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Antioxidant activity, nitric oxide scavenging activity and phenolic content of

Ocimum gratissimum leaf extract

Article  in  Journal of Medicinal Plant Research · December 2010

DOI: 10.5897/JMPR10.262

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Journal of Medicinal Plants Research Vol. 4(24), pp. 2479-2487, 18 December, 2010
Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2010 Academic Journals

Full Length Research Paper

Antioxidant activity, nitric oxide scavenging activity

and phenolic contents of Ocimum gratissimum leaf
extract
Francis M. Awah1* and Andrew W. Verla2
1
Department of Biochemistry, Madonna University, Elele Campus, Rivers State, Nigeria.
2
Department of Chemistry, Madonna University, Elele Campus, Rivers State, Nigeria.
Accepted 9 July, 2010

Plant derived antioxidant could be useful as food additives to prevent food deterioration and also to
impart human health and prevent oxidative stress associated diseases. In this study, the free radical
scavenging potential of a methanol extract of the leaves of Ocimum gratissimum was assessed by
.
measuring its capability for scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH ) radical, superoxide anion
.– . .
radical (O2 ), hydroxyl radical ( OH), nitric oxide radicals (NO ), as well as its ability to inhibit lipid
peroxidation, using appropriate assay systems compared to natural and synthetic antioxidants. Total
phenolic, flavonoid and flavonol contents were determined by spectrophotometric methods. The extract
.–
significantly inhibited DPPH radical with an IC50 value of 12.3± 1.95 µg/ml, inhibited O2 (IC50 = 82.9 ±
.
5.12 µg/ml), OH radical (IC50 = 38.9 ± 2.8 µg/ml) and non-enzymatic lipid peroxidation (IC50 = 270.5 ± 8.2
µg/ml) and also inhibited the accumulation of nitrite in vitro. The plant extract yielded 0.839 ± 0.097 mg
gallic acid equivalents phenolic content and 39.12 ± 2.43 mg rutin equivalents flavonoid content. The
observed antioxidant potentials and phenolic content of the extract suggest that an ethanol extract of O.
gratissimum leaves is a potential source of natural antioxidants and could be useful in the food industry
to retard oxidative degradation of lipids and thereby improve food quality. However, isolating the active
principles and in vivo studies are warranted.
.
Key words: Ocimum gratissimum extract, antioxidant activity, NO scavenging activity, phenolic content.

INTRODUCTION

Although, reactive oxygen species (ROS) and reactive
nitrogen species (RNS) play an important roles in many
biological processes and are involved in host defense,
overproduction of these species such as hydroxyl radical
. .–
( OH),hydrogen peroxide (H2O2),superoxide anions (O2 ),
.
and nitric oxide (NO ), as well as peroxyl nitrite contri-
butes to the immunopathology of a vast variety of condi-
tions including inflammatory diseases, cancer, athero-
sclerosis, diabetes mellitus, hypertension, AIDS, and
aging (Darley-Usmar et al., 1995; Lee et al., 2000) and
also contribute to food deterioration. Antioxidants
*Corresponding author. E-mail: awambuh@yahoo.com. Tel:
(234)-805-743-1113.
however, can delay or inhibit the oxidation of lipids or
other molecules by inhibiting the initiation or propagation
of oxidative chain reactions (Velioglu et al., 1998; Gülçin
et al., 2010).
Oxidative degradation of lipids is a major factor limiting
the shelf life of foods. The free-radical reaction of lipid
peroxidation is generally responsible for the deterioration
of lipid-containing foods. Use of antioxidants during the
manufacturing process can minimize the extent of lipid
peroxidation (Aruoma, 1993; Shahidi and Wanasundara,
1992). Recently, there has been a great increase of
interest in natural antioxidant phytochemicals of plant
origin since they are viewed as promising therapeutic
drugs for free radical pathologies and also found to be
useful in the food industries as nutraceuticals due to their
good antioxidant potentials and impact on the status of
2480 J. Med. Plant. Res.
human health (Jayaprakasha and Jaganmohan, 2000;
Kitt et al., 2000; Nogochi and Nikki, 2000). Exposure to
oxygen and sunlight are the two main factors in the
oxidation of food. Oxidation of food is a destructive pro-
cess, causing loss of nutritional value and changes in
chemical composition. Oxidation of fats and oils leads to
rancidity and, in fruits such as apples; it can result in the
formation of compounds which discolour the fruit (Zheng
and Wang, 2001). Antioxidants are added to food to slow
the rate of oxidation and, if used properly, they can
extend the shelf life of the food in which they have been
used. The protection afforded by natural products has
been attributed to various phenolic antioxidants which are
increasingly becoming of interest in the food industry
because they retard oxidative degradation of lipids and
thereby improve food quality (Gülçin, 2010).
The perennial plant Ocimum gratissimum L
(Lamiaceae) (scent leaf) is widely distributed in the
tropics of Africa and warm temperature regions. Scent
leaf is a traditional vegetable condiment used in Nigeria
and elsewhere to enhance food flavour and is widely
used in food and oral care products. The volatile aromatic
oil from the leaves consists mainly of thymol and
eugenol; and also contains xanthones, terpenes and lac-
tones which, posses antiseptics, antibacterial and
antifungal activities (Dubey et al., 2000; Ezekwesili et al.,
2004). The antinociceptive property of the essential oil of
the plant has been reported (Rabelo et al., 2003). The
Ocimum oil is also active against several species of
bacteria and fungi (Akinyemi et al., 2004; Janine de
Aquino et al., 2005; Lopez et al., 2005). Nutritional impor-
tance of this plant centers on its usefulness as a sea-
soning because of its aromatic flavour (Ezekwesili et al.,
2004). Its antioxidant potential however, is yet to be
investigated. The present work has been designed to
evaluate the antioxidant potential and quantitative total
phenolic contents of a methanol extract of O. gratissimum
(MEOG) in different assay systems to explore its possible
use in the food industry as an antioxidant flavouring
agent. O. basicilum a member of the Lamiaceae family
has also been reported to possess substantial antioxidant
activity (Gülçin et al., 2007).
MATERIALS AND METHODS
Chemicals
L-ascorbic acid, and 2,2-diphenyl-1-picrylhydrazyl (DPPH.) radical
were purchased from Fluka Chemicals, 2-deoxy-D-ribose, sodium
nitroprusside (SNP), sodium nitrite, sulpanilamide, phosphate buffer
saline (PBS), phosphoric acid, potassium dihydrogen phosphate
(KH2PO4), potassium hydroxide (KOH), ferric chloride (FeCl3+),
naphthylethylenediaminedihydrochloride, ethylenediaminetetra-
acetic acid (EDTA), nitro blue tetrazolium (NBT), sodium carbonate
(Na2CO3), aluminium trichloride, perchloric acid (HClO4), butylated
hydroxyltoluene (BHT), polyvinylpolypyrrolidone, riboflavin, ferrous
sulphate, hydrogen peroxide (H2O2), thiobarbituric acid (TBA),
Folin-ciocalteu reagent (FCR) and trichloroacetic acid (TCA) were
all purchased from Sigma Chemical Co. (St. Louis, MO). All plastics
were from Falcon Labware and all other unstated chemicals and
reagents were of analytical grade.
Crude extract preparation
O. gratissimum leaves obtained from Owerri, Imo State, Nigeria
were air-dried at room temperature and reduced to fine powder by
milling. The resulting powder was subjected to extraction with 80%
methanol. The methanol extract was concentrated using a rotary
evaporator and stored at 4˚C until used (Gülçin, 2005; Elmastas et
al., 2006).
Antioxidant activity assays
Qualitative DPPH.radical-scavenging assay using thin-layer
chromatography
Qualitative screening for antioxidant activity was done using the
DPPH.radical assay according to the method of Takao et al., 1994).
Briefly, a thin-layer chromatogram of the extract on silica gel plates
(Merck) was developed using hexane–methanol–ethyl acetate
(2:10:2, v/v) as mobile phase. DPPH. radical test was performed
directly on thin-layer chromatography (TLC) plates by spraying with
DPPH. [0.2% (w/v)] in ethanol to reveal the antioxidant activity of the
extract (Cuendet et al., 1997).
Quantitative DPPH.radical-scavenging assay
Scavenging activity on DPPH. free radicals by the extract were
assessed according to the method reported by Gyamfi et al. (1999)
with slight modifications (Awah et al., 2010). Briefly, a 2.0 ml
solution of the extract at different concentrations diluted two-fold in
methanol was mixed with 1.0 ml of 0.3 mM DPPH. in methanol. The
mixture was shaken vigorously and allowed to stand at room
temperature in the dark for 25 min. Blank solutions were prepared
with each test sample solution (2.0 ml) and 1.0 ml of methanol while
the negative control was 1.0 ml of 0.3 mM DPPH solution plus 2.0
ml of methanol. L-ascorbic acid was used as the positive control.
Thereafter, the absorbance of the assay mixture was measured at
518 nm against each blank with a UV-visible spectrophotometer
(Talaz et al., 2009). DPPH. radical inhibition was calculated using
the equation:
A0 − As
% Inhibition = × 100%
A0
Where, A0 is the absorbance of the control, and As is the
absorbance of the tested sample. The IC50 represented the
concentration of the extract that inhibited 50% of radical.
Hydroxylradical (.OH)-scavenging assay
The 2-deoxyribose assay was used to determine the scavenging
effect of the extract on the .OH radical, as reported by Halliwell et
al. (1987) with minor modifications (Awah et al., 2010). Each
reaction mixture contained, the following final concentrations of
reagents in a final volume of 1.0 ml: 2-deoxyribose (2.5 µM),
potassium phosphate buffer (pH 7.4, 20 mM), FeCl3 (100 µM),
EDTA (104 µM), H2O2 (1 mM), and L-ascorbic acid (100 µM). The
mixtures were incubated for 1 h at 37˚C, followed by addition of 1.0
ml of 1% (w/v) TBA in 0.05 M NaOH and 1.0 ml of 2.8% (w/v) TCA.
The resulting mixture was heated for 15 min at 100˚C. After cooling

on ice, absorbance was measured at 532 nm. Inhibition of 2-
deoxyribose degradation expressed in percentage was calculated
as per the equation:
A0 − As
% Inhibition = ×100%
A0
Where, A0 is the absorbance of the control, and As is the
absorbance of the tested sample. The IC50 represented the
concentration of the extract that inhibited 50% of radical.
Superoxide radical (O2.-)-scavenging assay
This assay was based on the capacity of the extract to inhibit the
photochemical reduction of nitro blue tetrazolium (NBT) as
described by Martinez et al. (2001) with slight modifications (Awah
et al., 2010). Briefly, each 3.0 ml reaction mixture contained 0.05 M
PBS (pH 7.8), 13 mM methionine, 2 M riboflavin, 100 M EDTA,
NBT (75 M) and 1.0 ml of test sample solutions. The tubes were
kept in front of a fluorescent light (725 lumens, 34 W) and
absorbance was read at 560 nm after 20 min. The entire reaction
assembly was enclosed in a box lined with aluminium foil. The
inhibition of superoxide anion was estimated by the equation:
A0 − As
% Inhibition = ×100%
A0
Where, A0 is the absorbance of the control, and As is the absor-
bance of the tested sample. The IC50 represented the concentration
of the extract that inhibited 50% of radical.
In vitro nitric oxide radical (NO.) scavenging assay
NO. generated from sodium nitroprusside (SNP) was measured
according to the method of Marcocci et al. (1994). Briefly, the
reaction mixture (5.0 ml) containing SNP (5 mM) in phosphate-
buffered saline (pH 7.3), with or without the plant extract at different
concentrations, was incubated at 25˚C for 180 min in front of a
visible polychromatic light source (25W tungsten lamp). The NO.
radical thus generated interacted with oxygen to produce the nitrite
ion (NO. ) which was assayed at 30 min intervals by mixing 1.0 ml of
incubation mixture with an equal amount of Griess reagent (1%
sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylene-
diaminedihydrochloride). The absorbance of the chromophore
(purple azo dye) formed during the diazotisation of nitrite ions with
sulphanilamide and subsequent coupling with naphthylethylene-
diaminedihydrochloride was measured at 546 nm. The nitrite
generated in the presence or absence of the plant extract was
estimated using a standard curve based on sodium nitrite solutions
of known concentrations. Each experiment was carried out at least
three times and the data presented as an average of three
independent determinations.
Lipid peroxidation assay
A modified thiobarbituric acid-reactive species (TBARS) assay
(Awah et al., 2010) was used to measure the lipid peroxide formed,
using egg yolk homogenates as lipid-rich media (Ruberto et al.,
2000). Briefly, egg homogenate (500 l of 10%, v/v in PBS (pH 7.4)
and 100 l of samples were added to a test tube and made up to
1.0 ml with distilled water. Then, 50 l of FeSO4 (0.075 M) and 20 l
Awah and Verla 2481
of L-ascorbic acid (0.1 M) were added and all were mixed and
incubated for 1 h at 37˚C to induce lipid peroxidation. Thereafter,
0.2 ml of EDTA (0.1 M) and 1.5 ml of TBA reagent (3 g TBA, 120 g
TCA and 10.4 ml 70% HClO4 in 800 ml of distilled water) were
added in each sample and heated for 15 min at 100˚C. After
cooling, samples were centrifuged for 10 min at 3,000 rpm and
absorbance of supernatant was measured at 532 nm. Lipid
peroxidation inhibition was calculated as per the equation:
A0 − As
% Inhibition = ×100%
A0
Quantitative phytochemical analysis
Determination of total phenolic contents
Total phenolics were determined using Folin-ciocalteu reagent
(FCR) as described by Velioglu et al. (1998), with slight modi-
fications. Briefly, 100 l of the extract dissolved in methanol (1
mg/ml) was mixed with 750 l of FCR (diluted 10-fold) and allowed
to stand at 22˚C for 5 min; 750 l of Na2CO3 (60 g/l) solution was
then added to the mixture. After 90 min the absorbance was
measured at 725 nm. Results were expressed as gallic acid
equivalents.
Determination of tannin contents
Tannin content in each sample was determined using insoluble
polyvinyl-polypirrolidone (PVPP), which binds tannins as described
by Makkar et al. (1993). Briefly, 1 ml of extract dissolved in
methanol (1 mg/ml), in which the total phenolics were determined,
was mixed with 100 mg PVPP, vortexed, left for 15 min at 4˚C and
then centrifuged for 10 min at 3,000 rpm. In the clear supernatant
the non-tannin phenolics were determined the same way as the
total phenolics (Velioglu et al., 1998). Tannin content was calcu-
lated as a difference between total and non-tannin phenolic content.
Determination of flavonoids and flavonols
The flavonoids content was determined according to the method
described by Kumaran and Karunakaran (2006) with slight modi-
fications. Briefly, 100 l of plant extracts in methanol (10 mg/ml)
was mixed with 100 l of 20% aluminium trichloride in methanol and
a drop of acetic acid, and then diluted with methanol to 5 ml. The
absorbance at 415 nm was read after 40 min. Blank samples were
prepared from 100 l of plant extracts and a drop of acetic acid, and
then diluted to 5 ml with methanol. The absorption of standard rutin
solution (0.5 mg/ml) in methanol was measured under the same
conditions. The amount of flavonoids in the plant extract in rutin
equivalents (RE) was calculated by the following formula:
A × m0
Flavonoid content =
A0 × m
Where, A is the absorbance of plant extract solution, Ao is the
absorbance of standard rutin solution, m is the weight of plant
extract, mg and mo is the weight of rutin in the solution, mg. The
content of flavonols was also determined as described by Kumaran
and Karunakaran (2006) with slight modifications. Briefly, 1 ml of
methanolic extract (10 mg/ml) was mixed with 1 ml aluminium
trichloride (20 mg/ml) and 3 ml sodium acetate (50 mg/ml). The
2482 J. Med. Plant. Res.
Figure 1. TLC chromatograms of the O. gratissimum:
antioxidant components of the crude extract revealed by 0.2%
(w/v) DPPH radical solution.
absorbance at 440 nm was read after 2.5 h. The absorbance of
standard rutin solution (0.5 mg/ml) in methanol was also measured
under the same conditions. The amount of flavonols in the extract
was calculated by the same formula for flavonoids.
Statistical analysis
The results were analyzed using the Statistical Package for Social
Sciences (SPSS) version 10.0 for Windows. All the data are
expressed as mean ± SEM (n = 3). Student’s t-test was used to
compare means, and values were considered significant at p <
0.05.
RESULTS AND DISCUSSION
Antioxidants are used as food additives, to stabilize foods
that by their composition would in the presence of oxygen
and other reactive oxygen species undergo significant
loss in quality such as the development of rancidity from
oxidation of unsaturated fats resulting in off-odours and
off-flavours and discolouration from oxidation of pigments
or other components of the food. In this study, we
investigated the in vitro antioxidant potential of O.
gratissimum a common spice used to enhance food
flavour in Nigeria and elsewhere.
.
Inhibitory effect of MEOG on DPPH Radicals
Scavenging the stable DPPH radical model is a widely
used method to effectively evaluate antioxidant activities
of extracts (Gulcin et al., 2004). Thin layer chroma-
tography (TLC) analysis showed that the extract posse-
ssed a good antioxidant activity. The antioxidant com-
pounds in the extract and the standard neutralized the
.
free radical character of DPPH by transferring either elec-
.
trons or hydrogen atoms to DPPH (Naik et al., 2003),
thereby changing the colour from purple to the yellow
coloured stable diamagnetic molecule diphenylpicryl-
hydrazine (Figure 1). The degree of discoloration indica-
ted the scavenging potential of the extracts in term of
hydrogen donating ability (Mosquera et al., 2007).
Gradient elution in column chromatography showed that
the ethyl acetate: methanol (40:60, v/v) fraction of the
extract was the fraction with antioxidant high antioxidant
activity. The ability of the methanol extract of O.
.
gratissimum to scavenge DPPH radicals was further
determined quantitatively. The addition of the extract to
the DPPH solution caused a rapid decrease in the optical
density at 517 nm indicating the good scavenging capa-
city of the extract. The extract showed substantial
antioxidant activity in a dose-dependent manner similar to
 Awah and Verla 2483

Table 1. DPPH radical scavenging activity (%) of methanol extract of

O. gratissimum (MEOG)

Concentration ( g/ml) Inhibitory activity (%)

‡
MEOG Ascorbic acid
2.0 12.3 ± 1.0 14.3 ± 1.8
4.0 17.1 ± 1.6 36.7 ± 2.3
8.0 27.9 ± 1.0 67.5 ± 2.9
16.0 54.4 ± 1.4 92.5 ± 3.7
32.0 78.9 ± 2.7 93.6 ± 2.9
62.5 91.5 ± 3.9 93.9 ± 4.2
125.0 92.2 ± 3.1 94.1 ± 2.2
IC50 12.3 ± 1.95 4.9 ± 0.4
Data represented as Mean ± SD (n = 3).
‡
Standard antioxidant.

similar to that of ascorbic acid which was used as a
control standard antioxidant. Almost complete inhibition
.
of the DPPH radical activity was observed when 50 µg/ml
of the extract was used. The concentration of O.
gratissimum extract required to achieve a 50% reduction
.
in DPPH radicals (IC50), which was calculated using the
concentration vs activity curve, was 12.3 ± 1.95 µg/ml,
compared to that of ascorbic acid 4.9 ± 0.4 µg/ml (Table
1). The DPPH test measures the hydrogen atom or elec-
tron donating capacity of the extract to the stable radical
.
DPPH formed in solution (Tepe et al., 2005).
.–
Inhibitory effect of MEOG on superoxide anion (O2 )
radicals
.–
Superoxide anions (O2 ) are the most common free
radicals whose concentration increases under conditions
of oxidative stress and are generated either by auto-
oxidation processes or by enzymes and produces other
cell damaging free radicals and oxidizing agents (Liu and
.–
Ng, 2000). The ability of the extract to scavenge O2
radical generated from the photochemical reduction of
riboflavin resulted in a decrease in the absorbance of the
blue formazan solution at 560 nm. As shown in Table 2,
the scavenging activity was dose-dependent. The IC50
value of the MEOG was 82.9 ± 5.12 µg/ml compared to
the standard antioxidant rutin 3.3 ± 0.2. At 250 g/ml, the
percentage inhibition of the plant extract was 81.0 ± 3.3%
.-
whereas that of rutin was 98.3 ± 3.1%. The O2 radical
scavenging effect of the extracts could culminate in the
. .-
prevention of OH radical formation since O2 and H2O2
.
are required for OH radical generation. The present
study cannot completely exclude the possibility that the
MEOG affects processes other than redox regulation.
The scavenging potential will depend on the number and
locations of the hydroxyl groups in the phenolic com-
pounds present in the extract. The radical scavenging
activity is also consistent with the high level of phenolic
compounds observed in the plant extract (Table 5) since
phenolic compounds such as flavonoids are known to
.-
posses high O2 anion scavenging abilities (Robak et al.,
1988). Collectively, these results suggest that the extract
effectively scavenges ROS and could protect against
oxidative damage.
Inhibitory effect of MEOG on
3+
Fe -dependent
.
hydroxyl radicals ( OH)
.
Hydroxyl radicals ( OH) are the major active oxygen
species causing oxidation of polyunsaturated fatty acid in
food and enormous cellular and tissue damage (Aurand
et al., 1977). The effect of the methanol extract of O.
gratissimum (MEOG) on hydroxyl radicals generated by
3+
Fe ions was measured by determining the degree of
deoxyribose degradation, an indicator of thiobarbituric
acid–malonaldehyde (TBA-MDA) adduct formation. As
shown in Table 3 the extract inhibited hydroxyl radical-
induced deoxyribose degradation in a concentration
dependent manner with a maximal inhibition of 76.7 ±
1.4% observed at a concentration of 500 µg/ml of extract.
The antioxidant components in the plant extracts com-
.
peted with deoxyribose against the OH radical generated
3+
from the Fe dependent system and prevented the
reaction. The antioxidant(s) in the extract could be acting
3+
as chelators of the Fe ions in the system thereby pre-
venting them from complexing with the deoxyribose, or
simply donating hydrogen atoms and accelerating the
conversion of H2O2 to H2O (Wang et al., 2007). The
.
observed ability of the extracts to scavenge or inhibit OH
radical indicates that the extracts could significantly
.
inhibit lipid peroxidation since OH radicals are highly
implicated in peroxidation.
2484 J. Med. Plant. Res.

Table 2. Superoxide anion (O2.-) radical-scavenging activity (%) of

extract at different concentrations.

Inhibitory activity (%)

Concentration ( g/ml) ‡
MEOG -tocopherol
10 10.5 ± 1.1 60.6 ± 2.7
25 18.4 ± 1.6 75.9 ± 3.5
50 31.6 ± 1.8 89.4 ± 2.9
125 57.9 ± 2.3 97.5 ± 2.6
250 81.0 ± 3.3 98.3 ± 3.1
IC50 82.5 ± 2.63 3.3 ± 0.2
Data represented as Mean ± SD (n = 3)
‡
Standard antioxidant

Table 3. Hydroxyl radical inhibitory activity (%) of extracts in the 2-

deoxyribose assay

Inhibitory activity (%)

Concentration ( g/ml) ‡
MEOG -tocopherol
5 25.7 ± 2.2 23.5 ± 1.7
10 37.2 ± 2.9 36.3 ± 1.4
20 40.4 ± 9.6 55.2 ± 1.7
50 49.9 ± 6.7 64.0 ± 2.4
100 60.3 ± 2.1 67.3 ± 3.1
200 67.7 ± 3.2 74.8 ± 2.3
500 77.9 ± 1.3 76.7 ± 1.4
IC50 38.9 ± 2.8 25.5 ± 1.7
Data represented as Mean ± SD (n = 3).
‡
Standard antioxidant.

Table 5. Total flavonols, flavonoids and phenol contents of plant extract.

Plant extract Total flavonols ‡ Total flavonoids ‡ Phenolic contents *

Total Phenols Non-tannins Tannins
MEOG 5.11 ± 1.21 39.12 ± 2.43 0.839 ± 0.097 0.538 ± 0.041 0.301 ± 0.012
Data represented as Mean ± SD (n = 3).
‡ Expressed as mg rutin equivalents / g dry weight plant extract.
* Expressed as mg gallic acid equivalents / mg dry weight plant extract.

.
Scavenging effect of MEOG on NO production concentration dependent with 250 µg/ml scavenging most
efficiently. The MEOG in SNP solution significantly
. +
Nitric oxide (NO ) released from SNP has a strong NO inhibited (p < 0.05) the accumulation of nitrite, a stable
.
character which can alter the structure and function of oxidation product of NO liberated from SNP in the
many cellular components. The methanol extract of O. reaction medium with time compared to the standard -
. .
gratissimum (MEOG) exhibited good NO scavenging tocopherol (Figure 2). The toxicity of NO increases when
activity leading to the reduction of the nitrite concentration it reacts with superoxide to form the peroxynitrite anion
. . -
in the assay medium. The NO scavenging capacity was ( ONOO ), which is a potential strong oxidant that can
 Awah and Verla 2485

Figure 2. Effect of ethanol extract of O. gratissimum leaves on the accumulation of nitrite upon decomposition of sodium
nitroprusside (SNP; 5 mM) at 25˚C. Data are mean ± SEM (n = 3).

Table 4. Inhibition (%) of lipid peroxidation by different concentration of

plant extract

Inhibitory activity (%)

Concentration ( g/ml) ‡
MEOG Butylated hydroxytoluene
15 19.6 ± 0.6 44.9 ± 1.8
30 23.5 ± 1.1 53.5 ± 0.6
60 32.0 ± 1.1 56.9 ± 1.9
125 43.5 ± 2.0 63.6 ± 1.1
250 49.8 ± 1.9 68.2 ± 2.9
500 50.3 ± 2.2 72.2 ± 1.4
1000 66.5 ± 1.3 76.8 ± 1.9
IC50 270.5 ± 8.2 24.3 ± 1.92

Data represented as Mean ±SD (n = 3).

‡
Standard antioxidant.
2486 J. Med. Plant. Res.
.
decompose to produce OH and NO2 (Pacher et al.,
2007). The present study shows that a MEOG has a
potent nitric oxide scavenging activity.
Inhibitory effect of MEOG on lipid peroxidation
Lipid peroxidation involves the formation and propagation
of lipid radicals, which eventually destroy membrane
lipids and could lead to food deterioration in the food
industry. The TBARS formation assay was used to
2+
assess the inhibition of Fe -induced lipid peroxidation by
the extract. The MEOG showed a very good
concentration-dependent inhibition of lipid peroxidation
(IC50 = 270.5 ± 8.2 g/ml) compared to the standard anti-
oxidant butylated hydroxyl toluene (BHT) (IC50 = 24.3 ±
1.92 g/ml) (Table 4). This corroborates the observed
.
potent OH radical scavenging activity of the extract and
suggests that the extract may afford a protective effect.
Total phenolics, tannins, flavonoids and flavonols
contents
Due to the potent free radical scavenging activity of the
MEOG, it was subjected to some phytochemical analysis.
As shown in Table 5, phenolic compounds were a major
class of bioactive components in the extract. Phenolic
compounds may contribute directly to antioxidative
action. The total phenolic content was 0.839 ± 0.097 mg
gallic acid equivalents/mg dry weight plant extract. The
total flavonoid content and the flavonol subclass of the
MEOG were 39.12 ± 2.43 and 5.11 ± 1.21 mg rutin
equivalents/g dry weight plant extract. The antioxidant
activity of plant phenolic compounds are attributed to
their redox properties, which allow them to act as redu-
cing agents, hydrogen donators, singlet oxygen quen-
chers and metal chelators (Cook and Samman, 1996).
Conclusion
Considering the observed antioxidant potential of the
investigated methanol extract of O. gratissimum and the
. . .–
potent DPPH radical, OH radical, O2 radical, NO
scavenging activity, it could be presumed that this extract
is able to prevent lipid peroxidation and further suggest
that the extract is a potential therapeutic agent for the
control of oxidative and non-oxidative damage caused by
reactive oxygen and nitrogen species. The results have
demonstrated that the scent leaf possesses antioxidant
and nitric oxide scavenging abilities, which indicates that
the plant contains certain compounds which are potential
anti-oxidants. Since reactive oxygen species are thought
to be associated with food deterioration and the patho-
genesis of chronic infections, and inflammatory diseases,
the observed inhibitory potential may partially explain the
helpful effects of O. gratissimum in treating different
disease conditions. The results further suggest that scent
leaf could be used in the food industry as a natural
antioxidant and food flavour. The antioxidants present in
this plant extract may function by combining with oxygen
or preventing oxygen from reacting with components of
the food. It is however, worthwhile to further investigate
the in vivo potentials of this plant and also isolate the
active components which could ultimately lead to their
application in the food industry as an antioxidant flavour
or in pharmaceutical formulations.
ACKNOWLEDGEMENT
The authors are grateful to Prof. P. N. Uzoegwu
(Department of Biochemistry, University of Nigeria,
Nsukka) for useful suggestions.
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