2.2. DPPH Radical Scavenging Activities

DPPH radical is an oil-soluble free radical that becomes a stable product after accepting an electron or hydrogen from an antioxidant. DPPH radical is stable in methanol and show maximum absorbance at 517 nm. When DPPH encounters a proton-donating substance such as an antioxidant, the radical would be scavenged and the absorbance is reduced. Hence the antioxidant activity of the substance can be expressed as its ability in scavenging the DPPH radical. Figure 1 shows results of ability of APH and its membrane fractions to scavenge DPPH radical. GSH had the highest DPPH scavenging activity (42.29%) when compared to those of APH and its membrane fractions. There was no significant difference (p > 0.05) between the DPPH scavenging activities of <1 kDa and <3 kDa peptides. The result indicates that the DPPH radical Scavenging activities of the peptides are molecular size dependent. The lower -molecular weight (LMW) peptides, especially <1 kDa exhibited better DPPH radical scavenging activities than the high-molecular weight (HMW), especially the 3–5 kDa and 5–10 kDa peptides. This result is in agreement with the previous results reported by Girgih et al. [3] and also by Aluko and Monu [25], which showed that LMW peptide fractions had higher DPPH scavenging activities than HMW peptides. Increased hydrophobic character of peptides derived from protein sources have been shown to correlate with higher DPPH or other radical scavenging activities [4,7] when compared with peptide fractions of lower hydrophobic content. The highest DPPH radical scavenging activities that was found in the <1 kDa fraction may be due to its having the highest contents of total HAA and AAA. Kim et al. [26] reported that hydrophobic amino acids act as antioxidants by increasing the solubility of peptides in non-polar environments thereby facilitating better interaction with free radicals in order to terminate their activities. The results suggest that the high DPPH scavenging properties of LMW peptides could make them useful ingredients to that can be used to prevent oxidative deterioration of foods.

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Figure 1

DPPH radical scavenging activities of African yam bean seed protein hydrolysate (APH) and its ultrafiltration membrane fractions.

2.3. Superoxide Radical Scavenging Activities

Superoxide radicals are generated by a number of biological reactions. Although superoxide radicals do not directly initiate lipid oxidation, they could promote oxidative reactions due to its ability to reduce transition metals, release protein-bond metals and form perhydroxyl radicals which initiate lipid oxidation [27]. For cytoprotection against this reactive oxygen, superoxide dismutase (SOD), which catalyzes the neutralization of superoxide anion to hydrogen peroxide, is one of the defense mechanisms in the living cell. Not only superoxide anion radicals but also their derivatives are celldamaging, which can cause damages to the DNA and cell membrane lipids [28]. Therefore, it is of great importance to scavenge superoxide anion radicals. The results of superoxide scavenging activities of APH and its membrane fractions are shown in Figure 2. GSH was significantly (p < 0.05) a more effective superoxide scavenger than the APH and peptide fractions. The results indicate that APH and its membrane fractions displayed moderate superoxide scavenging activity with the <3 kDa exhibited the significantly strongest (p < 0.05) superoxide activity of 43% and the APH exhibited the lowest superoxide scavenging activity of 31.3%. These scavenging activities are similar to those reported for chickpea protein hydrolysate (35–69%) [7] at 2.0 mg/mL peptide concentration while the present results were obtained at 1 mg/mL peptide concentration. In addition, Li et al. [7] also reported that a low molecular weight fraction from chickpea protein hydrolysate with strong superoxide radical scavenging activity was observed to have higher concentrations of Phe, Ile, Leu and Val in comparison to other fractions and it was therefore suggested that the superoxide scavenging activity was related to the hydrophobic amino acid. However, in the present work, there was no strong relationship between hydrophobic amino acid content and the superoxide scavenging activity of the peptide fractions. The results suggest that the peptides may not be as effective as GSH for the inhibition of superoxideinduced damage to cells. However, depending on degree of bioavailability, the peptides could offer some level of protection to cells against the toxic effects of superoxide free radicals.

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Figure 2

Superoxide radical scavenging activities of African yam bean seed protein hydrolysate (APH) and its ultrafiltration membrane fractions.

2.4. Hydroxyl Radical Scavenging Activities

Among the oxygen radicals specifically, the hydroxyl radical is the most reactive specie and can be formed from superoxide anion and hydrogen peroxide, in the presence of metal ions, such as copper or iron. When a hydroxyl radical reacts with aromatic compounds, it can add onto a double bond, resulting in hydroxycyclohexadienyl radical. The resulting radical can undergo further reactions, such as reaction with oxygen, to give peroxyl radical, or decompose to phenoxyl-type radical by eliminating water [29]. Hydroxyl radical could severely damage adjacent biomolecules such as all proteins, DNA, polyunsaturated fatty acids, nucleic acids and almost all biological molecules that it comes in contact with. These hydroxyl radical-mediated damages can lead to premature aging, and the development of cancer and several diseases [5]. Therefore, removal of hydroxyl radical is one of the most effective defenses of a living body against various diseases. Hydroxyl radical scavenging activity of APH and its membrane fractions is shown in Figure 3. The result shows that <1 kDa displayed significantly strongest (p < 0.05) hydroxyl radical scavenging activity of 28.21% when compared to 1–3 kDa, 3–5 kDa, 5–10 kDa and APH. APH and its membrane fractions exhibited significantly lower (p < 0.05) hydroxyl radical activity when compared to that of GSH. Thus, GSH will likely provide better protection (when compared to peptides) to cells against damage by hydroxyl radicals. Since some degree of scavenging was obtained for the peptides, they have the potential to be developed as hydroxyl radical scavengers within cells. A number of studies have demonstrated a good correlation between certain amino acids residue or short peptides with radical scavenging activity of protein hydrolysates or peptides. Silver carp protein hydrolysates derived from alcalase was reported to possess stronger hydroxyl radical scavenging activity and contained higher concentrations of hydrophobic amino acids in comparison to a flavourzyme hydrolysate [30]. Li et al. [7] reported that a lower molecular weight fraction from chickpea protein hydrolysate with strong hydroxyl radical scavenging activity was observed to have higher concentration of hydrophobic amino acid, a result that is similar to the present result. Pownall et al. [4] also reported a strong correlation between the hydroxyl radical scavenging activity and total percentage of hydrophobic acid of the pea protein hydrolysate factions. Thus, the small peptide size in conjunction with high contents of HAA and AAA may have contributed to the superior hydroxyl radical scavenging property of the <1 kDa peptides when compared to the other peptide fractions with >1 kDa peptide size.

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Figure 3

Hydroxyl radical scavenging activities of African yam bean seed protein hydrolysate (APH) and its ultrafiltration membrane fractions.

2.5. Metal Chelating Activity

Transition metal ions, such as Fe²⁺ and Cu²⁺ can catalyze the generation of reactive oxygen species which accelerates lipid oxidation. Fe²⁺ can also catalyze the Haber-Weiss reaction and induce superoxide anions to form more hazardous hydroxyl radicals. These hydroxyl radicals react with adjacent biomolecules to cause severe tissue damage [13], especially lipid oxidation. Therefore, the chelation of transition metal ions by antioxidative peptides could retard the oxidation reaction. Figure 4 shows the ability of APH and its membrane faction to chelate the transition metal ion, Fe²⁺. APH and its membrane fractions displayed similar metal chelating activities which were significantly stronger (p < 0.05) than that of GSH. The chelating activity of peptides in hydrolysates could enhance ability of tissues to reduce rate of deteriorative metal-catalyzed lipid oxidation. The peptides may also serve as good agents to prevent metal ion-dependent oxidative damage to food lipids and thereby serve as food preservatives. Carboxyl and amino group in the side chains of the acidic (Glx and Asx) and basic (Lys, His and Arg) amino acids are thought to play an important role in chelating metal ions [31]. Presumably, peptide cleavages lead to enhanced Fe²⁺ binding due to increased concentration of carboxylic (COO⁻) and amine groups in acidic and basic amino acids, thus removing the pro-oxidative free metal ions from the hydroxyl radical system. His residues have also been reported to contribute to the metal chelating effect of protein hydrolysates, which is largely related to its imidazole ring [23]. Although the percentage of His was very low, glutamic acid + glutamine, aspartic acid + asparagine, Gly, Leu, Lys, Ala and Phe are the most predominant amino acids in APH and the membrane fractions. These amino acids could have contributed to the metal ion chelating activity observed for the protein hydrolysates.

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Figure 4

Metal chelating effects of African yam bean seed protein hydrolysate (APH) and its ultrafiltration membrane fractions.

2.6. Ferric Reducing Power Activity (FRAP)

The reducing capacity of a given compound may serve as a significant indicator of its potential antioxidant activity. An electron-donating reducing agent is able to donate an electron to a free radical. As a result, the radical is neutralized and the reduced species subsequently acquires a proton from the solution [32]. In this study, the reducing power of the APH and its membrane fractions was measured at the absorbance of 700 nm by transformation of the Fe³⁺/ferricyanide complex to ferrous form. An increased in absorbance indicate better reducing power of the test sample. Figure 5 shows that the reducing power activities of the APH and its membrane fractions were significantly lower (p < 0.05) than that of GSH. The 1 kDa fraction had significantly higher (p < 0.05) reducing power when compared to those of APH as well as the 1–3, 3–5, 5–10 kDa peptide fractions. The results indicate that smaller size peptides exhibited better reducing power than high molecular weight fractions. In contrast, Girgih et al. [3] reported an increase in reducing power of hemp seed protein hydrolysate with increasing peptide size. However, the present results are similar to the reducing power observed in smooth hound muscle protein hydrolysate and chickpea protein hydrolysate [7,33]. The correlations between the total hydrophobic amino acid and the reducing power of various hydrolysates have been reported [4]. Zhang et al. [15] reported that the rapeseed fractions that displayed the strongest reducing power also contained a higher amount of hydrophobic amino acids, which were suggested to be responsible for the strong reducing power of these peptides. The presence of amino acids such as Leu, Lys, Met, Tyr, Ile, His and Trp have been attributed to strong reducing power observed in protein hydrolysate fractions [34]. You et al. [35] suggested that the high amount of Tyr, Met, His, Lys and Trp that were found in the loach peptide hydrolysate could have contributed to the observed strong reducing power. However, recent report from multivariate analysis suggests that only sulphur-containing and acidic amino acids have strong contributions to the reducing power of food protein hydrolysates whereas positively charged and AAA may actually have negative effects [16]. The weak FRAP properties of the peptides indicate that they may not be as useful as GSH as electron donors when present within cells.

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Figure 5

Ferric reducing power of African yam bean seed protein hydrolysate (APH) and its ultrafiltration membrane fractions.

3.2. Preparation of AYB Seed Protein Isolate (API)

API was produced from AYB seeds according to the method described by Adebowale et al. [41] with slight modifications. Briefly, AYB flour was dispersed in deionized water (1:20, w/v), and the dispersion was adjusted to pH 10.0 with 2 M NaOH to solubilize the proteins. The resultant dispersion was stirred at 37 °C for 2 h followed by centrifugation (7000 × g at 4 °C) for 1 h. The pellet was discarded, and the supernatant filtered with cheesecloth and adjusted to pH 5.0 with 2 M HCl to precipitate most of the proteins. Thereafter, the mixture was centrifuged (7000 × g at 4 °C) for 45 min, the resultant precipitate was re-dispersed in deionized water, adjusted to pH 7.0 with 2 M NaOH and freeze-dried to produce API powder. The protein content of API was determined using the modified Lowry method [42].

3.3. Preparation of AYB Protein Hydrolysate (APH) and Membrane Fractions

APH was produced according to the previous method reported by Omoni and Aluko [43]. Briefly, a 5% (w/v) API slurry was adjusted to pH 9.0 and heated to 50 °C followed by addition of alcalase (4% w/w, on the basis of protein content of API). The digestion was carried out for 4 h at 50 °C and reaction mixture was maintained at pH 9.0 by adding 2 M NaOH when necessary. The reaction was terminated by adjusting the mixture to pH 4.0 with 2 M HCl after which the reaction mixture was placed in boiling water for 15 min to ensure complete denaturation of enzyme protein and coagulation of undigested proteins. The mixture was allowed to cool to room temperature and centrifuged (7000 × g at 4 °C) for 30 min and the resulting supernatant (APH) was sequentially passed through ultrafiltration membranes with molecular weight cut-off (MWCO) of 1, 3, 5, and 10 kDa in an Amicon stirred ultrafiltration cell. Thus, the retentate from 1 kDa membrane was passed through 3 kDa membrane whose retentate was passed through 5 kDa and the last retentate was then passed through 10 kDa membrane to give permeates that have peptide sizes of <1, 1–3, 3–5, and 5–10 kDa, respectively. The permeate from each MWCO membrane was collected, lyophilized, and stored at −20 °C until needed for further analysis. The protein contents of the freeze-dried APH and membrane fractions were determined using the modified Lowry method [42].

3.4. Amino Acid Analysis

An HPLC system was used to determine the amino acid profiles after samples were hydrolyzed for 24 h with 6 M HCl according to the method previously described by Bidlingmeyer et al. [44]. The cysteine and methionine contents were determined after performic acid oxidation [45], and tryptophan content was determined after alkaline hydrolysis [46].

3.5. DPPH Radical Scavenging Assay

The scavenging activity of APH and its fractions against DPPH was determined as previously described [4] using a 96-well clear flat bottom plate. Peptide fractions were dissolved in 0.1 M sodium phosphate buffer, pH 7.0 containing 1% (w/v) Triton X-100. DPPH was dissolved in methanol to a final concentration of 100 μM. A blank control consisted of only DPPH and sodium phosphate buffer. Appropriate dilutions of the samples (100 μL) were mixed with 100 μL of DPPH solution in the 96-well plate to a final assay concentration of 1 mg/mL and incubated at room temperature in the dark for 30 min. Thereafter, the absorbance of the sample (As) and control (Ac) was read at 517 nm. The scavenging activity of the peptide fractions was compared to that of GSH (1 mg/mL). The percent scavenging activity of GSH and the samples was calculated using the following equation:

3.6. Assay of Metal Ion Chelation

The metal chelating activity was measured using a slightly modified version of a previous method Pownall et al. [4]. Peptide sample solution or GSH (final concentration of 1 mg/mL) was combined with 0.05 mL of FeCl₂ (2 mM) and 1.85 mL distilled water in a reaction tube. Thereafter, 0.1 mL of 5 mM Ferrozine [3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″-disulfonic acid sodium salt] solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 10 min followed by removal of 200 μL aliquot of the reaction mixture and added to a clear bottom 96-well plate. The control experiment contained all the reaction mixtures except that distilled water was used to replace the peptide. Absorbance of sample (As) and control (Ac) was measured using a spectrophotometer at 562 nm and the metal chelating activity of the sample was compared to that of GSH. The percentage chelating effect (%) was calculated using the following equation:

3.7. Ferric Reducing Power Assay

Reducing power was measured according to a method reported by Girgih et al. [3] with slight modifications. Peptide samples (250 μL) or GSH was prepared in 0.2 M sodium phosphate buffer (pH 6.6), mixed with 250 μL of buffer and 250 μL of 1% potassium ferricyanide solution dissolved in distilled water. The final peptide or GSH concentration in the assay mixture was 1 mg/mL while the control reaction contained buffer and ferricyanide only. Each resulting mixture was heated at 50 °C and incubated for 20 min, which was followed by addition of 250 μL 10% aqueous trichloroacetic acid. Subsequently, a 250 μL aliquot of the reaction mixture was combined with 50 μL of 0.1% aqueous ferric chloride solution and 200 μL of distilled water was added. The mixture was allowed to stand at room temperature for 10 min and then centrifuged at 1,000 × g for 10 min to obtain a supernatant, which was used to determine absorbance at 700 nm.

3.8. Hydroxyl Radical Scavenging Assay

The hydroxyl radical scavenging assay was modified based on a method described by Girgih et al. [3]. APH, peptide fractions, GSH and 1,10-phenanthroline (3 mM) were each separately dissolved in 0.1 M sodium phosphate buffer (pH 7.4) while FeSO₄ (3 mM) and 0.01% hydrogen peroxide were each separately dissolved in distilled water. An aliquot (50 μL) of APH, peptide fractions or GSH (equivalent to a final assay concentration of 1 mg/mL) or buffer (control) was first added to a clear, flat bottom 96-well plate followed by additions of 50 μL of 1, 10-phenanthroline and 50 μL of FeSO₄. To initiate reaction in the wells, 50 μL of hydrogen peroxide (H₂O₂) solution was added to the mixture, which was then covered and incubated at 37 °C for 1 h with shaking. Thereafter, the absorbance of the mixtures was measured at 536 nm every 10 min for a period of 1 h. The absorbance was also determined for a blank (does not contain peptides or H₂O₂) and a control (does not contain peptides). The OH^· scavenging activity was calculated as shown by described by Girgih et al. [3].

3.9. Superoxide Scavenging Assay

The superoxide radical scavenging activity was determined according to the method described by Pownall et al. [4]. An aliquot (80 μL) of peptide fraction or GSH (equivalent to a final assay concentration of 1 mg/mL) was mixed with 80 μL of 50 mM Tris–HCl buffer (pH 8.3) containing 1 mM EDTA in a clear bottom 96-well plate. Then, 40 μL of 1.5 mM pyrogallol (dissolved in 10 mM HCl) was added to each well. The reaction rate (ΔA/min) was monitored as increase in absorbance at 420 nm for 4 min at room temperature. The control mixture contained the buffer but no peptide or GSH was added. The superoxide radical scavenging activity of the samples was calculated as shown by Pownall et al. [4].

3.10. Inhibition of Linoleic Acid Oxidation

Linoleic acid oxidation was measured using a slight modification of the method described by Girgih et al. [3]. Peptide fractions or standard compounds (GSH and BHT) at final assay concentrations of 0.25, 0.5, and 1.0 mg/mL were each dissolved in 1.5 mL of 0.1 M sodium phosphate buffer, pH 7.0. Each mixture was added to 1 mL of 50 mM ethanolic linoleic acid and stored in a glass test tube kept at 60 °C in the dark for 7 days. On a daily basis, 100 μL of the sample mixture was removed and mixed with 4.7 mL of 75% aqueous ethanol, 0.1 mL of ammonium thiocyanate (30%, w/v) and 0.1 mL of 0.02 M acidified ferrous chloride (dissolved in 1 M HCl). An aliquot (200 μL) of the resulting solution was added to a clear bottom 96-well plate and the degree of color development was measured using the spectrophotometer at 500 nm after 3 min incubation at room temperature.

Funding for this work was provided by the Natural Science and Engineering Research Council of Canada (NSERC), the Department of Foreign Affairs and International Trade Canada (DFAIT) grants to REA and the Education Trust Fund of Nigeria (ETF) grant to CFA.