World Journal of Pharmaceutical research

Oluwafemi Ojo et al.

World Journal of Pharmaceutical Research
Volume 2, Issue 6, 1899-1912.
Research Article
ISSN 2277 7105

IN-VITRO ANTIOXIDANT AND FREE RADICAL SCAVENGING

ACTIVITIES OF OCIMUM GRATISSIMUM
*Oluwafemi Ojo1, Omotade Oloyede2, Olaide Olarewaju2, Adebola Ojo2,
Basiru Ajiboye1, Sunday Onikanni1
1

Department of Chemical Sciences, Biochemistry Unit, Afe Babalola University

2

Article Received on
26 August 2013,
Revised on 29 Sept. 2013,
Accepted on 24 October 2013

Department of Biochemistry, Ekiti State University, Nigeria.

ABSTRACT
The present study was carried out to evaluate the antioxidant and free
radical scavenging activity of aqueous, ethyl acetate and ethanolic
(1:1:1) extract of Ocimum gratissimum (AEEOG) in various systems.
DPPH radical, nitric oxide radical, iron chelating ability, ferric

*Correspondence for
Author:

reducing antioxidant ability and hydroxyl radical scavenging assays

Oluwafemi Ojo

were carried out to evaluate the antioxidant potential of the extract.

Department of Chemical

The antioxidant activity of aqueous, ethyl acetate and ethanolic extract

Sciences, Biochemistry Unit,

increased in a dose dependent manner. The effect of AEEOG on

Afe Babalola University

reducing power increases in a dose dependent manner, indicating some

Nigeria.
oluwafemiadeleke08@gma

compounds in Ocimum gratissimum is both electron donors and could

react with free radicals to convert them into more stable products and

il.com

to terminate radical chain reactions. The ethyl acetate extract had

significantly (P 0.05) higher chelating effect than ethanolic and aqueous extracts. IC50
values for chelating effect of ethanolic, ethyl acetate and aqueous extracts of O. gratissimum
were 6.81, 5.51 and 6.34 mg/ml, respectively. In DPPH radical scavenging assay the IC50
value for DPPH scavenging by the ethanolic, ethyl acetate and aqueous extracts of O.
gratissimum were 2.47, 1.58 and 5.29 mg/ml, respectively. AEEOG was found to inhibit the
nitric oxide radicals generated from sodium nitroprusside, the IC50 values for nitric oxide
scavenging ability of ethanolic, ethyl acetate and aqueous extracts of O. gratissimum were
7.51, 13.17 and 21.73 mg/ml, respectively. AEEOG was also found to inhibit the hydroxyl
radical generated by the deoxyribose method, the IC50 values for hydroxyl radical effect of
ethanolic, ethyl acetate and aqueous extracts of O. gratissimum were 3.98, 6.48 and 8.83
mg/ml, respectively. The total phenolic contents of Ocimum gratissimum were also
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determined in this study and is found to be 4.000.00 mg GAE/g, 10.340.47 mg GAE/g and
1.560.06 mg GAE/g for aqueous, ethyl acetate and ethanolic respectively. The results
obtained in this study indicate that the AEEOG can be a potential source of natural
antioxidant.
Key words: Ocimum gratissimum, antioxidants, DPPH radical, nitric oxide radical, iron
chelating ability, hydroxyl radical, reducing ability.
INTRODUCTION
In living cells, reactive oxygen species (ROS) are continuously produced in numerous
processes such as mitochondrial respiration, metabolism of Xenobiotics by cytochromes
P450, inflammation, and phagocytosis. Exposure to UV and gamma radiation also generates
ROS. ROS damage cellular macromolecules (lipids, proteins, nucleic acids) leading to
oxidative stress. It has been demonstrated that oxidative stress is involved in many diseases
such as cardiovascular diseases, rheumatoid arthritis, neurodegenerative diseases, alcoholic
and non-alcoholic steatohepatitis, diabetes mellitus, and cancer. As the incidence of these
diseases is constantly increasing the research in the field of natural and synthetic antioxidants
is still of high interest [1, 2]. Numerous in vitro and in vivo studies have reported that
polyphenolic compounds protect against oxidative stress [2, 3]. Some of these medicinal
plants used in ethno medicine for the treatment and management of many of these diseases
have been investigated for their antioxidative properties [4, 5]. Many of the metabolites from
these medicinal plants especially flavonoids exhibited potent antioxidant activity in vitro and
in vivo [6, 7, 8]. Most of the free radical scavenging potential in herbs and spices is due to
the redox properties of phenolic compounds which allow them to act as reducing agents,
hydrogen donators and singlet oxygen quenchers [9, 10]. The importance of free radicals and
reactive oxygen species (ROS) has attracted increasing attention. ROS and free radical
mediated reactions are involved in degenerative or pathological processes such as aging,
cancer, rheumatoid arthritis, coronary heart disease and Alzheimers disease. Many
antioxidants compounds, naturally occurring in plant sources have been identified on free
radical or active oxygen scavenger. Many synthetic antioxidant components have shown
toxic or mutagenic effects, which have shifted the attention onto the naturally occurring
antioxidants [11]. Free radicals include super oxide radical (SOR), hydroxyl radical (OH),
hydroperoxyl radical (HPR), alkoxy radical (AR), peroxyl radical (PR) and nitric oxide
radical (NOR). Other molecules that act like free radicals are singlet oxygen, hydrogen
peroxide (H2O2), and hypochlorous acid (HOCl). Collectively free radicals and non-free

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World Journal of Pharmaceutical Research

radicals are called oxidants or reactive oxygen species [12]. Ocimum gratissimum Linn
(Labiatae) is grown for the essential oils in its leaves and stems. Eugenol, thymol, citral,
geraniol and linalool have been extracted from the oil [13]. Essential oils from the plant have
been reported to possess an interesting spectrum of antifungal properties [14]. The
antinociceptive property of the essential oil of the plant has been reported [15]. The whole
plant and the essential oil are used in traditional medicine especially in Africa and India. The
essential oil is also an important insect repellant. The present work has been designed to
evaluate the antioxidant potential of extracts from the leaves of O. gratissimum and to
explore the basis for its traditional use.
MATERIAL AND METHODS
Collection and preparation of extract
Fresh leaves of O. gratissimum were bought in the market at Ado, Nigeria. The plant was
identified and authenticated by a plant scientist in the Department of Plant Science, Ekiti
State University, Ado-Ekiti, Nigeria and a voucher specimen was deposited accordingly at
the herbarium of the Department of Plant Science, Ekiti State University, Ado-Ekiti, Nigeria.
The fresh leaves of the plant were air-dried, pulverized and extracted exhaustively in distilled
water. The filtrate was concentrated and evaporated to dryness at 60C, using rotary
evaporator. The yield was calculated and the dry extract was stored in a refrigerator at -4C
until use for the experiments.
Chemicals and reagents
Chemicals and reagents used such as 1,10-phenanthroline, gallic acid, FolinCiocalteaus
reagent were procured from SigmaAldrich, Inc., (St. Louis, MO), trichloroacetic acid
(TCA) was

sourced from SigmaAldrich, Chemie GmbH

(Steinheim, Germany),

dinitrophenyl hydrazine (DNPH) from ACROS Organics (New Jersey, USA), hydrogen
peroxide, methanol, acetic acid and Fecl3were sourced from BDH Chemicals Ltd., (Poole,
England), CuSO45H2O, H2SO4, sodium carbonate, AlCl34, potassium acetate, TrisHCl
buffer, sodium dodecyl sulphate, FeSO4, potassium ferricyanide and ferric chloride were of
analytical grade while the water was glass distilled.
Phytochemical screening
Phytochemical analysis of the major phytoconstituents of the plant extracts were undertaken
using standard qualitative methods (color tests and/or TLC) [16].

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Antioxidant studies
Reductive ability
The reducing property of the ginger extracts were determined by assessing the ability of the
extract to reduce a FeCl3 solution as described by [17]. A 2.5 mL aliquot were mixed with 2.5
mL, 200 mM sodium phosphate buffer (pH 6.6) and 2.5 mL, 1% potassium ferricyanide. The
mixtures were incubated at 50 C for 20 min, and then 2.5 mL, 10% TCA were added. These
were then centrifuged at 650 g for 10 min. A 5 mL of the supernatant were mixed with an
equal volume of water and 1 mL, 0.1% ferric chloride. The same treatments were performed
to a standard ascorbic acid solution and the absorbance taken at 700 nm. The reducing
powere were then calculated and expressed as ascorbic acid equivalent.
DPPH radical scavenging activity
The free radical scavenging activity of the hydroalcoholic extract of Ocimum gratissimum
was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH) using the method of Blois; a method
based on the reduction of a methanolic solution of the coloured DPPH radical. [18-19] Used
as a reagent, DPPH evidently offers a convenient and accurate method for titrating 0.1 mM
solution of DPPH in methanol was prepared and 1 mL of this solution was added to 3 mL of
the extract suspension in water at different concentrations (1, 2, 3, 4 and 5 mg). After 30
minutes of incubation, absorbance was measured at 517 nm. Ascorbic acid was used as
reference material. Lower absorbance of the reaction mixture indicated higher free radical
scavenging activity. All the tests were performed in triplicate and the results averaged. The
percentage reduction in absorbance was calculated from the initial and final absorbance of
each solution. [20-21] The percentage inhibition was calculated by comparing the absorbance
values of control and samples. Percentage scavenging of DPPH radical was calculated using
the formula,
[Absorbance of Control- Absorbance of Test] 100
% Scavenging of DPPH =
[Absorbance of Control]
Iron Chelation Ability
The Fe2+ chelating ability of both extracts were determined using a modified method of [22]
with a slight modification by [23]. Freshly prepared 500 mol L-1 FeSO4 (150 L) were
added to a reaction mixture containing 168 L of 0.1 mol L-1 TrisHCl (pH 7.4), 218 L
saline and the extracts (025 L). The reaction mixtures were incubated for 5 min, before the
addition of 13 L of 0.25% 1, 10-phenanthroline (w/v). The absorbance were subsequently

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measured at 510 nm in a spectrophotometer. The Fe2+ chelating ability were calculated with
respect to the control.
Percentage Fe2+ chelating ability (%) = Abscontrol Abstest sample

x 100

Abscontrol
where Abscontrol = absorbance of the control (reacting mixture without the test sample) and,
Abstest sample = absorbance of reacting mixture with the test sample.
Hydroxyl radical scavenging effect
Hydroxyl radical scavenging activity was measured by studying the competition between
deoxyribose and test compounds for hydroxyl radical generated by Fe3+-Ascorbate-EDTA
H2O2 system (Fenton reaction) according to the method of Kunchandy and Rao. [24] The
hydroxyl radicals attack deoxyribose that eventually results in TBARS formation. The
reaction mixture contained in a final volume of 1.0 mL, 100 l of 2-deoxy-2-ribose (28 mM
in KH2PO4-K2HPO4 buffer, pH 7.4), 500l solutions of various concentrations of
hydroalcoholic extract (1, 2, 3, 4 and 5 mg) in KH2PO4 -KOH buffer (20 mM, pH 7.4), 200 l
of 1.04 mM EDTA and 200 M FeCl3 (1:1 v/v), 100 l of 1.0 mM H2O2 and 100 l of 1.0
mM ascorbic acid was incubated at 37 oC for 1 h. The free radical damage imposed on the
substrate, deoxyribose was measured as TBARS by the method of Ohkawa et al. [25] 1.0 mL
of thiobarbituric acid (1%) and 1.0 mL of trichloroacetic acid (2.8%) were added to the test
tubes and the incubation was continued at 100 oC for further 20 min. After cooling,
absorbance was measured at 532 nm against control containing deoxyribose and buffer.
Nitric oxide radical scavenging effect
Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH
interacts with oxygen to produce nitrite ions which were measured by the Griess reaction.
[26-27] Scavengers of nitric oxide compete with oxygen leading to reduced production of
nitric oxide [28]. The reaction mixture (3 ml) containing sodium nitroprusside (10 mM) in
phosphate buffered saline (PBS) and the extract in different concentrations (1, 2, 3, 4 and 5
mg) were incubated at 25 oC for 150 min. At every 30 min interval, 0.5 mL of the incubated
sample was removed and 0.5 ml of Griess reagent (1% sulphanilamide, 0.1%
naphthylethylene diamine dihydrochloride in 2% H3PO4) was added. The absorbance of the
chromophore formed was measured at 546 nm. All the analyses were performed in triplicate
and the results were averaged. The percentage inhibition of nitric oxide generated was
measured by comparing the absorbance values of control and test.

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Total phenolic content

The extractable phenol content were determined on the extracts using the method reported by
[29]. Appropriate dilutions of the extracts were mixed with 2.5 mL of 10% Folin
Ciocalteaus reagent (v/v) and neutralized by 2.0 mL of 7.5% sodium carbonate. The reaction
mixtures were incubated for 40 min at 45 C and the absorbance were measured at 765nm in
the spectrophotometer. The total phenol content were subsequently calculated using gallic
acid as standard. The absorbance of the blue colour that developed were read at 765 nm. The
concentrations of total phenols were expressed as mg/gm of dry extract.
The concentrations of total phenolic compounds in the extract were determined by using the
formula:
T=CV/M
Where, T= Total phenolic content mg/gm of plant extract in GAE,
C= Concentration of Gallic acid from the calibration curve,
V= volume of the extract in ml,
M= wt of the pure plant extract.
Statistical analysis
Statistical analysis of difference between groups was evaluated by one-way ANOVA
followed by student t test. The values P < 0.05 were regarded as significant.
RESULTS AND DISCUSSION
Phytochemical investigation
It was found that the extracts contained proteins and amino acids, saponins, phenolic
compounds and tannins.
Reductive ability
The antioxidant activity has been reported to be concomitant with the development of
reducing power [30]. The reducing power of extract might be due to its hydrogen donating
ability, as described by Shimada et al. [31]. The ethyl acetate extracts of all the seeds show
significantly (P < 0.05) higher reducing power than ethanol and water extracts of Ocimum
gratissimum (Fig 5). IC50 value for reducing power by the ethanolic, ethyl acetate and
aqueous extracts of O. gratissimum were 0.94, 1.55 and 0.49 mg/ml, respectively. This result
shows that the aqueous extract of O. gratissimum has a higher reducing power than ethanolic
and ethyl acetate extract.

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Ferric Reducing Antioxidant

properties (%)

Oluwafemi Ojo et al.

World Journal of Pharmaceutical Research

80
Ethanolic extract

70

Ethyl acetate

Aqueous extract

60
50
40
30
20
10
0
0

concentration of extracts (mg/ml)

Fig I: Ferric reducing antioxidant properties of ethanolic, ethyl acetate and aqueous
extracts of Ocimum gratissimum.
DPPH radical scavenging effect
DPPH scavenging activity has been used by various researchers as a quick and reliable
parameter to assess the in vitro antioxidant activity of crude plant extracts [32-33]. In DPPH
test the ability of a compound to act as donor for hydrogen atoms or electrons was measured
spectrophotometrically. The scavenging activities of DPPH exerted by each extract as well as
ascorbic acid were summarized in figure II. The scavenging effect of extracts in the range 1
5 mg/ml on the DPPH radical increased with an increasing concentration of OG extracts (Fig
II). IC50 value for DPPH scavenging by the ethanolic, ethyl acetate and aqueous extracts of O.
gratissimum were 2.47, 1.58 and 5.29 mg/ml, respectively. In the present investigation
Ocimum gratissimum at different doses demonstrated significant DPPH scavenging activity
indicating their abilities to act as radical scavengers.

DPPH free radical scavenging

ability (%)

80

Ethanolic extract

Ethyl acetate

Aqueous extract

70
60
50
40
30
20
10
0
-10 0

concentration of extracts (mg/ml)

Fig II: DPPH free radical scavenging ability of ethanolic, ethyl acetate and aqueous
extracts of Ocimum gratissimum.

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Iron Chelating Ability

The metal chelating effect of ethanol, ethyl acetate, water extracts of Ocimum gratissimum
decreased in the order of ethyl acetate > ethanol > aqueous extract (P < 0.05) and were
increased with the increased concentration of Ocimum gratissimum extracts (Fig III).
Ocimum gratissimum ethyl acetate extract had significantly (P < 0.05) higher chelating effect
than ethanolic and aqueous extracts. IC50 values for chelating effect of ethanolic, ethyl acetate
and aqueous extracts of O. gratissimum were 6.81, 5.51 and 6.34 mg/ml, respectively. This
shows that ethyl acetate extract of O. gratissimum has a higher chelating ability than aqueous
and ethanolic form.

Fe2+ chelating ability (%)

60

Ethanolic extract
Aqueous extract

50

Ethyl acetate

40
30
20
10
0
0

concentration of extract (mg/ml)

Fig III: Iron chelating ability of ethanolic, ethyl acetate and aqueous extract of Ocimum
gratissimum.
Hydroxyl radical scavenging effect
Hydroxyl radicals are the major active species causing lipid oxidation and enormous
biological damage [34-35]. The deoxyribose method is a simple assay to determine the rate
constants for reactions of hydroxyl radicals [36]. Ferric-EDTA was incubated with H2O2 and
ascorbic acid at pH 7.4. Hydroxyl radicals were formed in free solution and were detected by
their ability to degrade 2-deoxy-2-ribose in to fragments that on heating with TBA at low PH
form a pink chromogen [37-38]. Any hydroxyl radical scavenger added to the reaction would
compete with deoxyribose for the availability of hydroxyl radicals, thus reducing the amount
of MDA formation. IC50 values for hydroxyl radical effect of ethanolic, ethyl acetate and
aqueous extracts of O. gratissimum were 3.98, 6.48 and 8.83 mg/ml, respectively.

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OH free radical scavenging ability

(%)

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World Journal of Pharmaceutical Research

Ethanolic extract
Aqueous extract

60

Ethyl acetate

50
40
30
20
10
0
0

concentration of extract (mg/ml)

Fig IV: Hydroxyl free radical scavenging ability of ethanolic, ethyl acetate and aqueous
extracts of Ocimum gratissimum.
Nitric oxide radical scavenging effect
Nitric oxide (NO) is an important chemical mediator generated by endothelial cells,
macrophages, neurons, etc. and is involved in the regulation of various physiological
processes [39]. Excess concentration of NO is associated with several diseases [40-41].
Oxygen reacts with the excess nitric oxide to generate nitrite and peroxynitrite anions, which
act as free radicals [42-43]. In the present study, the extract competes with oxygen to react
with nitric oxide and thus inhibits generation of the anions. Figure V illustrates the
percentage inhibition of nitric oxide generation by the different form of extract of Ocimum

NO free radical scavenging ability

(%)

gratissimum.
120

Ethanolic extract
Aqueous extract

100

Ethyl acetate

80
60
40
20
0
0

concentration of extracts (mg/ml)

Fig V: Nitric oxide free radical scavenging ability of ethanolic, ethyl acetate and
aqueous extracts of Ocimum gratissimum.
The extract of Ocimum gratissimum showed significant free radical scavenging activity on
nitric oxide (NO)-induced release of free radicals. Different concentrations (1mg/ml, 2mg/ml,

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3mg/ml, 4mg/ml and 5mg/ml) exhibited different percentage of inhibition. From Fig V, the
concentration of the extract increased, as the absorbance decreased. IC50 values for nitric
oxide scavenging ability of ethanolic, ethyl acetate and aqueous extracts of O. gratissimum
were 7.51, 13.17 and 21.73 mg/ml, respectively.
Total Phenolic content
Phenols are very important plant constituents because of their scavenging ability due to their
hydroxyl groups [44]. The total phenolic content of aqueous, ethyl acetate and ethanolic
extract of Ocimum gratissimum is 4.000.00 mg GAE/g, 10.340.47 mg GAE/g and
1.560.06 mg GAE/g respectively. The phenolic compounds may contribute directly to
antioxidative action [45].
CONCLUSION
Medicinal herbs are known to contain a variety of antioxidants. Each herb generally
contained different phenolic compounds, and each of these compounds possesses differing
amounts of antioxidant activity. It is rather difficult to characterize every compound and
assess or compare their antioxidant activities. The most detailed investigations so far were
concerned with reactions involving phenolic compounds ranging from polymer chemistry to
biochemistry and food chemistry [46]. It has been revealed that various phenolic antioxidants
such as flavonoids, tannins, coumarins, xanthones and more recently procyanidins scavenge
radicals dose dependently, thus they are viewed as promising therapeutic drugs for free
radical pathologies [47]. The results of the present study, which demonstrate the radical
scavenging of different extracts, indicate that the use of Ocimum gratissimum for the
treatment of various neurogenerative and inflammatory diseases, and cancer seems quite
useful and reasonable. The antioxidant and free radical scavenging activities of Ocimum
gratissimum might be due to the presence phenolic compounds in extracts.
ACKNOWLEDGEMENT
The Authors wish to acknowledge Mr. Omotayo of the Department of Plant Science, Ekiti
State University, Ado-Ekiti, Nigeria in helping to identify the plant and providing the
voucher number as well as the Biochemistry Laboratory at Afe Babalola University for
providing the necessary facilities to carry out this study.

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