Phytochemical Screening and Evaluation of the Antioxidant Activity of Ficus capreifolia Leaf Extract via in-vitro Models

Aims: To carry out preliminary phytochemical screening and determine the antioxidant ability of Ficus capreifolia leaf extract. Study radicals and also reduced ferrous ions significantly when compared to vitamin C. Conclusion: The leaf extract exhibited significant antioxidant activities which could be ascribed to antioxidant phytochemicals such as flavonoids, polyphenols which were detected. This corroborates the reported traditional use of the plant and can be further exploited pharmacologically.


INTRODUCTION
The utilization of oxygen is key to aerobic metabolism however it is a paradox that reactive oxygen species (ROS) are responsible for the pathogenesis of many disorders [1]. These ROS (both free radicals and other non-radical species) damage membrane lipids, cause DNA mutations and protein damage which could be sporadic. Metabolically, the body has efficient mechanisms to mask, scavenge or dismutate these ROS. The production of these ROS is in fact indispensable for life hence there is a balance between ROS production and antioxidation. Should an imbalance occur (in favour of ROS production), a condition known as oxidative stress occurs and the consequences could be cancer, aging, cardiovascular disease, liver injury etc [2][3][4]. Plants are rich sources of potential antioxidant compounds hence the intake of diets rich in plants (especially fruits, cereals, vegetables) has been encouraged.  [5]. The roots and leaves of the plant have been used to treat Schistosomal infections and diabetes [6]. Despite being of potential medicinal value, the plant is grossly underutilized coupled with the limited literature available on studies of the plant. Based on this, this work investigates the antioxidant potential of the Ficus capreifolia leaves using some in vitro models. The leaves were also screened for the presence of some phytochemicals.

Chemicals and Reagents
All chemicals and reagents were of analytical grade thus used without further purification. All buffers and solutions were prepared using deionized water and used within 24 hrs.

Plant Collection and Identification
The leaves of Ficus capreifolia were harvested from its natural environment in Oto-Udu, Udu

Preparation of Extract
Fresh leaves of Ficus caprecfolia were properly washed with deionized water to remove dirt. The leaves were sundried and pulverized using a warring blender. The extraction was done using Batch Extraction Method with methanol as the extracting solvent. The powdered sample (200 g) was soaked in 1 L of absolute methanol for 72 hours. The supernatant was collected and filtered using Whatman filter paper and concentrated using a rotary evaporator set at 40°C to give a residue. Different concentrations (0.25, 0.50, 1.00 and 2.00 mg/ml) of the extract were prepared and subjected to further analysis.

Preliminary Phytochemical Analysis
The extract was screened for the presence of alkaloids, flavonoids, polyphenols, saponins, steroids, tannins, cardiac glycosides and carotenoids according to the methods of Bera et al. [4] and Edeoga et al. [7].

Hydrogen Peroxide Scavenging Activity
Hydrogen peroxide scavenging activity was performed by replacement titration as reported by Zhao et al. [8] with some modifications. Briefly 1 ml of hydrogen peroxide (0.1 M), 1 ml of the extract, 3 ml of ammonium molydate (3%), 10 ml of 2 M sulphuric acid and 7 ml of potassium iodide (1.8 M) were introduced into a conical flask. This was then titrated against sodium thiosulphate of (5.09 M). Disappearance of the yellow colour indicated end point of the reaction. The hydrogen peroxide scavenging activity of was expressed as a percentage thus:

Hydroxyl Radicals Scavenging Activity
Hydroxyl radical scavenging activity was investigated using the method described by Bera et al. [4]. Briefly, 1 ml of phosphate buffer (0.2 M, pH 7.2), 1 ml of test solution, 0.02 ml of ferric chloride (0.02 M) and 0.05 ml of phenanthroline (0.04 M) were introduce into a test tube. Reaction was initiated by the addition of 0.05 ml of 7 mM hydrogen peroxide. After 5 min of incubation at room temperature, absorbance was measured at 560 nm using a UV spectrophotometer. Hydroxyl radical scavenging activity was expressed as relative scavenging activity and calculated using the relation:

Reducing Ability
The ability of the extract to reduce Fe 3+ was investigated according to Oyaizu [9] as modified by Okoko and Ere [10]. Test solution (0.5 ml) was mixed with 0.5 ml of phosphate buffer (0.2M pH 6.6) and 0.5 ml of potassium ferricyanide (1%) and incubated at 50°C. After incubation for 20 minutes, 0.5 ml of trichloroacetic acid (10%) was added and centrifuged for 10 minutes at 3000 rpm. A portion of the upper layer (0.5 ml) was mixed with 0.5 ml distilled water and 0.1 ml ferric chloride (0.1%). After 10 min incubation at room temperature, absorbance was measure at 700 nm. Increase in absorbance indicated greater reducing ability.

Statistical Analysis
Representative readings were expressed as mean ± S.E from four replicates. Data were analyzed using either a student's t-test (for comparison between two groups) or analysis of variance (ANOVA) for multiple comparisons. Confidence exhibited at p < 0.05 was considered statistically significant. Vitamin C was used as the reference control for the antioxidant assessment.

RESULTS
Phytochemical analysis of the Ficus capreifolia (leaves) revealed the presence of tannins, carotenoids, alkaloids, steroids, polyphenols, flavonoids and cardiac glycosides. However, saponins were not detected (Table 1). The hydrogen peroxide scavenging ability, hydroxyl radical scavenging ability and the ability of the extract to reduce ferric ion are shown in Figs. 1, 2 and 3. The extract possessed significant ability to scavenge hydrogen peroxide, hydroxyl radical and to reduce ferric ions when compared to the reference compound (vitamin C). Though the responses seem to be concentration-dependent in all cases, the variations were not significant in many cases. The variation of hydrogen peroxide scavenging ability between 0.25 mg/ml and 0.5 mg/ml was significant (p < 0.05) while it was not significant (p > 0.05) from 0.5 mg/ml to 2.0 mg/ml. For the hydroxyl radical scavenging activity, there was no statistical difference among the various concentrations (p > 0.05). For the reducing ability, the difference between 0.25 mg/ml and 0.5 mg/ml was significant (p < 0.05), not significant between 0.5 mg/ml and 1.0 mg/ml but significant difference (p < 0.05) was exhibited between 1.0 mg/ml and 2.0 mg/ml.

DISCUSSION
The production of ROS and antioxidation are not mutually exclusive as long as life exists. Various physical and chemical processes induce and activate oxidant producing enzymes such as NADPH oxidase, xanthine oxidase etc. These enzymatic reactions produce ROS such as superoxide radical, nitric oxide, hydrogen peroxide, peroxynitrates [11]. The body has efficient mechanisms (both enzymatic and nonenzymatic) to keep these reactive species in check since they are products of essential processes. However, these ROS could be over produced due to a number of factors and the consequences are damage to tissues, DNA, proteins which are predisposing to cancer, cardiovascular diseases, diabetes, ageing, neuro degradative disorders, stroke, gastrointestinal diseases, etc [12][13][14][15]. Thus antioxidant supplementation is necessary to maintain redox balance. Plants are the main sources of these antioxidants.
Phytonutrients have been recommended to be effective substitutes for most synthetic products since there are reported lessfrequent side effects following the consumption of plants or plant-derived products [16]. Hydrogen peroxide is a potent oxidant produced via phagocytosis and in reactions catalyzed by xanthine oxidase, superoxide dismutase [17]. Though converted to water by catalase or gluthathione peroxidase, overproduction damages various cellular targets due to its diffusible nature [17][18][19].
The study revealed that Ficus capreifolia leaf scavenged hydrogen peroxide which was significant when compared to vitamin C (Fig. 1).
The hydroxyl radical is the most harmful in the biosystem because of its non-selective reactivity [20][21]. In the presence of transition metal ions (such as Fe 3+ and Cu 2+ ), hydrogen peroxide could generate hydroxyl radical via the Fenton reaction [22]. Thus the reduction of these redox active transition metal ions and the scavenging of the hydroxyl radical are indispensible at reducing free radical induced damage. Ficus capreifolia extract significantly scavenged hydroxyl radical and also reduced ferric ions. This ability could be due to important phytochemicals inherent in the plant. Preliminary phytochemical screening revealed the presence of tannins, carotenoids, alkaloids, steroids, polyphenols, flavonoids and cardiac glycosides (Table 1). Flavonoids and other polyphenols have been reported to possess immense antioxidant potential by scavenging hydroxyl radicals, hydrogen peroxide and also possess metal reducing abilities [23][24][25][26]. Thus the intake of diet rich in flavonoids (fruits and vegetables) has been inversely associated with the development of coronary heart disease, cancer, cerebrovascular disease etc [27]. In addition to polyphenols, the antioxidant activities of alkaloids, tannins and carotenoids have also been reported [28][29][30][31][32]. Thus the observed in-vitro antioxidant activity could be due to synergy exhibited by the various phytochemicals. However, this is yet to be investigated.

CONCLUSION
Ficus capreifolia extract exhibited high in-vitro antioxidant and free radical scavenging potentials when compared to vitamin C so could be exploited both pharmacologically and neutraceutically. The various in-vitro models indicate that Ficus capreifolia extract is a significant source of natural antioxidants, which could be helpful in preventing the progress of various diseases due to oxidative stress. Followup studies using various in-vivo models are recommended.