The antioxidant activities of Ocimum basilicum and Ocimum gratissimum consumed as spices in Nigeria were evaluated in a bid to provid e better scientific basis for increased domestication efforts on them. Total phenol and flavonoid con tents of the spice extracts ranged from 9.09 - 27.41 μg GAE/mg and 5.38 - 22.88 μg RE/mg respectively. The DPPH and hydroxyl radical scavenging activities of the spice extracts ranged from 58.43% - 92.37% and 6.27% - 16.67% re spectively. The total antioxidant capacities and reducing powers of the extracts (measured as absorbance values) ranged from 0.137 - 0.160 and 0.130 - 0.158 respectively. Generally, Ocimum basilicum maintained superior antioxidant ac tivities to O. gratissimum in all the test assays, and all the extracts showed dose-dependent antioxidant activities. Ascorbic acid, Gallic acid and Rutin used as reference compounds generally showed higher antioxidant activities to the spice extracts except in the hydroxyl radical scavenging assay. Put together, these results confirm that Ocimum basili cum and O. gratissimum extracts possess appreciable natural antioxidant potentials, thereby providing good justification for their increased domestication and consumption.
Spice plants have important uses in food and medicine and interestingly, the Nigerian ecology has been adjudged as favourable for the production of a wide range of these spices [
Ocimum basilicum L. and Ocimum gratissimum L. are two popular spices used widely in many Nigerian cuisines. Commonly known as scent leaf in Nigeria, Ocimum gratissimum L. is also used in traditional medicine for treatment of several ailments such as urinary tract and gastro intestinal infections [
Among the medicinal benefits of plants, antioxidant properties have received increasing attention due to their role in preventing or down regulating myriads of oxidative damages caused by free radicals in the body [
Spices are harmless sources for natural antioxidants [
Fresh leaves of Ocimum basilicum and Ocimum gratissimum (Plate 1) were procured from Watt market in Calabar (located at 4˚59'36''N, 8˚19'05''E), Cross River State, Nigeria and authenticated by the plant taxonomist in Botany Department, University of Calabar. The plant materials were freed from debris, dried at room temperature for one week and milled separately into fine powder using a blender (5 speed kitchen-aids 5KSB655CCS0). Ten (10) grams of each milled sample were extracted by soaking in 100mls of 90% methanol for 72 h at room temperature with intermittent shaking. The samples were subsequently filtered using Whatman No. 1 filter paper at the end of the extraction period and concentrated under vacuum in a rotary evaporator at 45˚C for complete solvent removal. A sample stock solution of 3000 µg/ml was prepared for each extract by accurately weighing 0.3 g of concentrated crude extract and dissolving in 100 ml of distilled water. Working solutions of each extract were
Plate 1. Shoots of Ocimum gratissimum (a) and O. basilicum (b).
prepared as desired by appropriate dilutions of the stock solutions.
DPPH (1,1-diphenyl-2-picryl hydrazyl) radical and Rutin were purchased from Sigma Aldrich Chemical Company, USA; Folin and Ciocalteau’s Phenol reagent and Trichloroacetic acid (TCA) from Qualikems Fine Chemical Pvt. Ltd., New Delhi, India; Gallic acid monohydrate from Kem Light Laboratories Pvt. Ltd., Mumbai, India. Solvents and other chemicals used for this study were of analytical grade, while water was glass distilled.
The total phenol contents of Ocimum basilicum and Ocimum gratissimum leaf extracts were determined by the Folin-Ciocalteau method according to [
The total flavonoid contents of the leaf extracts of Ocimum basilicum and Ocimum gratissimum were determined using the aluminium chloride colorimetric method according to [
Antioxidant activity of the leaf extracts of Ocimum basilicum and Ocimum gratissimum were measured in this assay as ability to scavenge stable DPPH radicals according to [
Percentage DPPH scavenging activities of the extracts and reference standards were determined using the formula:
where As = Absorbance of sample (extract or reference standard), Ab = Absorbance of blank and Ac = Absorbance of negative control [
The total antioxidant capacity (TAC) of the Ocimum basilicum and Ocimum gratissimum leaf extracts were determined by the phosphomolybdate method according to [
The hydroxyl radical scavenging activity of the leaf extracts of Ocimum basilicum and Ocimum gratissimum were determined by the Fenton reaction using the orthophenanthroline method according to the modified procedure of [
where As = Absorbance of reaction mixture with sample or standard, Ao = Absorbance of damage control and Ab = Absorbance of blank.
Antioxidant activity of the leaf extracts of Ocimum basilicum and Ocimum gratissimum were determined in this assay as their Fe3+ reducing ability according to the method of [
The experiment was laid out in a Completely Randomized Design (4 × 4 factorial arrangement) and data were analyzed using a one-way analysis of variance (ANOVA), except results for total phenol and flavonoid contents of the extracts that were analyzed using the student t-test and presented as means ± standard error. All measurements were replicated five times. Significant means were separated using the Least Significant Difference (LSD) analysis.
The total phenol contents of the leaf extracts of Ocimum basilicum and Ocimum gratissimum were estimated quantitatively from a linear regression curve (y = 0.4982 + 0.0101x, r2 = 0.948) of Gallic acid, a standard phenol, and expressed in micrograms Gallic acid equivalents per milligram of sample (µg GAE/mg sample). The results, as presented in
The total flavonoid contents of the leaf extracts of Ocimum basilicum and Ocimum gratissimum were estimated quantitatively from a linear regression curve (y = 0.0937 + 0.0008x, r2 = 0.954) of Rutin, a standard flavonoid, and presented in microgram Rutin equivalents per milligram of sample (µg RE/mg sample). The results, as presented in
In this assay, the ability of the investigated extracts to act as donors of hydrogen atoms or electrons in transformation of DPPH radical into its reduced form was investigated. The results indicated that Ocimum basilicum extract had a mean scavenging activity of 92.37% which was significantly higher (p < 0.001) than that of Ocimum gratissimum with a mean scavenging activity of 58.43% (
aValues are means of triplicate determinations; bMeans with different superscripts within each column differ significantly (p < 0.01) from each other.
and gallic acid (95.01%) used as reference standards. DPPH scavenging activities of all the tested samples were observed to be dose-dependent, with higher concentrations of each sample showing higher scavenging activities (
The ability of the constituent antioxidant compounds in Ocimum basilicum and Ocimum gratissimum extracts to reduce Mo (VI) ions to Mo (V) was taken as an indication of total antioxidant capacity and results were reported as absorbance values at 695 nm. Ocimum basilicum extract had a mean absorbance value of 0.160 which was significantly higher (p < 0.001) than that of Ocimum gratissimum with a mean value of 0.137 (
aMeans with different superscripts within each column differ significantly (p < 0.001) from one another; bGallic acid was used as a second reference compound for only DPPH assay, while Rutin was used for the remaining assays.
aMeans with different superscripts within each column differ significantly (p < 0.001) from one another; bHydroxyl radical assay was tested at concentrations of 1500, 2000, 2500 and 3000 µg/ml.
Ascorbic acid and rutin used as reference compounds were superior to the extracts in this assay (0.645 and 0.370 respectively). Generally, total antioxidant capacities of all the test samples were dependent on concentration. Higher concentrations of each sample showed significantly higher activity as evident from
The hydroxyl radical scavenging abilities of Ocimum basilicum and Ocimum gratissimum extracts, compared with ascorbic acid and rutin are presented in
The reducing abilities of Ocimum basilicum and Ocimum gratissimum extracts, compared with ascorbic acid and rutin are presented as absorbance values in
Spices and herbs have been mentioned as one of the most important targets to search for natural antioxidants from the point of view of safety [10,18]. The abundant locally consumed spice plants in Nigeria may therefore be potential rich reservoirs of antioxidants to be harnessed if studied and established. The activities of antioxidants have been attributed to various mechanisms including prevention of chain initiation, decomposition of peroxides, radical scavenging and reducing capacity [
Phenols and flavonoids represent two phytochemicals whose relative abundance in plant extracts has been profusely linked to antioxidant activities. Phenol compounds constitute one of the most diverse and widespread groups of natural compounds, possessing a broad spectrum of biological activities including antioxidant and radical scavenging properties [
DPPH radical scavenging assay provides an easy, rapid, and convenient method to evaluate antioxidants and radical scavengers [
nated by an antioxidant compound, the DPPH is decolorized, and this effect can be quantitatively measured from the changes in absorbance [
Reduction of metal ions is an important mechanism of antioxidant action and a potent antioxidant often acts as a potent reductant [
The hydroxyl radical scavenging activities of both spice extracts evaluated in this study and those of the well-studied antioxidant compounds used for comparison were generally low at the concentrations tested. However, it is remarkable that Ocimum basilicum extracts scavenged more of the hydroxyl radicals than the rest of the samples. This may suggest that the extracts may also prevent bio membranes and bio molecules from being attacked by free radicals [
In the reducing power assay, the presence of antioxidants in the samples would result in the reduction of Fe3+ to its lower valence state, Fe2+, by donating an electron. Amount of Fe2+ complex can then be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Increasing absorbance at 700 nm indicates an increase in reductive ability [26,28]. The reducing properties are generally associated with the presence of reducetones which have been shown to exert antioxidant action by breaking the free radical chain through electron donation [
The results of the present study indicate that the spice plants—Ocimum basilicum and Ocimum gratissimum consumed locally in Nigeria possess significantly different but appreciably potent antioxidant activities that cannot be neglected. The study therefore not only reveals the spices as accessible reservoirs of natural antioxidants to be utilized nutritionally and pharmaceutically, but very importantly, provides good scientific justification for increased domestication of these plants.
The authors are grateful to Science Technology Education Post Basic (STEP-B) in Nigeria for the provision of some of the equipment used in this research.