Antioxidant activity , nitric oxide scavenging activity and phenolic contents of Ocimum gratissimum leaf extract

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.


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 (H 2 O 2 ),superoxide anions (O 2

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), and nitric oxide (NO .), as well as peroxyl nitrite contributes to the immunopathology of a vast variety of conditions including inflammatory diseases, cancer, atherosclerosis, diabetes mellitus, hypertension, AIDS, and aging (Darley-Usmar et al., 1995;Lee et al., 2000) and also contribute to food deterioration.Antioxidants 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 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 process, 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 lactones 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 importance of this plant centers on its usefulness as a seasoning 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).

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).

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: 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 2deoxyribose degradation expressed in percentage was calculated as per the equation: 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 µM riboflavin, 100 µM EDTA, NBT (75 µM) 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: 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.

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 phosphatebuffered 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% naphthylethylenediaminedihydrochloride).The absorbance of the chromophore (purple azo dye) formed during the diazotisation of nitrite ions with sulphanilamide and subsequent coupling with naphthylethylenediaminedihydrochloride 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 µl of 10%, v/v in PBS (pH 7.4) and 100 µl of samples were added to a test tube and made up to 1.0 ml with distilled water.Then, 50 µl of FeSO4 (0.075 M) and 20 µl 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:

Determination of total phenolic contents
Total phenolics were determined using Folin-ciocalteu reagent (FCR) as described by Velioglu et al. (1998), with slight modifications.Briefly, 100 µl of the extract dissolved in methanol (1 mg/ml) was mixed with 750 µl of FCR (diluted 10-fold) and allowed to stand at 22 ˚C for 5 min; 750 µl 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 calculated 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 modifications.Briefly, 100 µl of plant extracts in methanol (10 mg/ml) was mixed with 100 µl 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 µl 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: 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 Antioxidant compound(s) 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 chromatography (TLC) analysis showed that the extract possessed a good antioxidant activity.The antioxidant compounds in the extract and the standard neutralized the free radical character of DPPH .by transferring either electrons or hydrogen atoms to DPPH .(Naik et al., 2003), thereby changing the colour from purple to the yellow coloured stable diamagnetic molecule diphenylpicrylhydrazine (Figure 1).The degree of discoloration indicated 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 capacity of the extract.The extract showed substantial antioxidant activity in a dose-dependent manner similar to 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 (IC 50 ), 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 electron donating capacity of the extract to the stable radical DPPH . formed in solution (Tepe et al., 2005).

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) are the most common free radicals whose concentration increases under conditions of oxidative stress and are generated either by autooxidation 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 O 2 .radicalgenerated 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 IC 50 value of the MEOG was 82.9 ± 5.12 µg/ml compared to the standard antioxidant rutin 3.3 ± 0.2.At 250 µg/ml, the percentage inhibition of the plant extract was 81.0 ± 3.3% whereas that of rutin was 98.3 ± 3.1%.The O 2 .
-radical scavenging effect of the extracts could culminate in the prevention of .OH radical formation since O 2 .
-and H 2 O 2 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 O 2 .
-anion scavenging abilities (Robak et al., 1988).Collectively, these results suggest that the extract effectively scavenges ROS and could protect against oxidative damage.

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 Fe 3+ 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 radicalinduced 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 competed with deoxyribose against the .OH radical generated from the Fe 3+ dependent system and prevented the reaction.The antioxidant(s) in the extract could be acting as chelators of the Fe 3+ ions in the system thereby preventing them from complexing with the deoxyribose, or simply donating hydrogen atoms and accelerating the conversion of H 2 O 2 to H 2 O (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.2).The toxicity of NO .increases when it reacts with superoxide to form the peroxynitrite anion ( .ONOO -), which is a potential strong oxidant that can  decompose to produce .OH and NO 2 (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 assess the inhibition of Fe 2+ -induced lipid peroxidation by the extract.The MEOG showed a very good concentration-dependent inhibition of lipid peroxidation (IC 50 = 270.5 ± 8.2 µg/ml) compared to the standard antioxidant butylated hydroxyl toluene (BHT) (IC 50 = 24.3 ± 1.92 µg/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 reducing agents, hydrogen donators, singlet oxygen quenchers 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, O 2 .-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 pathogenesis 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.

Figure 1 .
Figure 1.TLC chromatograms of the O. gratissimum: antioxidant components of the crude extract revealed by 0.2% (w/v) DPPH radical solution.

Figure 2 .
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 5 .
Total flavonols, flavonoids and contents of plant extract.

Table 4 .
Inhibition (%) of lipid peroxidation by different concentration of plant extract Data represented as Mean ±SD (n = 3).‡Standard antioxidant.