[AJPSci.

Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 1, Pg 15-18

ISSN- 22315640 (Print)

ISSN- 22315659 (Online)

www.asianpharmaonline.org

RESEARCH ARTICLE

In vitro Antioxidant Activity of Premna serratifolia Linn

P. Muthukumaran*, S. Salomi and R. Umamaheshwari
P.G .Department of Biochemistry, Meenakshi Chandrasekaran College of Arts And Science, Pattukkottai-614
626, Thanjavur, Tamil Nadu
*Corresponding Author E-mail: muthubabi_p@yahoo.co.in; kumaran.bio82@yahoo.com

ABSTRACT
The aim of this study was to investigate the antioxidant effect of Premna serratifolia Linn . The antioxidant activity
was evaluated by various antioxidant assays, including 1, 1-diphenyl-2- picrylhydrazyl (DPPH), 2, 2-azino-bis (3ethylbenzthiazoline-6-sulfonic acid) (ABTS), and hydrogen peroxide scavenging method. The antioxidant activities
were compared to standard antioxidant ascorbic acid. Premna serratifolia Linn wood extract showed a significant
antioxidant activity in DPPH, ABTS and H2O2 scavenging methods. The findings of the present study suggest that
Premna serratifolia Linn could be a potential source of natural antioxidant that could have greater importance as
therapeutic agent in preventing or slowing oxidative stress related degenerative diseases.

KEYWORDS: Premna serratifolia Linn, antioxidant activity.

INTRODUCTION:
Oxygen derived free radicals, such as the superoxide anion
and hydroxyl radicals are cytotoxic and promote tissue
injury. Antioxidants act as a major defence against radical
mediated toxicity by protecting the damages caused by free
radicals. Furthermore although medicinal plants are used as
antioxidants in traditional medicine, their claimed
therapeutic properties could be due, in part, to their capacity
for scavenging oxygen free radicals. Reactive oxygen
species (ROS) including free radicals such as superoxide
anion radicals, hydroxyl radicals, singlet oxygen and nonfree-radical species such as hydrogen peroxide are various
forms of activated oxygen and often generated by oxidation
product of biological reactions or exogenous factors.
ROS have aroused significant interest among scientists in
the past decade. Their broad range of effects in biological
and medicinal systems has drawn on the attention of many
experimental works. In living organism, various ROS can
form by different ways.
Normal aerobic respiration stimulates polymorphonuclear
leukocytes and macrophages, and Peroxisomes appear to be
the main endogenous sources of most of the oxidants
produced by cells. Exogenous sources of ROS include
tobacco smoke, certain pollutants, organic solvents, and
pesticides (1, 2, 3). ROS can cause lipid peroxidation in
foods, which leads to the deterioration of the food (4, 5).
Received on 18.09.2012
Accepted on 26.12.2012
Asian Pharma Press All Right Reserved
Asian J. Res. Pharm. Sci. 3(1): Jan.-Mar. 2013; Page 15-18

In addition, it is well known that ROS induce some

oxidative damage to biomolecules like lipids, nucleic acids,
proteins, amines, deoxyribonucleic acid and Carbohydrates.
Its damage causes ageing, cancer, and other many diseases
(6, 7). As a result of this, ROS have been implicated in
more than hundred diseases, including malaria, acquired
immunodeficiency syndrome, heart disease, stroke,
arteriosclerosis, diabetes, and cancer. ROS are continuously
produced during normal physiologic events, and removed
by antioxidant defence mechanisms (8). There is a balance
between generation of ROS and antioxidant system in
organisms. In pathological condition, ROS are
overproduced and result in lipid peroxidation and oxidative
stress. The imbalance between ROS and antioxidant
defence mechanisms leads to oxidative modification in
cellular membrane or intracellular molecules (9). Various
endogenous antioxidant defence mechanisms play an
important role in the elimination of ROS and lipid
peroxides to protect the cells against toxic effects of ROS
and lipid peroxides (10).
Many antioxidant compounds, naturally occurring from
plant sources, have been identified as free radical or active
oxygen scavengers (11, 12). Recently, interest has increased
considerably in finding naturally occurring antioxidant for
use in foods or medicinal materials to replace synthetic
antioxidants, which are being restricted due to their side
effects such as carcinogenicity. Natural antioxidants can
protect the human body from free radicals and retard the
progress of many chronic diseases as well as retard lipid
oxidative rancidity in foods (13).

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Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 1, Pg 15-18

Premna serratifolia Linn., (Verbenaceae) is an important

plant belonging to the family Verbenaceae, and is one of the
most widespread large shrubs in the forests of India, usually
occurring in deciduous forests. The whole plant possesses
medicinal properties, useful in the treatment of
cardiovascular diseases, skin diseases, inflammatory
diseases, arthritis, gonorrhea, rheumatism, anorexia and
jaundice. It is an important Ayurvedic medicinal herb and
its synonym is Premna integrifolia. It is popularly known
asMunney in Tamil, and Agnimantha in Ayurvedic
system of medicine. Root forms an ingredient in well
known Ayurvedic formulation Dasamula which is used
for variety of affections (14). It is widespread throughout
tropical Pacific and tropical Asia. It is common along the
Indian and Andaman coasts. Infusion of the leaves is
administered with pepper in cold and fever. Leaves are used
to cure "weakness of limbs" and the leaves and leaf sap
were used to alleviate headache (15).
Premna serratifolia Linn, has cardiotonic (16), anticoagulant (17), anti-inflammatory (18), anti hyperglycaemic
(19), anti-parasitic (20), antioxidant (21) and antimicrobial
(22) properties. Most of the plant parts of Premna
serratifolia Linn., have been used in the traditional system
of medicine in India to treat various infectious diseases.
Therefore we undertook the present investigation to
examine the antioxidant activity of ethanolic extract of
Premna serratifolia Linn., through various in vitro models.

detection of various constituents using conventional

protocol.
DPPH radical scavenging activity
Free radical scavenging activity of extracts of pericarp of
Premna serratifolia Linn were tested by its ability to bleach
the stable 1,1-diphenyl 2-picryl-hydrazyl (DPPH) radical. A
stock solution of DPPH 0.3mM in methanol) was prepared
such that 1ml of it in 3ml methanol gave an initial
absorbance of 0.9.Decrease in absorbance in the presence of
Ethanolic extract at different concentration(50-500 mg/ml)
were noted after 15 min. scavenging activity was expressed
as the %inhibition. (23,24),
Radical scavenging activity (%) =OD control - OD sample x 100
OD control

ABTS radical cation decolourisation assay

ABTS (54.8 mg) was dissolved in 50 ml of distilled water
to 2 mM concentration and potassium
persulphate (17 mM, 0.3 ml) was added. The reaction
mixture was left to stand at room temperature overnight in
dark before use. To 0.2 ml of various concentrations of the
extracts or standards, 1.0 ml of distilled DMSO and 0.16 ml
of ABTS solution was added to make a final volume of 1.36
ml. Absorbance was measured spectrophotometrically, after
20 min at 734 nm. The assay was performed in
triplicate(25, 26)

Scavenging of hydrogen peroxide

It can be generated through a dismutation reaction from
Plant material
superoxide anion by superoxide dismutase. It can generate
Fresh wood (without bark) of Premna serratifolia Linn., the hydroxyl radical in the presence of metal ions and
were collected in April, 2012, from the waste lands in the superoxide anion (27).
villages of Thanjore , Tamil Nadu, India

MATERIALS AND METHODS:

Preparation of the extract

The freshly collected wood was chopped, shade dried and
coarsely powdered (40 mesh size). The powder was
defatted with petroleum ether (60 - 80 C) and then
extracted with 90% ethanol in a Soxhlet extractor. The
extract was dried under reduced pressure using a rotary
vacuum evaporator and the percentage yield was 7.90%
w/w. The obtained ethanol extract was suspended in 5%
gum acacia for the pharmacological screening.
Chemicals
1, 1 - diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis
(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were
obtained from Sigma Aldrich Co, St Louis, USA. Ascorbic
acid and rutin were obtained from SD Fine Chemicals Ltd.,
Mumbai, India. Ethanol and dimethyl sulphoxide were
obtained from Ranbaxy Laboratories Ltd., Punjab, India.
Hydrogen peroxide (30%) was obtained from Qualigen Fine
Chemicals, Mumbai, India. All chemicals used were of
analytical grade.

O 2 + H 2O 2

OH- + OH+ + O2

A solution of hydrogen peroxide (20mM) was prepared in

phosphate buffered saline (PBS, pH 7.4).Various
concentrations of 1ml of the extracts or standards in
methanol were added to 2 ml of hydrogen peroxide
solutions in PBS. The absorbance was measured at 230 nm,
after 10 min against a blank solution that contained extracts
in PBS without hydrogen peroxide (28).

RESULTS:
The results of the preliminary phytochemical screening of
the ethanol extract of Premna serratifolia Linn., revealed
the presence of phytoconstituents such as alkaloids,
steroids, flavonoids, phenolic compounds, tannins and
glycosides specifically iridoid glycosides.

The antioxidant activity of Premna serratifolia Linn was

evaluated by DPPH, ABTS and Hydrogen peroxide radical
scavenging methods. Premna serratifolia Linn showed a
dose dependent scavenging activity and free radical
inhibition of DPPH, ABTS and H2O2 comparable to free
Phytochemical studies
radical
scavenging activity of ascorbic acid.(Table 1-3,Fig
Freshly prepared Premna serratifolia Linn wood extract
1).
was subjected to phytochemical screening tests for the

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DISCUSSION:
Reactive oxygen species are continuously formed in cells as
consequence of oxidative biochemical reactions and
external factors. However they become harmful when they
are produced in excess under certain abnormal conditions
such as inflammation, ischemia and in presence of iron
ions. Under these conditions the endogenous antioxidants
may be unable to counteract ROS formation. Reactive
oxygen species formed may cause cellular damage and this
damage may involve in etiology of diverse human diseases.
Exogenous antioxidant supplement is helpful to overcome
this severe problem of free radicals, which may scavenge
these free radicals.
T he free radical scavenging activity of natural compounds
can be evaluated through their ability to quench the
synthetic free radicals, in which the absorbance of the
reaction mixture is taken in visible range to know whether
the compound is having antioxidant activity.

cell membrane rapidly. Once inside the cell, H2O2 can

probably react with Fe2+and possibly Cu2+ to form hydroxyl
radical and this may be the origin of many of its toxic
effects. It is therefore biologically advantageous for cells to
control the amount of hydrogen peroxide that is allowed to
accumulate. The decomposition of H2O2 by root extract of
Premna serratifolia Linn results from its antioxidant and
free radical scavenging activity.

CONCLUSION:
The results obtained in the present study indicate that
Premna serratifolia Linn wood extract exhibits free radical
scavenging activity. The overall antioxidant activity of
Premna serratifolia Linn wood extract might be attributed
to its polyphenolic content and other phytochemical
constituents. The findings of the present study suggest that
Premna serratifolia Linn could be a potential source of
natural antioxidant that could have greater importance as
therapeutic agent in preventing or slowing oxidative stress
related degenerative diseases.

DPPH assay is based on the measurement of the scavenging

ability of antioxidant towards the stable DPPH radical. Table 1.DPPH free radical Scavenging
DPPH is relatively stable nitrogen centered free radical that extract Premna serratifolia Linn
can accept an electron or hydrogen radical to become a
Sample
Concentration
stable diamagnetic molecule. DPPH radicals react with
g /mL
suitable reducing agent as a result of which electron become
100
paired off forming the corresponding hydrazine. The
150
solution therefore looses color stoichiometrically depending
200
300
on the number of electrons consumed which is measured
500
Ethanolic Extract
spectrometric ally at 517 nm (29). From the results it may
Ascorbic acid
be postulated that the wood extract of Premna serratifolia
Linn have hydrogen donors, thus scavenge the free radical
DPPH.

activity of ethanolic wood

Percentage
Inhibition
43.25
50.84
57.80
72.89
81.64

IC50

155
g /ml

16g/ml

Table 2.ABTS Scavenging activity of Premna serratifolia Linn

ABTS assay is relatively recent one, which involves a more

Sample
Concentration
Percentage
IC50
drastic radical, chemically produced and, is often used for
g /mL
Inhibition
screening complex antioxidant mixture such as plant
100
20.30
150
43.34
211
extracts, beverages and biological fluids. The solubility in
200
52.72
g /ml
both the organic and aqueous media and the stability in a
300
54.47
wide pH range raised the interest in the use of ABTS radical
Ethanolic Extract
500
68.4
for the estimation of the antioxidant activity. The principle
Ascorbic acid
258g/ml
behind the technique involves the reaction between ABTS
and potassium persulphate to produce the ABTS radical
cation (ABTS+) a blue green chromogen. In the presence of Table 3.Hydrogen peroxide scavenging activity of Premna
antioxidant reductant, the colored radical is converted back serratifolia Linn
to colorless ABTS, the absorbance of which is measured at
Sample
Concentration
Percentage
IC50
734nm.
The extract of Premna serratifolia Linn possessed an
antioxidant activity with IC50 value being 211 g /ml;
suggest the free radical scavenging activity of Premna
serratifolia Linn wood extract.
Hydrogen peroxide is a weak oxidizing agent and can
inactivate a few enzymes directly, usually by oxidation of
essential thiol (-SH) groups. Hydrogen peroxide can cross

17

Ethanolic Extract
Ascorbic acid

g /mL
100
150
200
300
500

Inhibition
18.82
33.26
46.12
58.26
72.48

619
g /ml

405g/ml

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Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 1, Pg 15-18

Fig1: Free Radical scavenging activity of Premna serratifolia linn Extractat different
concentration by DPPH,ABTS and H2O2 Scavenging activity

n
io
it
ib
h
n
i
f
o
e
ga
t
n
e
rc
e
P

90
80
70
60
50
40
30
20
10
0

DPPH free radical Scavenging

activity of ethano lic extract
Premna serratifo lia Linn
ABTS Scavenging activity of
Premna serratifo lia Linn

100

150

200

300

500

Hydrogen peroxide
scavenging activi ty of Premna
serratifolia Linn

Concentration ( g/ml)

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