Oxidative stress occurs as a result of imbalance between the productions of
Reactive Oxygen Species (ROS) and the ability of the biological system to neutralize
the free radicals (Hadi et al., 2007). Free radicals
such as superoxide anion (O2¯), hydrogen peroxide (H2O2),
hydroxyl radical (●OH) nitric oxide (NO) and organic hydroperoxide
(ROOH) are chemically unstable atoms due to the presence of lone pair electrons
in their outer shells. They have been implicated as underlying cause of several
degenerative diseases in humans by damaging vital macromolecules in the biological
system such as lipids, DNA and protein (Farber, 1994).
Some of these diseases include atherosclerosis, sickle cell anaemia, myocardial
infarction, heart failure and Parkinson disease (Lee et
al., 2001). However, the presence of enzymatic and non-enzymatic antioxidant
defence in the body system has been reported to prevent the pathological action
of these radicals by quenching or neutralizing the chain of reactions before
vital damages to the cells (Oboh, 2006). Examples of
these antioxidants are catalase, superoxide dismutase, glutathione reductase
and vitamin C and E. In the last three decades, attention has been shifted to
the use of natural antioxidants from plant origin due to the harmful effect
of commercially available synthetic antioxidants such as tertbutylhydroxyl toluene
(TBHQ), Butylated Hydroxyl Anisole (BHA) and Butylated Hydroxyl Toluene (BHT)
which has been implicated in liver damage and development of cancer cells (Oyetayo,
2009). Medicinal plants are well known to contain secondary metabolites
such as phenolics, flavonoids, flavonols and proanthocyanidins which have been
reported to possess potent antioxidant properties (Oboh and
Rocha, 2007). However, majority of these plants have not been investigated
for their possible antioxidant potential in the quest to retard or alleviate
the extent of oxidative deterioration.
Ocimum americanum L. (Lamiaceae) is a wild herb that grows in tropical
Africa (Steel, 2006). It is known as African basil but
popularly called "Efinrin elewe dudu" in south-western Nigeria. The leaf is
used in traditional folk medicine in Ghana for the treatment of diabetes (Hogarh,
1996). In Nigeria, it is used by traditional healers for the treatment of
constipation, diarrhoea, piles, dysentery and as insect repellent. The leaf
is rich in essential oils of therapeutic importance and mostly used for the
preparation of delicious local soups as well as flavouring agent in yam and
cocoyam porridges in the Yoruba tribe of Nigeria (Ekundayo
et al., 1989). It is also used as a local condiment because of its
aromatic properties (Bassole et al., 2005). Previous
work conducted on this plant reported the inhibitory activity of its acetone
extract on some neurotoxins induced brain damage in rats (Oboh,
There is dearth of scientific information on the antioxidant potentials of O. americanum leaves to justify its continuous use in traditional folk medicine. Therefore, the present study was explored to evaluate the polyphenolic contents and the free radical scavenging potentials of its leaves with a view to ascertain its potential health benefits.
MATERIALS AND METHODS
Collection and identification of plant sample: The leaves of Ocimum americanum were collected from a local farmland near Orin Ekiti South-Western Nigeria in the month of May, 2011. The plant was authenticated by Mr. Omotayo (herbarium curator) at the Department of Plant Science, University of Ado Ekiti, Nigeria where the voucher specimen (Aluko 09) was deposited.
Reagents: The reagents used in this study were purchased from Sigma-Aldrich Gmbh, Sternheim, Germany. This includes: tannic acid, quercetin, catechin, Folin-Ciocalteu reagent, aluminium chloride, sodium acetate, vallinin, 2,2 diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), potassium persulfate, hydrogen peroxide, sodium nitroprusside, sulphanilic acid, naphthylenediamine dichloride, 2-deoxy-D-ribose, ferric chloride, thiobabituric acid, trichloroacetic acid, potassium ferricyanide, ascorbic acid and butylated hydroxyl toluene. All other solvents used were of analytical grade.
Sample extraction: The leaves were air dried for 10 days and then pulverized into fine powder using an electric blender. Fifty gram of the powdered sample was defatted with 250 mL of n-hexane with constant shaking on an orbital shaker (Stuart Scientific Orbital Shaker SO1, Essex, UK) for 12 h. The solvent was removed by filtration using a Buchner funnel with Whatmans No. 1 filter paper and the residue obtained was extracted sequentially with 500 mL of ethyl acetate, n-butanol and ethanol, respectively with continuous shaking on an orbital shaker for 24 h. The ethyl acetate and ethanol extracts were concentrated to dryness under vacuum in a rotary evaporator thereafter; the extracts were collected in clean bottles and left opened in a laboratory fume hood for complete evaporation of residual solvent. Meanwhile, butanol extract was allowed to evaporate in a fume hood for 7 days. The percentage yield for ethyl acetate, ethanol and butanol extracts was 0.10, 0.12 and 0.11 w/w, respectively.
Determination of total phenolics: The method of Wolfe
et al. (2003) was adopted to determine total phenolic contents of
O. americanum leaves in different solvent systems. A reaction mixture
of 2.5 mL of 10% (v/v) Folin-Ciocalteu reagent and 2 mL of 7.5% (w/v) of sodium
carbonate was added to 0.5 mL (1 mg mL-1) of the extracts. The mixture
was vortexed and incubated at 40°C for 30 min after which the absorbance
was measured at 765 nm. The total phenolic content was calculated from the equation
obtained from the calibration curve of Tannic acid (Y = 0.1216x, R2
= 0.936512) and expressed as mg g-1 tannic acid equivalent where
x is the absorbance and Y is the tannic acid equivalent.
Determination of total flavonoids: Total flavonoids content of O.
americanum leaves was determined by the modified method of Ordonez
et al. (2006). The extract (1 mL in a final concentration of 0.1
mg mL-1) was mixed with 1 mL of 2% (w/v) aluminium chloride prepared
in ethanol and left in the dark at room temperature for 1 h. A yellow colour
was observed which was measured spectrophotometrically at 420 nm. The total
flavonoid content was calculated as mg g-1 quercetin equivalent from
the equation (Y = 0.0255x, R2 = 0.9812) obtained from the calibration
curve where x is the absorbance and Y is the quercetin equivalent.
Determination of total flavonols: The determination of the total flavonols
content of the extracts was done according to the method of Kumaran
and Karunakaran (2007). Two milliliters of the extract was mixed with 2
mL of 2% aluminium chloride in ethanol and 3 mL of 5% sodium acetate solution
and allowed to stand for 2.5 h at room temperature and later, measured the absorbance
at 440 nm. The flavonols content was expressed as mg g-1 quercetin
equivalent using the calibration equation (Y = 0.0255x, R2 = 0.9812)
from standard quercetin where x is the absorbance and Y is the quercetin equivalent.
DPPH scavenging assay: A volume of one milliliter of 0.135 mM of DPPH
in methanol was mixed with 1 mL of different concentrations (50-300 μg
mL-1) of the leaf extract, vitamin C and BHT. The mixture was vortexed
and kept in a dark cupboard for 30 min (Liyana-Pathirana
and Shahidi, 2005). The observed decolourisation was measured spectrophotometrically
at 517 nm and the scavenging ability of the extract was calculated as follows:
where, Abscontrol is the absorbance of DPPH radicals+methanol, Abssample is the absorbance of DPPH radical+sample or standard.
ABTS scavenging assay: The ABTS scavenging potential of the extract
was assayed using the method of Re et al. (1999)
with some modifications. The stock solution consisting of 7 mM ABTS solution
and 2.4 mM potassium persulfate (1:1) was allowed to react in the dark for twelve
hours at room temperature. The radical generated was mixed with methanol to
obtain an absorbance of 0.702±0.001 unit at 734 nm. One milliliter of
the resulting solution was added to 1 mL of the extract or standard (50-300
μg mL-1) and the absorbance was measured at 734 nm after 7 min.
The percentage scavenging ability was calculated from this equation:
where, Abscontrol is the absorbance of ABTS radicals+methanol, Abssample is the absorbance of ABTS radical+sample or standard.
Hydrogen peroxide scavenging assay: The modified method of Oyedemi
et al. (2010) was employed for the hydrogen peroxide scavenging assay
of the extracts of O. americanum leaves. The stock solution was 4 mM
hydrogen peroxide prepared in 0.1 M phosphate buffer (pH 7.4). The solution
(0.6 mL) was added to 2 mL of the extract and standard (50-300 μg mL-1)
and incubated for 15 min at room temperature. The absorbance was read at 230
nm and the percentage inhibition of hydrogen peroxide was calculated as:
where, Abscontrol is the absorbance of H2O2 radicals, Abssample is the absorbance of H2O2 radical+sample or standard.
Nitric oxide scavenging assay: The nitric oxide scavenging activity
of the extracts was evaluated by the method of Igbinosa
et al. (2011). Two milliliters of 10 mM sodium nitroprusside prepared
in 0.5 mM phosphate buffer saline (pH 7.4) was added to 0.5 mL of plant extract
or standard (50-300 μg mL-1) and vortexed. The mixture was incubated
for 2.5 h at 25°C and thereafter, 1 mL of the mixture was taken and mixed
with 1 mL of Griess reagent (equal volumes of 0.33% sulphanilic acid prepared
in 20% glacial acetic acid and 0.1% (w/v) naphthylenediamine dichloride) and
incubated at room temperature for 30 min. The absorbance was read at 540 nm
and the percentage nitric oxide inhibition by the extracts was calculated using
the following equation:
where, Abscontrol is the absorbance of NO radicals, Abssample is the absorbance of NO radical+sample or standard.
Hydroxyl radical scavenging assay: The method of Zhang
et al. (2011) was adopted for the evaluation of the hydroxyl radical
scavenging activities of the extracts of O. americanum leaves. Briefly,
100 μL of the extract or standard (50-300 μg mL-1) was
added to a reaction mixture of 500 μL 5.6 mM 2-deoxy-D-ribose in KH2PO4/NaOH
(0.05 M, pH 7.4), 100 μL ferric chloride (104 μM), 100 μL EDTA
(104 μM), 100 μL H2O2 (1 mM) and 100 μL
ascorbic acid (1 mM). The mixture was vortexed for 15 sec and incubated in a
water bath for 30 min at 50°C. After the incubation, 1 mL of 1% TBA and
1 mL of 2.8% TCA were added to the mixture and further incubated for 30 min.
The absorbance of the mixture was read at 532 nm and the percentage inhibition
was calculated as:
where, Abscontrol is the absorbance of OH•, Abssample is the absorbance of OH•+sample or standard.
Lipid peroxidation assay: The lipid peroxidation inhibitory activity
of the extract was evaluated by the method of Duh et
al. (2001). Egg lecithin (a creamy powder) was prepared by repeated
washing of egg yolk with acetone until the yellow colour was removed. A concentration
of 3 mg mL-1 of egg lecithin in phosphate buffer was added to 10
μL ferric chloride (400 mM) and 10 μL L-ascorbic acid (400 mM). Then,
100 μL of different concentrations of the extracts or standard drug was
added and incubated at 37°C for 1 h. The reaction was stopped by the addition
of 2 mL (1:1:1) TBA-TCA-HCL reagent (0.37% TBA, 15% TCA and 0.25N HCL). The
mixture was boiled for 15 min, cooled, centrifuged at 2000 rpm for 10 min and
absorbance of the supernatant was measured at 532 nm. The percentage inhibition
was determined as follows:
where Abscontrol is the absorbance of LPO, Abssample is the absorbance of LPO+sample or standard.
Determination of reducing power: The reducing power of the extracts
was determined by the modified method of Yen and Chen (1995).
Various concentrations (50-300 μg mL-1) of the extracts or standard
drugs in a volume of 0.5 mL was mixed with 1 mL of 0.2 M phosphate buffer (pH
6.6) and 1 mL of 1% (w/v) potassium ferricyanide. The mixture was incubated
for 20 min at 50°C. Thereafter; 1 mL of 10% (w/v) TCA was added and centrifuged
at 3000 rpm for 10 min. The supernatant was decanted and 1.25 mL of the solution
was allowed to react with 1.25 mL distilled water and 0.25 mL of 0.1% (w/v)
of ferrous chloride for 5 min and the absorbance was read at 700 nm. The observed
increase in absorbance with increasing concentration indicated the ferric reducing
potential of the extracts.
Statistical analysis: All results were expressed as Mean±standard
Deviation (SD) of three replicates and were subjected to Analysis of Variance
(ANOVA) using the student Minitab release version 12, Windows 95. Significant
levels were tested at p<0.05.
The various extracts of O. americanum were evaluated for their polyphenolic contents. The ethanol extract showed the highest polyphenolic content with total phenol (94.00 mg TE g-1), flavonoids (38.69 mg QE g-1) and flavonols (15.64 mgQE g-1). This was followed by butanol extract which contained total phenol (79.00 mg TE g-1), flavonoids (34.35 mg QE g-1) and flavonols (5.37 mg QE g-1). Generally, ethyl acetate extracts had the lowest concentration of the polyphenolic compounds. The findings of our study revealed high concentrations of total phenolics and flavonoids but low concentrations of flavonols in the three extracts of O. americanum leaves (Table 1). The high phenolics content in this plant might be responsible for the strong antioxidant activity observed in this study.
Figure 1 showed the DPPH radical scavenging activity of the extracts and standard antioxidants (BHT and Vitamin C). The extracts and drugs exhibited a concentration-dependent inhibition of DPPH radical. The observed percentage inhibition was in the ascending order of ethyl acetate<butanol<ethanol<BHT and vitamin C.
ABTS radical scavenging potential of the extracts at the concentrations investigated in this study was determined together with standard antioxidants (Fig. 2).
||Scavenging effects of extracts of O. americanum leaves
on DPPH radical. The results are Mean±SD (n = 3)
||Scavenging effects of extracts of O. americanum leaves
on ABTS radical. The results are Mean±SD (n = 3)
||Polyphenolic contents of ethanol, butanol and ethyl acetate
extracts of O. americanum leaves
|Data expressed as Mean±SD (n = 3). TE: Tannic acid,
QE: Quercetin, CE: Catechin equivalent
The inhibitory activities of both the extracts and reference drugs occurred at all the concentrations tested in an increasing order.
O. americanum extracts demonstrated hydrogen peroxide decomposition activity in a concentration-dependent manner (Fig. 3). The scavenging activities of the extracts were found to be lower than those of the standard drugs. At a concentration of 300 μg mL-1, the hydrogen peroxide radical inhibition exhibited by the extracts and known antioxidants followed the order: ethyl acetate<butanol<ethanol<BHT<vitamin C.
||Scavenging effects of extracts of O. americanum leaves
on hydrogen peroxide radical. The results are Mean±SD (n = 3)
||Scavenging effects of extracts of O. americanum leaves
on nitric oxide radical. The results are Mean±SD (n = 3)
Figure 4 illustrates the scavenging activities of the extracts and standard drugs against nitric oxide released by sodium nitroprusside. The inhibitory activities of the extracts and reference drugs were found in the following order: Vitamin C>BHT>butanol>ethanol>ethyl acetate extract.
The effect of O. americanum extracts on the inhibition of hydroxyl radical production was assessed by the iron (II)-dependent deoxyribose damage assay. Figure 5 presents the results of the effects of the extracts and standard drugs on OH* radical production. The extent of inhibition occurred in a dose-dependent manner. At a concentration of 300 μg mL-1, the ethanol and butanol extracts showed maximum inhibitory effect of 67.95 and 66.67%, respectively which was comparable to that of BHT (69.23%).
The anti-lipid peroxidation effect of the extracts and reference drugs is presented
in Fig. 6. The addition of Fe2+/ascorbate to the
lecithin of egg yolk caused increased lipid peroxidation which was inhibited
by the addition of the extracts and standard antioxidants in a concentration-dependent
||Scavenging effects of extracts of O. americanum leaves
on hydroxyl radical. The results are Mean±SD (n = 3)
||Inhibitory effects of extracts of O. americanum leaves
on lipid peroxidation. The results are Mean±SD (n = 3)
The highest inhibition of lipid peroxidation was exhibited by vitamin C while
the lowest inhibitory activity was demonstrated by the ethyl acetate extract
of O. americanum leaves.
The reductive capabilities of the extracts of O. americanum leaves as compared with standard antioxidants are shown in Fig. 7. The transformation of Fe3+-Fe2+ was investigated in the presence of the extract and reference drugs in a concentration-related manner. The presence of reductants was confirmed by the changes of yellow colour of the test solution to greenish-blue and the absorbance increased with increasing concentration. The reducing power is shown in the ascending order of ethyl acetate<ethanol<butanol<BHT< vitamin C.
||Ferric reductive abilities of extracts of O. americanum
leaves. The results are Mean±SD (n = 3)
Polyphenols are pharmacologically active components of plants which are capable
of neutralizing free radicals, chelating metal catalysts and inhibiting the
activity of oxidizing enzymes in biological systems (Miliauskas
et al., 2004). They are also capable of regenerating endogenous α-tocopherol
in the phospholipid bilayer of the membrane to its active antioxidant form.
This mechanism of antioxidant action confers health beneficial potentials on
polyphenolic compounds. Several workers have attributed therapeutic potential
of various medicinal plants to their significant antioxidant potential due to
the presence of phenolic compounds (Shahidi et al.,
1994; Oyedemi et al., 2010; Basma
et al., 2011). These compounds have been reported to reduce the risk
of cardiovascular diseases, cancer, urinary tract diseases and metabolic syndrome
(Wang et al., 1998). The high levels of phenolics
in the solvent extracts of O. americanum leaves could justify the ethnotherapeutic
usage of this plant by the traditional healers. Our study revealed that there
was a relationship between the polyphenolic contents and the antioxidant activities
of the extracts. These appreciable levels of polyphenols in the extracts of
the leaves might be responsible for the use of this plant for the treatment
of radical related problems such as diabetes and gastrointestinal lesions (Nyarko
et al., 2002).
DPPH is a stable radical commonly used to determine the antioxidant activity
of various compounds. It is a purple radical that decolorizes to either yellow
or colourless due to the antioxidant potential of test samples. The ethanol
extract of O. americanum leaves exhibited the highest inhibition of DPPH
radical which corresponds to its phenolic contents. This was followed by butanol
extract whereas, ethyl acetate extract showed the least inhibition. It is worth
noting that the inhibition of DPPH radical by vitamin C and BHT used as reference
drugs was significantly higher than those of the extracts. This observation
is in tune with the previous findings of Ganie et al.
(2011) on the antioxidant effect of ethyl acetate extract of Podophyllum
hexandrum. The strong inhibition displayed on DPPH radical could be linked
to polyphenolic compounds which are capable of donating electrons or transferring
hydrogen atom to neutralize free radicals and thus, could be a promising therapeutic
agent to treat stress induced pathological conditions.
ABTS is a blue green chromophore with characteristic absorbance maxima at 734
nm and mostly reactive toward phenolics, thiols and other antioxidants (Walker
and Everette, 2009). The reactions of the solvent extracts with the pre-formed
radical decolorized the chromophore with increasing concentrations. In this
study, ABTS radical scavenging activity of ethanol extract of the leaf of O.
americanum was higher than other solvent extracts but lower when compared
with vitamin C and BHT. At 300 μg mL-1, the percentage inhibition
of ABTS radicals was found to be 65.70, 56.98, 53.33 and 100% for ethanol, butanol,
ethyl acetate extracts, BHT and vitamin C, respectively. A similar trend for
the inhibition of DPPH radical was observed in the inhibitory activities of
the extracts on ABTS radical. This is contrary to the findings of Wang
et al. (1998), who reported that compounds which posses ABTS●+
activity may not exhibit DPPH● scavenging potential. Though,
the mechanism of action was not investigated in this study, but could be assigned
to the hydrogen proton donating ability of the extracts to ABTS●+.
The donor could be adduced to the polyphenolic contents. The observation from
this present study gives scientific credence to the traditional usage of O.
americanum leaves for the treatment of oxidative stress induced diseases
Hydrogen peroxide is an oxidant with capability of oxidizing biological compounds
by penetrating biological membranes. It can be reduced to hydroxyl radical through
the action of gluthatione peroxidase and catalase in the presence of iron or
copper. The formation of this radical is an underlying cause of tissue damage
and if not prevented may result to cell death (Reddy et
al., 2010). The hydrogen peroxide inhibition increased with increasing
concentration of the extracts and the standards. The ethanol extract showed
the highest scavenging potential against H2O2; followed
by butanol while the least inhibition was exhibited by ethyl acetate extract
at 300 μg mL-1. Although vitamin C and BHT showed better scavenging
activity, the inhibition of H2O2 by the extracts can be
attributed to the proton donating abilities of their polyphenolic contents.
Nitric Oxide (NO) radical is generated from sodium nitroprusside at physiological
pH. It is associated with inflammatory conditions such as atherosclerosis, arthritis,
ulcerative colitis and carcinomas (Hazra et al.,
2009). It is a highly reactive compound that is capable of changing the
structural and functional behavior of many cellular components (Ashokkumar
et al., 2008). The extract of O. americanum leaves inhibited
nitric oxide radical in a concentration dependent manner. The percentage inhibition
of ethanol, butanol and ethyl acetate extracts was lower than that of vitamin
C or BHT. The inhibitory potentials of the extracts against NO radical can be
attributed to their ability to compete with oxygen and its derivatives (Marcocci
et al., 1994).
Hydroxyl radicals have been implicated in the oxidative damage of DNA, proteins
and lipids (Spencer et al., 1994). The formation
of hydroxyl radicals in biological systems has been attributed to the interactions
of metal ions such as ferrous or copper with hydrogen peroxide (McCord
and Day, 1987). The hydroxyl radical generated by Fe2+-ascorbic
acid and EDTA-H2O2 system (Fentons reaction) was
scavenged by the extracts and standards in a concentration dependent manner.
At 300 μg mL-1, the percentage inhibition of ethanol and butanol
extract against OH radical was 67.95 and 66.67%, respectively which was comparable
to that of BHT (69.23%). Ethyl acetate extract depicted the least inhibition
(46.15%). This observation suggests that the extracts of O.americanum can
be used as an alternative remedy to synthetic antioxidants in combating the
oxidative activity of hydroxyl radical. In addition, the weak antioxidant potential
of ethyl acetate extract may be due to poor extraction of polyphenolic content.
The effect of lipid peroxidation on biological system has brought about an
increasing interest in herbal preparations capable of preventing lipid peroxidation
and oxidative damage in biological systems (Yagi, 1987).
In this study, lecithin extracted from egg yolk was employed as a lipid rich
media. The ethanol extract exhibited the highest inhibition of lipid peroxidation
followed by butanol while the least inhibition was shown by ethyl acetate extract.
Even though, BHT and vitamin C showed better inhibition against lipid oxidation
than the plant extracts, the inhibition of lipid peroxidation by the extracts
can be adduced to the presence of phenolic compounds. This supports the earlier
reports correlating the presence of polyphenolic compounds to antioxidant activity
of natural plant products (Oyedemi and Afolayan, 2011;
Abalaka et al., 2011; Aiyegoro
and Okoh, 2010).
The reducing capacity of a compound is as an indication of its potential antioxidant
activity due to the presence of reductants (Duh et al.,
1999). The ability of O. americanum extracts to reduce ferric ions
to its ferrous form is evident in the concentration dependent formation of Perls
Prussian blue which was monitored spectrophotometrically at 700 nm. At a concentration
of 300 μg mL-1, the reducing ability of butanol, ethanol and
ethyl acetate extracts of O. americanum leaf was significantly lower
than that of BHT and vitamin C. Nevertheless, our study revealed that O.
americanum contain components capable of electron transfer which can react
with free radicals to convert them to stable products and terminate the chain
of reactions that leads to oxidative stress.
This study revealed that the leaves of O. americanum contain appreciable amounts of polyphenolic compounds that are capable of eliciting potent antioxidant activities. The antioxidant profile of this plant can be harnessed to treat radical related pathological conditions. The mechanism of antioxidant action was based on the ability of its extracts to donate electrons, reduce ferric ions, scavenge nitric oxide, hydrogen peroxide and hydroxyl radicals. The antioxidant activity exhibited by the solvent extracts of O. americanum leaves could justify the ethnotherapeutic usage of this plant by the traditional healers. The antioxidative potential of the plant was dependent on the solvent of extraction which means that ethyl acetate may not be a good solvent of extraction in the exploitation of the antioxidant property of O. americanum leaves.
The authors thank the Govan Mbeki Research and Development Centre, University of Fort Hare and National Research Foundation of South Africa for their financial support.