1. Introduction

Our body is constantly exposed to a variety of oxidizing agents and the body is equally inbuilt with antioxidants to cater for the free radicals generated from the oxidants thus maintaining a balance between the production of free radicals and neutralization by antioxidants. When there is imbalance between formation and neutralization of free radicals by antioxidants, it results to oxidative stress[1-3]. Oxidative stress has been implicated in the etiology of diseases such as cardiovascular diseases and lung cancer[4-7]. Epidemiological studies have shown that regular consumption of fruits and vegetables reduces the risk incidence of chronic diseases.[8,9]. The protection that fruits and vegetables provide against diseases has been attributed to the various antioxidants contained in them. They are good sources of natural antioxidants which include carotenoids, vitamins, phenolic compounds, flavonoids, dietary glutathione, and endogenous metabolites and have been shown to scavenge singlet and triplet oxygen, free radicals, enzyme inhibitors and decompose peroxides[10,11]. Phenolic compounds are secondary metabolites in fruits and vegetables. They have been reported to exhibit antioxidant activity which allows them to scavenge both active oxygen species and electrophiles, to inhibit nitrosation and to chelate metal ions, to have the potential for autoxidation and the capability to modulate certain cellular enzyme activities[12-14]. Flavonoids, a class of phenolic compounds has been shown to possess anti-inflammatory, antiviral, anticarcinogenic, antithrombotic, antiallergic and hepatoprotective[15]. Carotenes have been proved to possess antioxidant activity due to their ability to quench singlet oxygen and inhibit lipid peroxidation[16]. Thus, diets rich in fruits and vegetables are believed to play an important role in preventing diseases but human choices of diet are driven by necessity and economy. Lack of knowledge on the importance of good diet and prevalence of poverty has influenced the composition of diets taken by Nigerians. Coupled with this, the exposure to different kinds of toxic substances such as smoke from generators, second-hand vehicles and pesticide which are capable of inducing oxidative stress through production of free radicals. Thus, this study was set to determine the antioxidant activity and phytochemical contents of commonly eaten fruits and vegetables and to determine the difference in antioxidants components due to ripeness.

2. Materials and Methods

2.1. Sampling Procedure

Ten fruits and vegetables (Table 1) used in this study were brought from various markets in Oshogbo and identified by Mrs. F.M Tairu at National Horticultural Research Institute (NIHORT), Ibadan.

Table 1. Botanical and common/ local names of plants used in this study Botanical names Vernacular/ Common names Zingiber officinale Ginger Allium sativum Ayu/Garlic Cucumis sativus Cucumber Lactuca sativa Lettuce Lycopersicon esculentum Tomati/Tomatoes (ripe) Lycopersicon esculentum Tomati/Tomatoes (unripe) Capsicum frutescens Sombo/Bellpepper (ripe) Capsicum frutescens Sombo/Bellpepper (unripe) Daucus carota Carrot Capsicum annuum Green pepper

2.1.1. Standards and Reagents

Standards: BHA (butylatedhydroxyanisol), Quercetin, Folin-ciocalteu’s phenol, DPPH (2,2-diphenyl-1-picrylhydrazyl), were all purchased from Sigma-Aldrich, Germany. Sodium carbonate, Aluminum chloride and Methanol were purchased from BDH Poole, England. All the chemicals used were of analytical grade. Deionized-Distilled water was used throughout the experiment. Jenway 6405 UV-Visible Spectrophotometer by Buch Scientific Inc.USA was used for analysis.

2.2. Extraction

The samples were rinsed with distilled water to remove sand, cut into pieces and lyophilized to remove the moisture content. Resulting dried samples were powdered using Moulinex blender. These ground samples were extracted twice with a total volume of 100 ml of 70% aqueous methanol. The mixture was shaken on an orbital shaker for 75 min at 250rpm and then filtered through Whatman No 1 filter paper. The combined methanolic extract was then evaporated at 55^oC using water bath and dried to powder in a lyophilizer.

2.3. Phytochemical Screening

2.3.1. Determination of Total Phenols by Folin-Ciocalteu Method

Total phenol content in the sample was determined using Folin-Ciocalteu method of Olajire and Azeez (2011)[17]. 0.5ml of the methanolic extract was added to 10ml distilled water and 2.5ml of 0.2N Folin-Ciocalteu phenol reagent. The mixture was allowed to stand at room temperature for 5min and then 2ml of 2% of sodium carbonate was added. The resulting solution was measured at 780nm. Quercetin was used as standard for the calibration curve.

2.3.2. Determination of Flavonoid Concentration

The AlCl₃ method of Jagadish et al, 2009[18] was used for determination of the flavonoid content of the sample extract. 1.5ml of extract was added to 1.5ml of 2% methanolic AlCl₃ solution. The mixture was vigorously shaken on orbital shaker for 5min at 200rpm and the absorbance was read at 367nm after 10min of incubation. Reagent blank using distilled water instead of sample was prepared. Quercetin was used as standard for the calibration curve.

2.3.3. Determination of β- Carotene and Lycopene

β- Carotene and Lycopene were determined according to the method of Nagata and Yamashita (1992)[19]. The dried methanolic extract (100mg) was vigorously shaken with 10ml of acetone – hexane mixture (4:6) for 1min. The absorbance of the filtrate was measured at λ = 453, 505, 645 and 663 nm. Contents of β- Carotene and Lycopene were calculated according to the following equations: lycopene (mg/100ml) = - 0.0458A₆₆₃ + 0.372A₅₀₅ + 0.0806A_453: β- Carotene (mg/100ml) = 0.216A₆₆₃ – 0.304A₅₀₅ + 0.452A_453. The values are expressed as µg/g of extract.

2.4. DPPH Radical Scavenging Capacity Assay

1ml of methanolic solution of the extract was added to 4ml of 0.1mmoll^-1 methanolic solution of DPPH. After 30min incubation in the dark at room temperature, the absorbance was read against a blank at 517nm. Inhibition of free radicals by DPPH in percent () was calculated using this formula:

(1)

Where is the absorbance of the control reaction and is the absorbance of the test compound. Quercetin and BHA were used as standard controls.

2.5. Statistical Analysis

All results are expressed as mean ± standard deviation. All results are means of three replicates. The data were correlated using Pearson correlation coefficient at p <0.05. IC₅₀ was calculated using linear regression analysis. SPSS 15 version was used for the statistical analysis.

3. Results and Discussion

3.1. Results

Table 2 presents the results obtained for antioxidant activity and IC₅₀ of ten fruits and vegetables used. Figures 1, 2, 3 and 4 present phenolic, flavonoid, lycopene and β-carotene contents of fruits and vegetables analyzed.

3.1.1. Antioxidant Activity

Antioxidants activity ranged from 4.32±0.02% for Allium sativum to 92.62±0.98% for Lactuca sativa. The decreasing order of antioxidant activity is Lactuca sativa > Zingiber officinale > Capsicum frutescens (ripe) > Capsicum frutescens (unripe) > Capsicum annuum > Lycopersicon esculentum (unripe) > Lycopersicon esculentum(ripe) > Cucumis sativus > Daucus carota > Allium sativum. IC₅₀ which is the inhibitory concentration at which 50% of free radicals are scavenged ranged from 0.26mg/ml for Lactuca sativa to 10.24mg/ml for Allium sativum. The IC₅₀ of Lactuca sativa (0.26mg/ml), Zingiber officinale (0.29mg/ml), ripe Capsicum frutescens (0.67mg/ml), and unripe Capsicum frutescens (0.74mg/ml) compared to standards: Quercetin (IC₅₀ = 0.83mg/ml), and BHA (IC₅₀ = 0.96mg/ml) are lower. Antioxidants activity in these samples poorly correlates with phenolic contents (r² = 0.31, p< 0.05), β-carotene (r² = 0.38, p< 0.05) and lycopene (r² = 0.05, p< 0.05) but significantly with flavonoid (r² = 0.63, p< 0.05). There was an increase in antioxidants activity increase due to ripeness in Capsicum frutescens but a decrease was obtained for Lycopersicon esculentum

Figure 1. Total phenolic content of fruits and vegetables studied

Table 2. Antioxidant activity (AA), flavonoid, phenolics, lycopene, β- Carotene, and the IC50 of the fruits and vegetable studied

3.1.2. Phytochemical Contents

Phenolic contents of fruits and vegetables as shown in figure 1 ranged from 106.67±11.55mg quercetin/g of extract for Capsicum annuum to 360.01±40.01mg quercetin/g of extract for Lycopersicon esculentum (unripe) while flavonoid contents (figure 2) ranged from 64.06±3.46mg quercetin/g of extract for Daucus carota to 482.29±1.73mg quercetin/g of extract for Zingiber officinale. Ripe Lycopersicon esculentum and Capsicum frutescens have higher phenolic and flavonoid values than unripe

Figure 2. Flavonoid contents of fruits and vegetables analyzed

Zingiber officinale had the highest content of β-carotene (66.30±1.45µg/g of extract) and Allium sativum had the lowest (2.37±0.27µg/g of extract). Ripe Lycopersicon esculentum (22.73±3.44µg/g of extract) had the highest lycopene and Allium sativum had the lowest (1.23±0.25µg/g of extract). There was an increase in the contents of lycopene and β-carotene due to ripeness in Lycopersicon esculentum but a decrease was obtained for Capsicum frutescens.

Figure 3. Lycopene contents of fruits and vegetables studied

Figure 4. β-carotene contents of fruits and vegetables analyzed

3.2. Discussion

3.2.1. DPPH Antioxidants

The electron donating ability of fruits and vegetables is a suitable parameter to establish the possession of oxidative stress quenching ability and their health studies have shown that Zingiber officinale, Lactuca sativa and Capsicum frutescens are good sources of antioxidants[20-22]. IC₅₀ of these vegetables and fruits show that they can scavenge more free radicals than BHA and quercetin which are standards. Zingiber officinale as obtained in this study has also been reported to inhibit better than quercetin[21]. Antioxidants capacity as found in Capsicum frutescens increased in ripe fruit and was in agreement with[20] who reported that antioxidant capacity increases with ripeness while our results for ripe and unripe Lycopersicon esculentum with decrease in antioxidant capacity due to ripeness was also in agreement with what was obtained by[23] for tomatoes with different degrees of ripeness. Though, high correlations have often been observed between phenolics and antioxidant capacity, our esults indicate that flavonoids which are a group in phenolic compounds had highest correlation coefficient with antioxidants capacity.

3.2.2. Phytochemical Contents

Phenolics and flavonoids have been shown to contribute significant to antioxidant ability of fruits and vegetables[20]. Especially flavonoids, have been reported to be anticancer, anti-inflammatory, antifugi, antimicrobial, antibacterial and antiviral[15]. High phenolic and flavonoid contents obtained in this study for some vegetables show that they could serve as nutritional sources for anticancer, antiviral and anti-inflammatory. Our results indicate that phenolic and flavonoid contents increased in Lycopersicon esculentum and Capsicum frutescens due to ripeness which were what Lee[24] obtained. Howard[25] also observed that flavonoid contents increased with ripeness. Our results were equally in agreement with Riadh[23] for an increase in phenolic contents due to ripeness.

Lycopene and β- Carotene possess the ability to scavenge singlet oxygen. Consumption lycopene and β- Carotene have been reported to be inverse with incidence of cancer[16]. Results obtained show that some of these vegetable contain high levels of lycopene and β- Carotene and an increase was observed for both contents in Lycopersicon esculentum due to ripeness while otherwise was observed for Capsicum frutescens. Our results are in agreement with AbdulHammed[26] and Riadh[23] who obtained increased amount of β- Carotene and lycopene contents in the ripe than the unripe tomato.

4. Conclusions

The antioxidant activities, total phenol, flavonoid, β- Carotene, and lycopene contents of ten fruits and vegetables commonly consumed in Nigeria were assessed. Some of the vegetables can be considered as good sources of natural antioxidants since their extract were found to possess high antioxidant activity. This study shows that consuming ripe Capsicum frutescens and Lycopersicon esculentum would add more nutritional benefits to foods as the phytochemical contents increased with ripeness.