Research Article - (2013) Volume 3, Issue 1
1Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali-3814, Bangladesh
2Faculty of Science, University Brunei Darussalam, Brunei BE141
The present study was conducted to evaluate the possible chemical groups present, total phenolic content, and antioxidant activity of ethanolic extract of leaves of Ocimum sanctum. In the study, presence of tannin and flavonoid were found significant in the crude extract. For investigating the antioxidant activity, three complementary test systems, namely DPPH free radical scavenging, reducing power assay and ferrous ion chelating ability were conducted. In DPPH free radical scavenging test, IC50 value for the crude extract was found fairly significant (7.23±0.05 μg/ml) while compared to that of the reference standards ascorbic acid and BHA (2.12±0.02 & 4.87±0.05 μg/ml respectively). In reducing power assay, the maximum absorbance for the extract was found 2.155 ±0.018 at 100 μg/ml while compared to 2.811 ± 0.013μg/ml and 2.031± 0.019 μg/ml for standard ascorbic acid and BHA respectively. The IC50 value of the extract as percentage of Fe++ ion chelating ability was determined as 19.05±0.08 μg/ml where EDTA showed 8.87±0.035 μg/ml. The total phenolic amount was also calculated as quite high in the extract 279.05±0.73 mg/g of gallic acid equivalent). Therefore, the obtained results tend to suggest potent antioxidant activities of the crude ethanolic extract of the leaves of O. sanctum and justify its folkloric use.
Ocimum sanctum, Antioxidant activity, DPPH, Total phenolic content, Reducing power assay
Ocimum sanctum Linn., commonly known as Holy basil in England and Tulsi in Bangladesh, is a herbaceous plant famous for its significant role in traditional medicine [1]. Recent studies with O. sanctum have suggested that the plant has hypoglycemic [2], adaptogenic[3], anticancer [4], radioprotective[5], analgesic and anti-inflammatory properties [6]. Earlier studies have indicated that leaves of O. sanctum also possess antizygotic, amti-implantation and early abortification effect in women and in experimental animals [7]. Fresh leaves of O. sanctum taken with black pepper are also applied as a prophylactic measure for malaria [8]. Mostly the leaves and sometimes the seeds of O. sanctum are used for medicinal purposes [9].
Antioxidants are molecules which are capable of hindering oxidation by counteracting reactive oxygen species [10]. Reactive oxygen species (ROS) are generated during cellular metabolism. Oxidative stress which is involved in many acute and chronic diseases is mediated by ROS. So the balance between antioxidation and oxidation is believed to be critical for proper maintaining of a healthy biological system [11-13].
Only few studies are presently available that documents its antioxidant potential. In recent times, focus on plant research has increased for the determination of their phenolic concentration and related total antioxidant potential [10, 12, 14]. It is suggested that some medicinal plants contain various types of natural antioxidants, such as phenolic acids, flavonoids and tannins. Therefore, this investigation was conducted to study the antioxidant activities and content of total phenolics of O. sanctum.
Chemicals
In the study, 1,1-Diphenyl-2-picryl hydrazyl (DPPH), Trichloro acetic acid (TCA), L- Ascorbic acid, Butylated Hydroxy Anisole (BHA), Gallic acid, Folin-ciocalteu phenol reagent, phosphate buffer (pH 6.6), Potassium ferricyanide [K3Fe(CN)6] (1%), distilled water, EDTA, Ferrozine, FeCl2 and FeCl3 (0.1%) of analytical grade ( Merck, Darmstadt, Germany) were used for antioxidant activity assay.
Plant material and Extract Preparation
For this present investigation the leaves of Ocimum sanctum were collected by the authors from the surrounding area of Noakhali, a coastal region of Bangladesh, in November, 2010. The plant was identified and authenticated by the expert botanist of Bangladesh National Herbarium, Mirpur, Dhaka (DACB Accession No. 39694).
500 gm of the dried and powdered samples were soaked each in 2500 ml of 80% ethanol (Merck KGaA, Darmstadt, Germany) in clean, sterilized and flat-bottomed glass container. The container with its contents was sealed and kept for a period of 15 days accompanying occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material and Whatman® filter paper no. 1. The resultant filtrate was then evaporated in water bath maintained 45°C to dryness and thus reddish black gummy concentrate of the extract was obtained. The gummy concentrate was designated as crude extract of ethanol.
Preliminary Phytochemical screening
The freshly prepared crude extract was qualitatively tested for the presence of chemical constituents [15]. In each test 10% (w/v) solution of the extract in ethanol was taken.
Determination of Total Phenolic Content
The total phenolic content of the extracts was determined by the modified Folin-Ciocalteu method [16]. Briefly, 0.5 mL of each extract (1 mg/ml) was mixed with 5 ml Folin-Ciocaltu reagent (1:10 v/v distilled water) and 4 ml (75g/L) of Sodium carbonate. The mixture was vortexed for 15 second and allowed to stand for 30 min at 40°C for color development .The absorbance was read at 765 nm with a spectrophotometer (UV-1800, Shimadzu, Japan). Total phenolic content was determined as mg of Gallic acid equivalent per gram using the equation obtained from a standard Gallic acid calibration curve.
DPPH free radical scavenging assay
The stable DPPH radical-scavenging activity was measured using the modified method described by using the modified method described by Chang et al., 2001 [17]. Stock solution (1mg/ml) of the different solvent fractions of ethanol extract of the leaves of O. sanctum prepared in respective solvent systems from which serial dilutions were carried out to obtain the concentrations of 5, 10, 20, 40, 60, 80, 100 μg/ml. In this assay, 2 ml of 0.1 mM ethanolic DPPH solution was added to 2 ml of extract solution at different concentrations and the contents were stirred vigorously for 15 seconds. Then the solutions were allowed to stand at dark place at room temperature for 30 min for reaction to occur. After 30 min, absorbance was measured against a blank at 517 nm with a double beam Analykjena UV-Visible spectrophotometer. The percentage of DPPH free radical-scavenging activity of each plant extract was calculated as:
DPPH free radical-scavenging activity (I %), = [(A0 – A) /A0 ]× 100, Where, A0 is the absorbance of the control solution (containing all reagents except plant extract); A is the absorbance of the DPPH solution containing plant extract.
The DPPH radical-scavenging activity (%) was plotted against the plant extract concentration (μg/ml) to determine the concentration of extract necessary to decrease DPPH radical-scavenging by 50% (called IC50). The IC50 value of each extract was estimated by sigmoid non-linear regression, using SigmaPlot 2000 Demo (SPSS Inc., Chicago, IL, USA). All determinations were performed in triplicate. Ascorbic acid was used as positive control standard.
Reducing power assay
The method of Dehpour et al., [18] was followed to determine the reducing power of O. sanctum leaves of ethanolic extract. 1 ml of extract solution of different concentrations (5, 10, 20, 40, 60, 80, 100 μg/ml) was mixed with 2.5ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassium ferricyanide [K3Fe(CN)6] (1% w/v). The mixture was incubated at 50°C for 20 minutes. The reaction was terminated by adding 2.5 ml of Trichloroacetic acid (10%, w/v), then the mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant solution (2.5 ml) was mixed with distilled water (2.5 ml) and ferric chloride (0.5 ml, 0.1% w/v) solution. Then the absorbance was measured at 700 nm against a blank using UV spectrophotometer. Increased absorbance value of the reaction mixture indicates increased reducing power. Three replicates were made for each test sample and average data was noted. Here, Ascorbic acid and BHA were used as positive control standard.
Ferrous ion chelating ability
The ferrous ions chelating activity of ethanolic extract of O. sanctum and standards were investigated according to the method of Dinis et al., [19]. Briefly, different concentrations of the extract (5-100 μg/ml) were added to 0.1ml solution of 2 mM ferrous chloride (FeCl2). Then, the reaction was initiated by the addition of 0.2ml of 5 mM Ferrozine and mixture was shaken vigorously and kept at room temperature for 10 min. After the mixture had reached equilibrium, the absorbance of the solution was then measured at 562 nm in spectrophotometer, wherein the Fe+2 chelating ability of extracts was monitored by measuring the ferrous ion-Ferrozine complex. The percentage of inhibition of ferrozine-Fe2+ complex formation was given in the following formula.
Ferrous ions chelating ability (%) = [(A0 - A) /A0] × 100
Where A0 is the absorbance of the control solution (containing all reagents except extract); A is the absorbance in the presence of the sample of plant extracts. All the tests were carried out in triplicate and EDTA was used as standard.
Chemical Group Test
Results of different chemical tests on the ethanolic extract of O. sanctum leaves ensured the presence of steroids, alkaloids, saponins, gums and significantly presence of flavonoids and tannins (Table 1).
Table 1: Presence of possible chemical groups
Total Phenolic Content
The amount of total phenolic content was determined as quite high in the ethanolic crude extract of O. sanctum (279.05±0.73 mg/g of gallic acid equivalent) (Table 2). Phytochemical compounds especially phenolic compounds such as flavonoids, phenyl propanoids, phenolic acids, tannins etc. are very important components for the free radical scavenging and antioxidant activities of plants. So, the relationship between phenolic content of medicinal plants and antioxidant activity is well documented [20, 21]. The result of present study revealed that the presence of high concentration of phenolic components in the extract may effectively eliminate radicals and they contribute directly to antioxidant effect of the system [22].
Table 2: Total phenolic content determination of ethanolic extract of leaves of Ocimum sanctum
DPPH Free Radical Scavenging Activity
DPPH free radical scavenging activity of the O. sanctum was found to be increased with the increase of concentration of the extract (Table 3). The extract showed 90.36±0.04 % radical inhibitions at 100 μg/ml whereas at the same concentration the standards ascorbic acid and BHA showed 95.82±0.09 and 93.09±0.06% inhibitions respectively. The IC50 value of extract of O. sanctum was determined as 7.23±0.05 μg/ml where Ascorbic acid & BHA showed 2.12±0.02 and 4.87±0.05 μg/ml respectively showed at table 3. A method which is based on the scavenging of the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) has been used extensively for the prediction of the antioxidant activities of extracts of plants [23, 20]. The high inhibition value of O. sanctum ethanol extract may be due to the presence of phytochemicals such as tannins and flavonoids in the extract. It has been reported that tannins and flavonoids, commonly found in plants have significant antioxidant activity [24].
Table 3: DPPH radical scavenging activity of the ethanolic extract of O. sanctum leaves and standards
Reducing Power Assay
Ascorbic acid and BHA were used as positive control for the determination of reducing power of ethanolic extract of O. sanctum. The maximum absorbance for the ethanolic extract of O. sanctum was found to be 2.155 ±0.018 at 100 μg/ml while that of standard ascorbic acid and BHA was found as 2.8111 and 2.031, respectively at the same concentration (Table 4). With the increase of concentration, the absorbance of the extract and standards were found to be increased gradually. Recently it has been reported that there is a direct correlation between antioxidant capacity and reducing power of certain plant extracts [21]. The reducing properties are generally related with the presence of reductones, which are responsible to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom [22].
Table 4: Reducing power assay of the ethanolic extract of O. sanctum leaves and standards
Fe++ Ion Chelating Ability
Fe++ ion chelating ability of ethanol extract of O. sanctum is shown in table 5. The extract showed 85.89±0.08% Fe++ ion chelating ability at 100μg/ml where as the standard EDTA showed 99.75±0.011% at the same concentration. The IC50 value of the extract was also found significant while compared to the IC50 value of the reference standard EDTA. In this assay, crude extract and standard compounds interfered with the formation of ferrous and ferrozine complex which suggest that they have chelating activity and are capable of capturing ferrous ion before the formation of ferrozine. Iron is responsible to generate free radicals through the Fenton and Haber– Weiss reaction. But metal ion chelating activity of an antioxidant compound retard this oxyradical generation and the consequent oxidative damage. So, metal ion chelating capacity plays a significant role in antioxidant mechanism since it reduces the concentration of the catalysing transition metal [25].
Table 5: Fe2+ ion chelating ability of ethanol extract of O. sanctum leaves and EDTA (Standard)
In conclusion it can be revealed that the crude ethanolic extract of O. sanctum leaves contain significant antioxidant activities. The significant potency of the extract of O. sanctum as antioxidant agents may be due to the presence of phytochemicals like tannins, flavonoids, phenolics etc and might be responsible for its activity and justify its use as a traditional folk remedy for toothache. However, extensive researches are required to search for active principles responsible for these activities.
The authors are thankful to BNH to identify the plant. The authors are also grateful to all the teachers of Dept. of Pharmacy, Noakhali Science and Technology University for their cooperation.
