Biosciences, Biotechnology Research Asia Vol.

5(2), 837-840 (2008)

Total antioxidant power and free radical

scavenging capacity of some medicinal plants

P. KOTESWARA RAO¹, VARAPRASAD BOBBARALA², D. BHASKAR RAO¹,

CH. RAVI KIRAN¹, K.V. RAGHAVA RAO¹ and T. RAGHAVA RAO¹

¹Department of Biochemistry, College of Science & Technology,

Andhra University Visakhapatnam - 03 (India).
²For U Biosciences, A/4A, Park lane Residency, East point colony,
Visakhapatnam, A. P - 530 017 (India).

(Received: October 28, 2008; Accepted: December 05, 2008)

ABSTRACT

Current research is now directed towards finding naturally occurring antioxidants of plant
origin. The present study was aimed to assess the In vitro antioxidant activity of methanolic leaf
extracts of some medicinal plants by carrying the determination of Total Antioxidant power (FRAP
Assay) and Free radical scavenging capacity (DPPH Assay) and results were compared with well
established antioxidants like Ascorbic Acid and Butylated Hydroxy Toluene(BHT). Our results clearly
showed that all the plants of present study showed potent Total Antioxidant power and Free radical
scavenging capacity. Of all the plants tested, the highest total antioxidant power and free radical
scavenging capacity was shown by X. mekongensis, B. cylindrica and E. agallocha with nearly
equal to antioxidant capacity to stable antioxidants like BHT and Ascorbic acid. This data will be
useful in estimating the total pharmacological and phytochemical properties of these plants.

Key words: Reactive oxygen species, DPPH Radical,

Ascorbic acid Equivalents, Free radical scavengers

INTRODUCTION preferred because they are safe and environmental

Reactive oxygen in particular, free radicals
are considered to induce oxidative damage in
biomolecules and to play an important role in aging,
cardiovascular diseases, cancer, and inflammatory
diseases1-3. In addition, they are also well known to
be major causers of material degradation and food
deterioration4. Consequently, antioxidants are now
known to be prospective protective or therapeutic
agents. In the past few years, addition of synthetic
antioxidants has begun to be restricted because of
their health risks and toxicity5. The importance of
exploiting natural antioxidants from various sources
and replacing synthetic antioxidants with natural
ingredients has attracted increasing attention.
Several natural compounds from plants exhibit
antioxidant / radical scavenger properties are
friendly. It has become clear that the direct free
radical scavenging effect and membrane protection
play an important role in the action mechanism of
several old established drugs. Several herbs and
spices have been reported to exhibit antioxidant
activity, including rosemary, sage, thyme, nutmeg,
turmeric, white pepper, chili pepper, ginger, and
several Chinese medicinal plants extracts6-9. The aim
of the present work was to prove the
phytotherapeutical significance of some popular
medicinal plants on the basis of their antioxidant
activity due to their influence on pathological free
radical reactions. The medicinal plants of present
study includes Borreria hispida (BH) belongs
Rubiaceae, Bruguiera cylindrica (BC) and Ceriops
decandra (CD) belongs to Rhizophoraceae, Eugenia
bracteata (EB) belongs to Myrtaceae, Excoecaria
838 Rao et al., Biosci., Biotech. Res. Asia, Vol. 5(2), 837-840 (2008)
agallocha (EA) belongs to Euphorbiaceae,
Glycyrrhiza glabra (GG) belongs to Fabaceae,
Picrorhiza kurrow (PK) belongs to Scrophulariaceae,
Trianthima decandra (TD) belongs to Aizoaceae,
Xylocarpus mekongensis (XM) belongs to
Meliaceae. The reason for selection is that, these
plants have a wide range of applications in traditional
medicines and are involved in the treatment of
various skin diseases, liver disorders, cancer, HIV,
cough, asthma, diarrohea, dysentery, ulcers and
controlling of blood pressure etc,. In addition, some
of these plants were also reported to be the potent
antioxidants. Hence, in the present study, we made
an attempt to study the total antioxidant power and
radical scavenging capacity of methanolic extracts
of these plants. This data will be used to estimate
the phytochemical and antioxidant intake of the local
population and to understand the therapeutic uses
of these plants.
MATERIAL AND METHODS
Diphenyl picryl hydrazyl (DPPH) was
obtained from Himedia laboratories Pvt. Ltd,
Mumbai, India. Ferric Chloride, 2,4, 6-tri pyridyl-s-
triazine, Butylated hydroxy toluene (BHT) were
obtained from Sisco research laboratories Pvt. Ltd.,
Mumbai, India. All other chemicals used were of
analytical grade obtained from commercial sources.
Plant collection and preparation of the extraction
The plants are collected from the near by
hilly region, Paderu and coastal region of
Visakhapatnam, Andhra Pradesh, India and
authenticated by Dr. M. Venkayya, Associate
professor, Dept. of Botany, Andhra University,
Visakhapatnam, Andhrapradesh. Plant leaves were
cleaned with deionized water and dried at 50ºC for
24 hours. The dried plants were ground and then
sieved 80mesh. The dried leaves were ground to a
powder using a milling machine. The dried powder
was then weighed and extracted with methanol, with
reflux on a water bath at 40°C for three consecutive
days. The extracts were filtered and evaporated
under vacuum to dryness with a rotary evaporator
and then placed in an oven at 60°C until constant
weight was obtained and the solutions were
prepared with a concentration 1mg/ml using
methanol.
Total Antioxidant power
Ferric reducing ability of Plasma (FRAP) Assay
The total antioxidant potential of a sample
was determined using the ferric reducing ability of
plasma i.e., FRAP assay as a measure of antioxidant
power by Benzie and Strain10. The assay was based
on the reducing power of a compound (antioxidant).
A potential antioxidant will reduce the ferric ion
(Fe3+) to the ferrous ion (Fe2+); the latter forms a
blue complex (Fe2+/TPTZ), which increases the
absorption at 593 nm. Briefly, the FRAP reagent was
prepared by mixing acetate buffer (300µM, pH 3.6),
a solution of 10 µM TPTZ in 40 µM HCl, and 20µM
FeCl3 at 10:1:1 (v/v/v). The reagent (300 µM) and
sample solutions (10 µL) were added to each well
and mixed thoroughly. The absorbance was taken
at 593 nm after 10 min. Standard curve was prepared
using different concentrations of FeCl3. All solutions
were used on the day of preparation. The results
were corrected for dilution (e.g.1000ml) and
expressed as ascorbic acid equivalents (µmoles/ml)
or FRAP units. All determinations were performed
in triplicates.
Free radical scavenging capacity
Di Phenyl Picryl Hydrazyl radical scavenging
assay (DPPH Assay)
The DPPH assay was carried out as
described by Cuendet et al., (11). 5.0 ml of DPPH
solution (0.004%) in methanol was added to 50 µl
of plant extract. After 30 min of incubation at 370C,
the absorbance was read against control at 517 nm.
Ascorbic acid and BHT were used as positive
controls. Percentage of inhibition = (absorbance of
control –absorbance of test / absorbance of control)
x100. All determinations were performed in triplicate.
RESULTS AND DISCUSSION
Recently much attention has been focused
on reactive oxygen species and free radicals, which
play an important role in the genesis of various
diseases such as inflammation, cataract, liver
cirrhosis and ischemia/reperfusion injury (12).
Herbal drugs containing radical scavengers are
gaining importance in the prevention and treatment
of such diseases¹³. Hence the present study is
focused on the determination of Total antioxidant
power by FRAP Assay and DPPH Assay.
 Rao et al., Biosci., Biotech. Res. Asia, Vol. 5(2), 837-840 (2008) 839

Total Antioxidant power
Ferric reducing ability of Plasma (FRAP) Assay
The FRAP assay measures the reduction
of Fe3+ (ferric iron) to Fe2+ (ferrous iron) in the
presence of antioxidants. Because the ferric-to-
ferrous ion reduction occurs rapidly with all
reductants with half reaction reduction potentials
above that of Fe3+/Fe2+, the values in the FRAP
assay will express the corresponding concentration
of electron-donating antioxidants (10). We elected
to use the FRAP analysis for several reasons. The
FRAP assay is the only assay that directly measures
antioxidants or reductants in a sample. The other
assays are indirect because they measure the
inhibition of reactive species (free radicals)
generated in the reaction mixture, and these results
also depend strongly on the type of reactive species
used. The FRAP assay, in contrast, uses antioxidants
as reductants in a redox-linked colorimetric reaction.
Furthermore, the other assays, but not the FRAP
assay, use a lag phase type of measurement. This
has been difficult to standardize in previous

Fig.1: Showing the Total Antioxidant power (FRAP Assay)

Fig. 2: Showing the Free radical scavenging capacity (DPPH Assay)

840 Rao et al., Biosci., Biotech. Res. Asia, Vol. 5(2), 837-840 (2008)
experiments and has generated varying results
among different laboratories. In the FRAP assay,
pretreatment is not required, stoichiometric factors
are constant and linearity is maintained over a wide
range. The results also indicated that the total
antioxidant power of the plant extracts was
determined by the FRAP method was observed to
be highest in X. mexoeugensis with 1601 FRAP
units followed by B. cylindrica(1471 FRAP Units),
E. agallocha (1237 FRAP Units), E. bracteata (1140
FRAP Units), G. glabra (862 FRAP Units),
T.decandra (720 FRAP Units), P. kurrow (645 FRAP
Units), B. hispida (600 FRAP Units) and C. decandra
(360 FRAP Units) These results were presented
graphically in Fig. 1.
Free radical scavenging capacity
Di Phenyl Picryl Hydrazyl radical scavenging
assay (DPPH Assay)
The free radical scavenging capacity of
methanolic leaf extracts of the plants of present study
were tested by its ability to bleach the stable DPPH
radical. Antioxidants react with DPPH, which is a
stable free radical, and convert it to 1,1-diphenyl-2-
(2,4,6- trinitrophenyl) hydrazine14. The degree of
decolourization indicates the scavenging potentials
of the antioxidant compounds. This assay provided
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