibility of foods2 . Recent research suggested that
synthetic antioxidants could promote tumor
formation as well as anticarcinogenic properties.
Due to these contradictory properties, the
application and exploration of natural antioxidants
has received more attention3 . Keeping these points in
mind an attempt has made to explore the leaves of
Diplazium esculentum (Retz) Sw. for free radical
scavenging and central nervous system effect in order
to establish its folklore use in Uttarakhand, India.
D. esculentum (Retz) Sw. (Dryopteridaceae),
commonly called Latawar is considered high
economic value fern amongst the natives of Tehri
hills, Uttarakhand, India. This plant can attain an
average height of 0.5 to 2.5 m and is eaten as ulam
or green edible leaves; usually consumed with hot
sauce4 . The plant contains steroids, triterpenoids,
phenols, flavones and flavonoids. The dried rhizomes
are used as insecticides and decoction is used to cure
haemoptysis and cough5 .

KAUSHIK et al: ANTIOXIDANT ACTIVITY AND EFFECT OF DIPLAZIUM ESCULENTUM ON CNS

Materials and Methods

Collection of the plant Material

The plant material of the species D. esculentum

(Retz) Sw. was collected from Mothrowala, Dehra
Dun, Uttarakhand, India and identified by
Dr. Prashant Chaddha, Scientist, Botanical Survey of
India (BSI), Dehra Dun, Uttarakhand, India (Ref.
No.BSI/NC-9/2007-08/Tech/1011)
(Plate
1).
Authentic sample was deposited in the department of
pharmacognosy of the institute.
Processing of sample

The fresh leaves of the plant were shade dried at

room temperature (25-35C) for 20-25 days. The
dried leaf were powdered in a grinder and weighed
before extraction for calculating the yield.
Preparation of plant extracts

Dried powder 200 g was taken and subjected to

successive extraction with petroleum ether (40-60C),
chloroform, acetone, methanol and water in a Soxhlet
apparatus using hot continuous percolation method.
The extracts were concentrated under reduced
pressure in rotary evaporator (Buchi, R 200
Switzerland). The yields of dry extracts were
(4.0, 3.5, 2.5, 4.0 and 5.0% w/w, respectively)
recorded and stored in a clean glass bottles for further
phytochemical analysis were carried out using well
established protocols and methods.
Evaluation of free radical scavenging power

The ferric complexes reducing ability of the

extracts by FRAP assay were measured at low pH.
Chemicals and reagents

All chemicals and reagents were of analytical

grade quality purchased from Sigma, Merck,

229

Germany and Hi-Media, Bombay, India. Acetate

buffer (300 mmol/L, pH 3.6), 10 mmol/L. 2,4,
6-tripyridyl-s-triazine (TPTZ) in 40 mmol/L HCl,
20 mmol/L FeCl3 6 H2O in distilled water, 25 mL
acetate buffer, 2.5 mL TPTZ solution and 2.5 mL
FeCl3 6H2O solutions (working solution), aqueous
solution of known Fe (II) concentration was used for
calibration (in a range of 100-1000 mol/L).
FRAP assay

FRAP (900 l) reagent was mixed with 90 l

of distilled water and 30 l of test
sample/methanol/distilled water/standard solutions.
The reaction mixture was then incubated at 37C for
10 min and absorbance was recorded at 595 nm, using
a spectrophotometer (UV-VIS 1700 Shimadzu,
Japan). The concentration of FeSO4 was in turn
plotted against concentrations of the standard
antioxidants (Trolox, Acros Organics)6 .
CNS stimulant activity

Effect of aqueous extract of D. esculentum (Retz)

Sw. was studied in mice using the digital
actophotometer (Space-001 Lab, Maharashtra, India).
The continuous beam of light falls on photoelectric
cells. When the reading is considered zero, any cut of
in the continuity of light by the animal, is recorded on
a digital counter in the forms of counts. Depending on
the CNS stimulant action of the drug, the animals
show increased locomotors activity7 .
Ethical approval

The animal experiment was carried out as per the

Committee for the Purpose of Control and
Supervision on Experiments on Animals (CPCSEA)
norms, mentioned by the NIH guidelines wide our
college Reg. No: 1145/a/07/CPCSEA, Dated; 02 Jan
2008 and Project resolution No.7.
Animals Used

Plate 1Diplazium esculentum

20 Albino mice (25-30 g) of either sex were

procured from the Animal House (Reg. No.:
1145/a/07/CPCSEA) of the institute were procured
from animal house, GRD (PG) IMT, Dehra Dun
(UK). The animals were housed in cages under
standard laboratory conditions (12:12 hour light/dark
cycle at 252C). They had free access to standard
commercial diet and water. The animals were divided
into four groups of five each. The 1st group served as
the control group, the 2nd and 3rd groups were used as
test groups and the 4th group was the standard group.

INDIAN J NAT PROD RESOUR, JUNE 2012

230
Preparation of doses

The control group 1 received the vehicle; groups 2

and 3 were treated with water extract at different dose
level (50 and 100 mg/kg, respectively) and group 4
with caffeine (standard CNS stimulant agent) as
10 mg/kg dissolved in normal saline (0.9% NaCl). All
the groups were given 0.2 ml of the respective drug
by the I.P. route.
Method
Group 1 received vehicle and served as control,
group 2 and 3 received 50 mg/kg and 100 mg/kg
(0.2 ml I.P.) of aqueous extract, respectively and used
as test, group 4 received (0.2 ml I.P.) of 10 mg/kg
caffeine. The basal activity scores of all the animals
were recorded 2 days before the start of study using a
photoactometer. On the day of the study all rats were
given their assigned treatments, 30 minutes after the
treatment each rat was retested for activity scores for
10 min and the difference in the activity scores were
compared with the control scores7 . The mean score
for each group as determined was compared with each
other. The test groups were compared with the
standard and the control group.
Statistical evaluation
Differences between means were assessed by one
way analysis of variance (ANOVA), followed by
Dunnetts test using Sigma stat software. P< 0.05 was
considered significant.
Results and Discussion
In the present study, free radical scavenging activity
was determined by the FRAP (ferric reducing ability of
Table 1Antioxidant power of different extracts of Diplazium
esculentum (Retz) Sw.
S. No.

Sample

Absorbance
( - Max)
S.E.M

FRAP
mM/dry
extract

1
2
3
4
5

Pet. Ether
Chloroform
Acetone
Methanol
Aqueous

0.805 0.008
0.793 0.0285
0.3342 0.0057
0.351 0.0014
0.666 0.0018

0.47
0.229
3.8
4.16
7.6

Power
wt of

plasma) method, which depends upon the reduction of

ferric tripyridyltriazine [Fe (III)-TPTZ) complex to the
ferrous tripyridyltriazine. (Fe (II) - TPTZ) by a
reductant at low pH. Fe (II)-TPTZ has an intensive
blue color and can be monitored at 593 nm. FRAP
values are obtained by comparing the absorbance
change at 593 nm in test reaction mixtures with those
containing range with antioxidant mixtures.
The free radical scavenging power of different
extracts of D. esculentum leaves increased with an
increasing amount of extract. As can be seen in Table 1,
there is a clear difference between the control and the
sample containing the extract. Among all the extracts
aqueous extract was showing maximum antioxidant
power (7.6 mM/dry wt. of extract) but methanol and
acetone extracts were having lesser extent of free
radical scavenging power (4.16, 3.8 mM/dry wt. of
extract, respectively). Petroleum ether and chloroform
extracts were containing FRAP power in very less
amount (0.47, 0.229 mM/dry wt. of extract,
respectively). The range of antioxidant power lies
between 0.229 to 7.6 mM/dry wt. of extract (Table 1).
Polyphenols are the major plant compounds with
antioxidant activity. This activity is believed to be
mainly due to their redox properties8 which play an
important role in adsorbing and neutralizing free
radicals, quenching singlet and triplet oxygen or
decomposing peroxides. The results strongly suggest
that phenolics are important components of the plant
and some of its pharmacological effects could be
attributed to the presence of its valuable constituents.
The results from this study showed that the
constituents of this plant are having antioxidant and
pharmacological effects. In vivo methods using intact
animals are considered to be the best method for
investigating the action of drugs on the CNS.
Determination of D. esculentum effect on the CNS in
mice model at different dose level with the help of
actophotometer7 . The CNS activity was determined
separately for each group (Table 2).
After observing the CNS effect on different groups,
it is clearly indicating that aqueous leaf extract of
D. esculentum significantly increased the loco-motor

Table 2CNS stimulant effect of aqueous extract of Diplazium esculentum (Retz) Sw.
S. No.

Groups

1
2
3
4

Control
Standard (Caffeine)
Test-I Aqueous extract
Test-II Aqueous extract

No. of Animals

Dose (mg/kg)

Mean SEM

Stimulant

5
5
5
5

1% Tween 20
10
100
50

103 4.405
137.6 5.767
142.6 5.680
102.6 3.286

NS
+++
+++
NS

+++: Strong stimulant, NS: Not showing, Result shows a statistically significant difference (P = <0.001)

KAUSHIK et al: ANTIOXIDANT ACTIVITY AND EFFECT OF DIPLAZIUM ESCULENTUM ON CNS

activity. Aqueous extract at the dose level of

100 mg/kg was showing an excellent CNS stimulant
effect (142.6 5.680) with respect to the standard
group (137.6 5.767), but the 50mg/kg dose level
(102.6 3.286) was fewer stimulants as compared to
standard caffeine 10 mg/kg (Table 2). The results were
dose dependent and statistically significant. As the
score scale was not isomorphic to the arithmetic scale
the mean value and standard error was used to analyze
difference between groups of mice. The differences in
the mean values among the treatment groups are
greater than would be expected by chance, there is a
statistically significant difference (P<0.001). To isolate
the group or groups that differs from the others use a
multiple comparison procedure. All pair wise multiple
comparison procedures (Dunnetts Method) was also
performed for authentication of the results.
Conclusion
The findings of this study highlighted several novel
and important aspects of D. esculentum derived
extracts with regard to their antioxidant and central
nervous system effects. More studies are required to
achieve the proper role of its extracts to find out more
specific biochemical, pharmacological and molecular
aspects of the targeted molecules within that may
have the broadest implication to society.

231

Acknowledgement
Authors are highly thankful to S. Raja Singh,
Chairman, GRD (PG) Institute of Management and
Technology for providing requisite facilities for the
research work.
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