Pineapple (Ananas cormosus) and orange (Citrus sinensis) fruits were harvested from their trees in August, 2014 from Ugheli in Edo state of Nigeria and Kubwa town in the FCT, Abuja, Nigeria, respectively. Based on visual observations of color, texture and flavor, the fruits were categorized into unripe (UR), about to ripe (AR) and ripe (RP). The peel of the fruits was removed and the fruits were sliced horizontally into halves with a sharp knife and cut into small sizes. The categorized and sliced fruit pulps were taken to the medicinal Department of National Institute of Pharmaceutical Research and Development (NIPRD) Idu, FCT- Abuja for further treatments and analyses.

The categorized and sliced fruits were weighed, homogenized using a domestic blender, filtered using a cheese cloth and freeze-dried. The freeze-dried samples were stored in a sample bottle and used for analysis.

Chemicals of analytical grade (British Drug House) were used for the analysis. These include: Folin-Ciocalteu’s phenol reagent, gallic acid, aluminium chloride, potassium acetate, 70 % aqueous methanol, 1, 1-diphenyl-2-picryhydrazyl (DPPH), ascorbic acid, rutin, absolute ethanol, sulphuric acid, sodium phosphate and ammonium molybdate.

The total phenolic content was measured using Folin-Ciocalteu reagent as described by Jagadish et al. [15]. 4.0 mg of each freeze-dried categorized sample was dissolved in 4.0 ml of 70 % aqueous methanol. 0.6 ml of the methanoic extracts was mixed with 0.5 ml of 1:10 dilution of Folin-Ciocalteu phenol reagents and 0.4 ml of 0.7 M Na₂CO₃ solution. The solution was incubated for 10 min at a constant temperature of 100 °C in a water bath. After incubation, the solution was ice cooled and diluted with 1.5 ml distilled water and measured at an absorbance of 760 nm. The total polyphenol in the fruits was obtained from a standard curve of gallic acid ranging from 0.001 to 0.5 mg/ml (Pearson’s correlation coefficient: r²=0.9250). Total phenol content was expressed as milligram of gallic acid equivalent (GAE) / g weight of each categorized fruit pulp tissue.

The total flavonoid was spectro-photometically determined as described by Grant [2]. 1.0 mg/ml methanoic extract of each categorized sample was mixed with 10 % 0.1 ml aluminium chloride; 0.1 ml of 1.0 M potassium acetate and 2.8 ml distilled water was added. The solution was vortexed and allowed to sit for 40 min at 25°C and the absorbance measured at 415 nm. The total flavonoid content of the fruits was quantified based on the standard curve prepared for rutin at concentrations of 0.004 to 1.0 mg/ml (Pearson’s correlation coefficient: r²=0.982). The total flavonoid was expressed as milligram of rutin equivalent/g weight of each categorized fruit pulp tissue.

A spectro-photometric method developed by Prior et al. [16] was used to determine the total lipid antioxidant capacity of the fruits. 0.2 ml of 1.0 mg/ml of each freeze-dried categorized sample was dissolved in 0.2 ml absolute ethanol. The ethanoic extract was mixed with 1.0 ml of phosphomolybdenium reagent (32 mM sodium phosphate + 4 mM ammonium molybdate and 0.6 M H₂SO₄). The mixture was homogenized and incubated at 95°C for 90 min and the absorbance measured at 695 nm. CALT is expressed as equivalents of α-tocophenol. A standard curve was constructed with different amounts of α-tocophenol dissolved in ethanol. An extinction coefficient (E) of 137 Nm^-1 (r²=0.9998) was used for quantification.

CALT (mmol α-tocophenol/g fruit pulp tissue) = A₆₉₅ E^-1 ERV x SV^-1 x EV x M^-1

Where: A₆₉₅ is the absorbance at 695 nm.

• E^-1 is the inverse of the extinction coefficient.
• RV is the overall reaction volume.
• SV is the sample volume used in the reaction.
• EV is the volume of solvent used in the extraction of the plant material analyzed.
• M is the amount (grams) of fresh plant material.

The scavenging ability of the samples on the stable free radical DPPH was evaluated using the method described by Grant [2]. 20.0 mg of each freeze-dried categorized samples was added to 0.078 mg/ml methanol solution of DPPH. The mixture was allowed to stand without light for 30 min and the absorbance was measured at 517 nm. The DPPH radical scavenging effect was expressed as a percent inhibition of the DPPH radical and determined according to the equation bellow:

Ascorbic acid solution was used as a positive control for antioxidant activity.

Data were expressed as means ± SD of three independent experiments. A probability P < 0.05 was considered significant. Coefficient of variation in % was also used.

The total phenolic contents are essential in the assay of antioxidant capacity of fruits and vegetables [7, 17]. Recent findings also suggest the role of polyphenolic compounds as protective agents against cardiovascular disease and breast, gastrointestinal and skin cancers due to their oxidative/antioxidative potential or their ability to act as free radical scavengers [18]. Table 1 shows the mean total phenolic concentrations of unripe (UR), about to ripe (AR) and ripe (RP) pineapple and orange fruit pulps. One milligram of the unripe (UR) fruit pulps contain concentrations ranging from 0.49 to 0.66 mg of gallic acid equivalents of phenolic compounds, about to ripe (AR) ranged from 0.36 to 0.63 mg of gallic acid equivalents of phenolic compounds and ripe (RP) ranged from 0.45 to 0.64 mg of gallic acid equivalents of phenolic compounds. These concentrations of polyphenols can play an important role in antioxidant ability of these fruits. This is because it has been suggested that up to 1 g of polyphenolic compounds (from a diet rich in fruits and vegetables ingested daily has inhibitory effects on mutagenesis and carcinogenesis in humans [7, 19]. There is no significant difference at P < 0.05 in the total phenolic content of the fruits though the polyphenols in orange pulp is greater than that in pineapple at all the levels of ripeness examined. The result showed that the total phenolic content of UR ≥ RP > AR for both pineapple and orange pulp extracts suggesting that as the fruit pulps ripens, the total polyphenols decreased. The coefficient of variation ranged from 3.13 % in orange to 16.28 % in pineapple.

Flavonoids are secondary metabolites which have been found to have antioxidant, antibacterial and antimicrobial properties [20-22]. Table shows the mean total flavonoid concentration values for unripe (UR), about to ripe (AR) and ripe (RP) pineapple and orange fruit pulps. The amount of total flavonoids (mg rutin/g tissue) for UR pineapple and orange was 0.03 and 0.06 respectively. AR (0.01 & 0.04) and RP (0.03 & 0.06) for pineapple and orange fruit pulps respectively. At P < 0.05, the total flavonoids content of the fruit pulps were significantly affected by their level of ripeness. The result showed that the fruits at different maturation stages are good sources of flavonoids and that the flavonoid content of the unripe fruit was highest, decreased when the fruit was about to ripe and increased again when the fruit was ripe. The coefficient of variation ranged from 16.60 % in orange to 50 % in pineapple.

The lipid-soluble antioxidant capacities of the fruits were studied using α-tocophenol as a standard. Table 1 shows the mean lipid-soluble antioxidant capacity of UR, AR and UR fruit pulps. There is no significant difference at P < 0.05 in the lipid soluble antioxidant capacity of the fruits at different levels of ripeness.The lipid soluble antioxidant capacity of the fruits decreased as the fruit ripened. This suggests that the fruits are likely to contain more water-soluble antioxidants as they ripen.

The scavenging ability of an antioxidant is measured by its ability to quench the stable DPPH radical with maximum absorption at 517 nm [2, 23]. Table 2 shows the DPPH free radical scavenging ability of pineapple and orange fruit pulps at different levels of ripeness. The results showed that pineapple and orange fruit pulps are good radical scavengers at different levels of ripeness. The unripe (UR) fruit pulp has the highest scavenging activities (65.80 % and 86.33 %) respectively for pineapple and orange fruit pulps. This was followed by RP with % reduction of 63.93 for pineapple and 77.77 for orange. AR had the least % reduction of 61.17 for pineapple and 76.33 for orange. The radical scavenging ability of the fruit pulps was significantly affected (P < 0.05) by their level of ripeness. The radical scavenging capacity of the fruit pulps examined decreased with ripening.