CONTACT : OLUWAFEMI EMMANUEL EKUN oluwafemi.ekun@aaua.edu.ng 101 Abstract Peptide base d antioxidant s from plant and animal pr oteins are being identifie d as food additives and also as potential alternatives in the reduction of oxidative stress. This study inve stigated the antioxidative potentials of pe ptide digests of Albizia lebbeck seed pr otein. The proteins were extracted and isolated from A. lebbeck by de fatting with n-hexane, followed by alkaline solubilization and acid precipitation of the seed meal. The protein isolate was then subjected to enzymatic hydrolysis using four pr oteases – pepsin, trypsin, papain a nd chymotrypsin. The resulting hydrolysates were the n evaluated for the ir abilities to reduce ferric ions, as we ll a s their effects on hydroxyl radicals, 1,1-diphe nyl-2- picrylhydrazyl (DPPH) and superoxide radicals. Hydrolysates obtaine d fr om peptic prote olysis demonstrated the best activitie s against DPPH radical a nd ferric ions (57.589 ± 1.286% and 52.000 ± 0.589 mM Fe2 + respectively), whereas chymotrypsin hydrolysates scave nged super oxide radicals and hydroxyl radicals better than other protein digests (74.520 ± 0.998% and 36.925 ± 1.880% respectively). The choice of enzyme use d and the pre sence of specific amino acid residue s at certain positions of pe ptides in the hydr olysates influence d their antioxidant capacities. It is conclude d that Albizia lebbeck seed proteins enc ode potentially bioactive pe ptides, w hich c ould be harne ssed for numerous therapeutic and nutraceutical benefits. ISSN : 2580-2410 eISSN : 2580-2119 Antioxidant Properties of Albizia lebbeck Seed Protein Hydrolysates Oluwafemi Emmanuel Ekun 1* 1 Department of Biochemistry, Faculty of Science, Adekunle Ajasin University, Akungba Akoko, Ondo State Nigeria. Introduction Free radicals have continuously played roles as both toxic and beneficial compounds in cellular respiration, immune response, drug detoxification, among other physicochemical processes (Pham-Huy et al., 2008). They are produced f rom the oxidation of biomolecules in the body through metabolism in vivo or from exposure to envi ronmental pollutants, toxicants and other xenobiotics. When there is an apparent shortfall in the ability of the body’s detoxification systems to combat an excess of these oxidants, they accumulate in the body, causing a state referred to as oxidative stress (Majhenic et al., 2007). Oxidative stress generally describes a state of imbalance between the systemic generation of reactive oxygen and nitrogen species, and the ability of a biological system to quickly neutralize these reactive compounds or to repair the damage caused (Chandra et al., 2015). Perturbations in the normal cellular redox states can give rise to toxic effects such as DNA strand breakage, nucleotide base damage, erythrocyte lysis, among other deleterious OPEN ACCESS International Journal of Applied Biology Keyword Albizia lebbeck; protein; hydrolysate; peptides; antioxidant Article History Received May 21, 2022 Accepted December 14, 2022 International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly c ited. mailto:oluwafemi.ekun@aaua.edu.ng International Journal of Applied Biology, 6(2), 2022 102 effects (Chandra et al., 2015). This is thought to occur through the production of free radicals such as singlet oxygen, peroxides, superoxides, that damage cellular components such as lipids, proteins and DNA. Furthermore, some oxidative species act as intracellular messengers in redox signaling; and as a result, oxidative stress can cause disruptions in normal mechanisms of cellular signaling. In humans, oxidative s tress is involved directly and/or indirectly in the progression of several diseases, which include diabetes mellitus (Olusola et al., 2018), cancer (Halliwell, 2007), Parkinson’s disease, Alzheimers disease (Valko et al., 2007, Pohanka, 2013) atheroscleros is, myocardial infarction (Dean et al., 2011; Ramond et al, 2011) among others. It is important that natural antioxidants are utilized as additives and supplements for the reduction of oxidative stress due to disease progression (Bains and Hall, 2012). As a result, attention has turned to harnessing newer and previously underutilized plant sources for their antioxidant potentials. Specifically, protein hydrolysate prepa rations and pepti des from previously underutilized plants are being investigated for their bi ological activities. Peptides obtained from hydrolysis of plant and animal proteins have been identified as safer alternatives to synthetic antioxidants. The increased interest of antioxidant peptides obtained from these sources is as a result of their potential roles as dietary supplements (Ajibola et al., 2011: Olusola et al., 2018). Peptide antioxidants have simpler structures than their parent proteins. This enhances greater stability in different situations (heat, protease activities), they elicit no immunoreactions and often ex hibit enhanced nutriti onal and functional properties in addition to their antioxidant activity (Xie, et al., 2008). For most peptides, the ability to scavenge free radicals is mostly dependent on the peptide size, with peptides of low molecular weight being more effective than those of larger molecular weight. (Alashi et al., 2014). One of the plants whose proteins and peptides have not been previ ously investigated is Albizia lebbeck. Albizia lebbeck is a large, deciduous, drought – resistant, leguminous tree belonging to the Mimosaceae family. It is commonly known as the Siris tree or Lebbeck tree. It is alternatively known as “women’s tongue” because of the rattling sound its mature seed-containing pods make when the wind blows (Verma et al., 2013). It is relatively ubiquitous as it grows well in a wide diversity of regions. It is native to a number of areas especially in the forests of sub – Saharan African and Asian countries. A. lebbeck is a plant whose leaves, pods, seeds, stem bark and roots are excellent sources of bioactive phytochemicals and other secondary metabolites of pharmacologic significance (Zia-Ul-Haq et al., 2013; Verma et al., 2013). Its leaves contain flavonoids, saponins, tannins, alkaloids and cyanogenic g lycosides (Bobby et al., 2012) just as its roots are reservoirs of lupeol, stigmasterol and echinocystic acid (Verma et al., 2013; Musa et al., 2020). The seeds of A. lebbeck are rich sources of minerals such as calcium, iron, magnesium and potassium; phyt onutrients which include ascorbic acid and niacin; as well as essential fatty acids (Verma et al., 2013). Zia-Ul-Haq and others (2013) found out that that its seeds contained 34.17% c rude protein, 49.07% carbohydrate and 5.62% c rude fibre. Its high protein content makes it a good source of potentially bioactive peptides. Proteins of A. lebbeck are rich in arginine, lysine, histidine, leucine, isoleucine, phenylalanine, valine, tyrosine, aspartate, glutamate, but limi ting in methionine and cysteine (Zia -Ul-Haq et al., 2013). Parts of the plant have been used as feed for rumina nt livestock (Hassan et al., 2007) and in traditional medicine (Chulet et al., 2010). Its leaves have been reported to possess antimicrobial, anticancer, anti-diarrheic activities (Bobby et al., 2012) Its stem bark extracts have also been reported to demonstrate anti-parasitic potentials, and its use in treating dental infections have been documented (Qadri et al., 2005; Umar et al., 2009). Despite its numerous biological activities, the antioxidative capacities of hydrolysate preparations of its International Journal of Applied Biology, 6(2), 2022 103 proteins have not been evaluated. As a result, this study is geared towards the antioxidant potentials of Albizia lebbeck seed protein hydrolysates. Materials and Methods Materials Collection of Seeds A. lebbeck seeds were obtained from its trees in the botanical garden of Adekunle Ajasin University Akungba Akoko, Ondo State. Nigeria. They were subsequently identified and voucher samples were deposited at the Department of Plant Science and Biotechnology, Adekunle Ajasin University, Akungba Akoko. Chemicals and Reagents Enzymes: Pepsin (from porcine gastric mucosa), trypsin (from bovine panc reas), phymotrypsin (from bovine pa ncreas), papain (from Carica papaya) were products of Sigma- Aldrich laboratories, Co-Artrim, United Kingdom. Other Reagents: 1-diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid, trichloroacetic acid (TCA), potassium ferricyanide, ferric chloride, pyrogallol, hydrogen peroxide, ethylene diamine tetraacetic acid (EDTA) These were a lso products of Sigma-Aldrich laboratories, Co- Artrim, United Kingdom. All chemicals and reagents used were of analytical grade. Methods Isolation of A. lebbeck Seed Proteins The seeds were dried, pulverized and kept in a dry container at 4oC. They were defatted using n-hexane as described by Olusola et al.,(2018). The meal was extracted twice with n-hexane using twenty grams (20 g) of seed meal suspended in 200 ml of n-hexane. The meal was then dried at 40oC in a vacuum oven and ground again to obtain a fi ne powder, termed defatted seed meal, which was stored at -10oC. The protein component of the defatted meal was extracted using the method reported by Ekun et al.(2022). Defatted A. lebbeck seed meal was suspended in 0.5 M NaOH pH 12.0 at a ratio of 1:10, and stirred for one hour for the purpose of alkaline solubilization, using a magnetic stirrer. This was centrifuged at 18°C and 3000 g for 10 min. One additional extraction of the residue from the centrifugation process was performed with the same volume of 0.5 M NaOH and the supernatants were pooled. The pH of the supernatant was adjusted to 4.0 to facilitate acid- induced protein precipitation using 0.5 M HCl solution; the precipitate formed was recovered by centrifugation. The precipitate was washed with distilled water, adjusted to pH 7.0 using 0.1 M NaOH, freeze-dried and the protein isolate was then stored at -10°C until required for further analysis. Preparation of A. lebbeck Seed Protein Hydrolysates The protein isolate was hydrolysed using the methods described by Olusola and Ekun (2019) with slight modifications. The conditions for hydrolysis were optimized for each enzyme for maximized activity. Hydrolysis was performed using each of pepsin (pH 2.2, 37ºC), trypsin (pH 8.0, 37ºC), chymotrypsin (pH 8.0, 37ºC and papain (pH 6.0, 60ºC). The protein isolate (5% w/v) was dissolved in the appropriate buffer (phosphate buffer, pH 8.0 for trypsin and chymotrypsin, glycine buffer, pH 2.2 for pepsin, phosphate buffer, pH 6.0 for papain). The enzyme was added to the slurry at an enzyme-substrate ra tio (E:S) of 1:50. Digestion was performed at the specified conditions for eight (8) hours. The enzyme was International Journal of Applied Biology, 6(2), 2022 104 then inactivated by boiling in water bath (100oC) for fifteen (15) minutes and undigested proteins were precipitated by adjusting the pH to 4.0 with 2 M HCl/2 M NaOH followed by centrifugation at 7000 g for 30 min. The supernatant containing the peptides were then collected. The protein content of samples were determined using biuret assay method with bovine serum albumin (BSA) as standard. Determination of DPPH Radical Scavenging Activity The DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging activity was measured by using the assay method described by Arise et al.(2016a) with slight modifications. 1 mL each protein hydrolysate at different concentrations (0.5 – 2.5 mg/ml) was added to 1 mL 0.1 mM DPPH dissolved in 95 % ethanol. The mixture was shaken vigorously and incubated in the dark and at room temperature for 30 min. The absorbance was read at 517 nm. Ethanol (95 %) was used as a blank. The control solution consisted of 0.1 mL of 95 % etha nol and 2.9 mL of DPPH solution. Analyses were carried out in triplicates. Percentage inhibition of DDPH radical was calculated as follows: % DPPH inhibition = (Abs control - Abssample) x 100 / (Abscontrol) EC50 values were estimated f rom percentage inhibiti on pl ot, using a non-linear regression plot. Determination of Ferric Reducing Antioxidant Property The reducing power of the protein hydrolysates was determined according to the method reported by Olusola et al.(2018) with slight modification. An aliquot of 1 ml of different concentrati ons (0.5 – 2.5 mg/ml) of the hydrolysates (0.2 M PBS, pH 6.6) was mixed with 1 ml of 1% potassium ferric cyanide solution. The mixture was then incubated at 50°C for 30 minutes followed by the addition of 1 ml 10% (w/v) TCA. 1 ml of the incubation mixture was added with 1 ml of distilled water and 0.2 ml of 0.1% (w/v) ferric chloride in test tubes. After a 10 min reaction time, the absorbance of the resulting solution was read at 700 nm. Higher absorbance suggested stronger reducing power. Ascorbic acid was used as the reference anti oxidant. An aqueous solution of known Fe(II) concentrations (FeSO4·7H2O; 2.0, 1.0, 0.5, 0.25, 0.125 mM) were used for calibration. Results were expressed as mM Fe2+/mg hydrolysate. All the tests were performed in triplicate. Determination of Hydroxyl Radical Scavenging Activity Determination of the ability of the hydrolysates to prevent Fe2+/H2O2 induced deoxyribose decomposition was carried out using the method described by Ogunwa et al.(2016) with some modifications. The reaction mixture contained 120 L of 20 mM deoxyribose, 400 L of 0.1 M phosphate buffer pH 7.4, 40 L of 20 mM hydrogen peroxide and 40 L of 0.5M i ron (II) tetraoxosulphate (VI). The hydrolysates were added to the mixture. Distilled water was then added to make the mixture volume up to 800 L. This was incubated at 37oC for 30 minutes. The reaction was stopped by the addition of 0.5ml of 2.8% thiobarbituric acid, TBA. The reaction tubes were inc ubated in boiling water f or 20 mins, after which they were centrifuged at 3000g for 10 minutes. The absorbance was taken at 532 nm using a spectrophotometer. Percentage inhibition of the hydroxyl radical was calculated as follows: % OH inhibition = (Abs control - Abssample) x 100 / (Abscontrol) International Journal of Applied Biology, 6(2), 2022 105 Determination of Superoxide Radical Scavenging Activity The method described by Xie et al. (2008) was used to determine the abilities of the hydrolysates to scavenge the superoxide radical. Samples were each dissolved in 50 mM Tris–HCl buffer, pH 8.3 containing 1 mM EDTA and 80 μL was transferred into a clear bottom microplate well; 80 μL of buffer was added to the blank well. This was followed by addition of 40 μL 1.5 mM pyrogallol (dissolved in 10 mM HCl) into each well in the dark and the change in the rate of reaction was measured immediately at room temperature over a period of 4 minutes using a microplate reader at a wavelength of 420 nm. Ascorbic acid was used as control. The superoxide scavenging activity was calculated using the following equation: Superoxide scavenging activity (%) = (ΔAbs/minb – ΔAbs/mins)/ΔAbs/minb x 100 where b and s are blank and sample, respectively. Statistical Analyses Results were expressed as mean of triplicate observations ± standard error of mean. The data were statistically analyzed using One Way Analysis of Variance (ANOVA) and Duncan’s multiple range tests. Differences were considered statistically significant at p<0.05 using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). Results and Discussion DPPH Radical Scavenging Activity The abilities of A. lebbeck seed protein hydrolysates are illustrated in Figure 1 and their values of 50% inhibition are also displayed in Figure 2. All peptide digests demonstrated significantly (p<0.05) reduced DPPH scavenging activities when compared to the control, ascorbic acid. Among the hydrol ysates, peptide digests obtained from peptic hydrolysis had the highest (p<0.05) DPPH scavenging activities at all the studied concentrations, reaching a maximal effect of 57.589 ± 1.286 % at a final concentration of 2.5 mg mL-1. It had the lowest (p<0.05) EC50 value (1.075 ± 0.021 mg mL-1) among the peptide digests. This was comparable to 56.39 ± 0.45% obtained for peptic hydrolysates of Citrullus lanatus seed proteins (Arise et al., 2016a), and higher than 48% for peptic digests of yellow fin protein hydrolysates (Naqash & Nazeer, 2013). The potential bioactivity of a peptide released from enzymatic hydrolysis has been found to be dependent on factors such as nature of proteolytic enzyme utilized, hydrolysis time, as well as identity and position of amino acid residues in the peptide (Udenigwe & Aluko, 2011; Ekun et al., 2022). Pepsin being an enzyme which cleaves polypeptides at C -terminals of hydrophobic and to a lesser extent, acidic residues may have cleaved the proteins into several dissimilar peptides with the required ami no acid residues to donate protons to, and consequently scavenge the DPPH radical. At higher concentrations, chymotrypsin hydrolysates demonstrated higher (p<0.05) scavenging activities than tryptic and papain digests, displaying an a bility of 37.385±0.887 % at a final concentration of 2.5 mg mL-1. This could be that the aromatic aminoacyl residues (tryptophan, tyrosine, phenylalanine) released during proteolysis took part in scavenging the DPPH radical. This is consistent with the report of Wu et al. (2011) who reported that, even free aromatic amino acids, tryptophan and tyrosine from egg yolk have antioxidant potentials in foods. International Journal of Applied Biology, 6(2), 2022 106 Figure 1. DPPH Radical Scavenging Activity of A. lebbeck Seed Protein Hydrolysates Bars are expressed as means ± standard error of mean of triplicate determi nations (n=3). Compa risons are made among samples of the same concentration only. Values with different letters are significantly different from one another. Bars carrying the same l etter or symbol are not significantly different (p<0.05). Figure 2. Fifty Percent Effective Concentration Values (EC50) of DPPH Radical Scavenging Activity of A. lebbeck Seed Protein Hydrolysates Bars are expressed as means ± standard error of mean (SEM) of triplicate determinations (n=3). Bars with the same letters do not differ significantly while bars with different letters are significantly different (p<0.05) from one another. International Journal of Applied Biology, 6(2), 2022 107 Ferric Reducing Antioxidant Property The relative abilities of A. lebbeck seed protein hydrolysates in reducing the ferric ion, in comparison to ascorbate, are depicted in Figure 3. All hydrolysates had significantly lower (p<0.05) reducing power when compared to ascorbic acid (control). They also demonstrated a concentration- dependent increase in reducing power. This is consistent with a previous study (Razali et al., 2015) which revealed a similar trend of results. The low ferric-reducing property of A. lebbeck seed protein hydrolysates when compared with ascorbate may be attributed to the relatively low a mount of sulfur-containing aminoacyl residues in the hydrolysates, which would have otherwise increased antioxidative activity by donating protons to ferric iron in the reaction medium (Lopez-Barrios et al., 2014). However, peptic digests exhibited significantly higher (p<0.05) reducing capacities than other hydrolysates, as it converted a maximum of 52.000 ± 0.589% mM of Fe3+ to Fe2+ at a final concentration of 2.5 mg mL-1. This was comparable to the reducing abilities of peptic digests of Moringa oleifera seed proteins (Olusola et al., 2018) but higher than those of peptic hydrolysates obtained from cowpea seed proteins(Olusola and Ekun, 2019). Udenigwe and Aluko (2011) previously reported that acidic amino acid residues are moderately strong contributors to the reduction of ferric ions. This could be the reason why peptic hydrolysates still exhibited high reducing antioxidant power, even with limiting amounts of sulfur – containg aminoacyl residues such as cysteine and methioni ne. Tryptic hydrolysates also reduced 22.483 ±2.265 mM of Fe3+ to Fe2+ and these were higher (p<0.05) than the reducing properties of chymotryptic and papain hydrolysates, and this is consistent with previous studies on unfractionated hydrolysates of watermelon seed proteins (Arise et al., 2016a), and this suggests the presence of some other aminoacyl residues within the peptides, which may have contributed to the reduction of ferric ions. Figure 3. Ferric Reducing Antioxidant Properties of A. lebbeck Seed Protein Hydrolysates Bars are expressed as means ± standard error of mean of triplicate determi nations (n=3). Compa risons are made among samples of the same concentration only. Values with different letters are significantly different from one another. Bars carrying the same letter or symbol are not significantly different (p<0.05). International Journal of Applied Biology, 6(2), 2022 108 Hydroxyl Radical Scavenging Activity The hydroxyl radical scavenging capacities of A. lebbeck seed protein hydrolysates are illustrated in Figure 4, while their values of 50% inhibition are displayed in Figure 5. All hydrolysates demonstrated varying hydroxyl radical scavenging activities, such that chymotrypsin hydrolysates displayed significantly higher scave nging property than other digests, attaining a maximum activity of 36.925 ± 1.880 % at 2.5 mg mL-1. There is relative paucity of information relating to the activities of protein hydrolysates on the hydroxyl radical, when compa red to reports on their effects on other free radical systems. The effects of A. lebbeck seed protein hydrolysates and on the hydroxyl radical indicated that these protein digests had lower OH radical scavenging activities than the standard antioxidant, ascorbic acid. However, chymotrypsin hydrolysates scavenged the OH radical better than the other hydrolysates. Chymotrypsin is a specific endoprotease that cleaves proteins at C - terminal ends of aromatic amino acid residues (Voet et al., 2016). Also, Li & Li (2013) had previously found that the bioc hemical nature of the amino acid(s) at the C -terminal of peptides is more important to its antioxidative ability than that of its N-terminal. In similar fashion, Siow and Gan (2016) reported that hydrophobic, as well as aromatic aminoacyl residues are strong contributors to the scavenging of free radicals owing to their electron - dense branch chain groups. It therefore f ollows that the C -terminal aromatic residues arising from chymotrypsin digestion may have contributed strongly to the quenching of the hydroxyl radical. Papain digests and trypsin hydrolysates also recorded maximal scavenging activities of 30.663 ± 0.824 % and 31.544 ± 0.464 % respectively. These activities were significantly higher when compared to the maximum activity of peptic hydrolysates. However, all hydrolysates demonstra ted reduced l ower (p<0.05) hydroxyl radical quenching properties when compared to control. These results translated to EC 50 values of 0.668 ± 0.114 mg mL-1, 6.148 ± 0.3348 mg mL-1, 3.227 ± 0.288 mg mL-1, 2.697 ± 0.098 mg mL-1, and 1.206 ± 0.045 mg mL-1 for ascorbic acid (control), peptic, tryptic, chymotrypsin hydrolysates and papain digests respectively. Among the protein digests, papain hydrolysates had the lowest (p<0.05) EC50 value. Papain digests also demonstrated hydroxyl ra dical scavenging activities, but these reduced at higher concentrations and this is exemplified by its low EC50 (1.206 ± 0.045 mg mL-1) when compared to other hydrolysates. Papain, being a non-specific enzyme capable of releasing quite a lot of short, dissimilar peptides (Naik, 2012; Voet et al., 2016) could have produced certain peptides which, in turn, might have antagonized the effects of the main biologically active peptides in solution. This phenomenon may have been responsible for the progressive reduction in thei r activities at higher concentrations. Going by the cleavage specificities of the enzymes used in the hydrolysis, it could be inferred that these hydrolysates contained peptides with these aminoacyl residues which could scavenge the hydroxyl radical, either by virtue of proton donation and/or by their high hydrophobicities. International Journal of Applied Biology, 6(2), 2022 109 Figure 4. Hydroxyl Radical Scavenging Activities of A. lebbeck Seed Protein Hydrolysates Dots on each line graph are expressed as means ± standard error of mean (SEM) of triplicate determinations (n=3). Dots belonging to different hydrolysate/fractions with different letters at the same concentration are significantly different (p<0.05) from one another. Figure 5. Fifty Percent Effective Concentration Values (EC50) of Hydroxyl Radical Scavenging Activities of A. lebbeck Seed Protein Hydrolysates Bars are expressed as means ± standard error of mean (SEM) of triplicate determinations (n=3). Bars with the same letters do not differ significantly while bars with different letters are significantly different (p<0.05) from one another. Superoxide Radical Scavenging Activity The effects of A. lebbeck seed protein digests on the superoxide radical and their values of 50% inhibition a re displayed in Figures 6 and 7 respectively. As with other antioxidant activities, all hydrolysates displayed significantly lower (p<0.05) abilities against International Journal of Applied Biology, 6(2), 2022 110 the superoxide radical when compared with the control. Peptide digests obtained from chymotrypsin hydrolysis had better (P<0.05) capabilities than other hydrolysates in scavenging the superoxide radical, such that it had a maximum activity of 74.520 ± 0.998% at 2.5 mg mL-1. However, there is a relative paucity of informa tion in the literature about the activities of chymotrypsin – hydrolyzed proteins against the superoxide radical. Peptides obtained by chymotrypsin digestion have a number of hydrophobic residues and aromatic residues toward their C-terminals (Voet et al., 2016; Ekun et al., 2022) and these may have contributed to their increased quenc hing of the superoxide radical as concentrations increased. Tryptic digests were also effective in quenching the superoxide radical (58.659 ± 0.334 % at 2.5 mg mL-1) better than peptic hydrolysates and these results were c onsistent with the reports of Olusola et al.(2018) in their findings with unfractionated M. oleifera seed protein hydrolysates. At the same time, it was higher than what was obtained for tryptic digests of watermelon seed proteins (< 20% scavenging activity)(Arise et al., 2016b). Tryptic hydrolysates displayed a concentration – dependent increase in superoxide scavenging activity, attaining a maximum quenching extent of 58.659 ± 0.334 %. Udenigwe and Aluko (2011) earlier stated that lysine residues are especially important contributors to the quenching of the superoxide radicals. This could explain why tryptic hydrolysates were able to scavenge the superoxide radical, owing to the fact that they contain several peptides having lysine residues on their C-terminals. Papain hydrolysates had its maximal scavenging activity of 47.698 ± 1.083 % at a low concentrati on of 0.5 mg mL-1, and the lowest EC50 of 0.970 ± 0.067 mg mL-1. This indicates that the activity of papain digests against the superoxide radical decreased with increasing concentration, and this could be that the continued presence of certain peptides in the solution could have antagonized the activities of peptides that could have contributed positively to the quenching of the superoxide radical. Figure 6. Superoxide Radical Scavenging Activities of A. lebbeck Seed Protein Hydrolysates Dots on each line graph are expressed as means ± standard error of mean (SEM) of triplicate determinations (n=3). Dots belonging to different hydrolysate/fractions with different letters at the same concentration are significantly different (p<0.05) from one another. International Journal of Applied Biology, 6(2), 2022 111 Figure 7. Fifty Percent Effective Concentration Values (EC50) of Superoxide Radical Scavenging Activities of A. lebbeck Seed Protein Hydrolysates Bars are expressed as means ± standard error of mean (SEM) of triplicate determinations (n=3). Bars with the same letters do not differ significantly while bars with different letters are significantly different (p<0.05) from one another. Conclusion The results of this study demonstrated that the proteins of A. lebbeck seed proteins were susceptible to enzymatic hydrolysis and yielded hydrolysate preparations which possessed antioxidant activities against free radical systems and ferric ions in vitro howbeit by differing mechanisms. Peptic hydrolysates demonstrated the highest activities against DPPH radical and ferric ions, while chymotrypsin hydrolysates scavenged both hydroxyl and superoxide radicals better than other digests. This implies that, depending on the choice of enzyme used, A. lebbeck seed proteins possess biologically active peptides with immense potentials as food additives and therapeutic benefits. This will contribute towards increased value-added use of A. lebbeck seeds, which are currently being under-utilized. International Journal of Applied Biology, 6(2), 2022 112 References Ajibola, C.F., Fashakin, J.B., Fagbemi, T.N., & Aluko, R.E. 2011. Effect of peptide size on antioxidant properties of African yam bean seed (Sphenostylis stenocarpa) protein hydrolysate fractions. Int J Mol Sci 12(10):6685–702. Alashi, A.M., Blanchard, C.L, Mailer, R.J, Agboola, S.O., Mawson, A.J., He, R., Girgih, A. & Aluko, R.E.2 014. Blood pressure lowering effects of Australian canola protein hydrolysates in spontaneously hypertensive rats. Food Research International 55: 281-287. Arise, R.O., Yekeen, A.A & Ekun, O.E. 2016b. In vitro antioxidant and -amylase inhibitory properties of watermelon seed protein hydrolysates. Environmental and Experimental Biology 14: 163–172. Arise, R.O., Yekeen, A.A. & Ekun, O.E. & Olatomiwa, O.J. 2016a. Protein hydrolysates from Citrullus lanatus seed: antiradical and hydrogen peroxide-scavenging properties and kinetics of angiotensin-I converting enzyme inhibition. Ceylon Journal of Science 45(2): 39-52. Bains, M. & Hall, E.D. 2012. Antioxidant therapies in traumatic brain and spinal cord injury. BBA Molecular Basis of Disease1822: 675-684. Bobby, M.D., Wesely, E.G. & Johnson, M. 2012. High performance thin layer chromatog raphy profile studies on the alkaloids of Albizia lebbeck, Asian Pacific Journal of Tropical Biomedicine S1-S6. 
 Chandra, K., Salman, A.S., Mohd, A., Sweety R., & Ali, K.N. 2015. Protection Against FCA Induced Oxidative Stress Induced DNA Damage as a Model of Arthritis and In vitro Anti-arthritic Potential of Costus speciosus Rhizome Extract. www.ijppr.com International Journal of Pharmacognosy and Phytochemical Research 7(2); 383-389. Chulet, R., Jhajharia, M., Pradhan, P. & Sharma, S. 2010 Analgesic and antipyretic ac tivity of Albizia lebbeck. Pharmacology online 3: 737-749. Dean, O.M., van den Buuse, M., Berk, M., Copol ov, D.L., Mavros, C. & Bush, A.I. 2011. "N- acetyl cysteine restores brain glutathione loss in combined 2-cyclohexene-1-one and D-ampheta mine-treated rats: relevance to schizophrenia and bipolar disorder". Neuroscience Letters 499 (3): 149–53. Ekun, O.E., Olusola, A.O., Sanni, J.A, Ishola, F. 2022. Peptide Fractions from Chymotrypsin- hydrolyzed Moringa oleifera Seed Proteins Inhibit α-amylase and α-glucosidase in vitro. Biology, Medicine and Natural Product Chemistry 11(1):7-16 Halliwell, B. 2007. Oxidative stress and cancer: have we moved forwa rd? Biochemistry Journal, 401 (1): 1–11. Hassan, L.J., Umar, K.G. & Atiku, I. 2007. Nutriti onal Evaluation of Albizia lebbeck (L.) Pods as Source of Feeds for Livestock, American Journal of Food Technology 2: 435-439. Li, Y., & Li, B. 2013. 2013. Characterization of structure–antioxidant activity relationship of peptides in free radical systems using QSAR models: Key sequence positions and their amino acid properties. Journal of Theoretical Biology 318:29–43 http://www.ijppr.com/ http://www.biochemj.org/bj/401/0001/4010001.pdf International Journal of Applied Biology, 6(2), 2022 113 Lopez-Barrios, L. & Gutierrez-Uribe, J.A. & Serna-Saldıvar, S.O. 2014. Bioactive Peptides and Hydrolysates from Pulses and Their Potential Use as Functional Ingredients. Journal of Food Science 79(3) 273-283 Majhenic, L.M., Skerget, M., & Knez Z. 2007. Antioxidant and anti microbial activity of guarana seed extracts. Food Chem; 104: 1258-1268. Musa, A., Ahmed, K. & Alagbe, O.J. 2020. Prelimnary phytochemical screening of Albizia lebbeck stem bark. Int. J. Int. Edu. 3(12):1-5 Naik P. 2012. Protein metabolism. In: Essentials of Biochemistry. Jaypee Brothers Medical Publishers ; pp. 226–257. Naqash, S.Y. & Nazeer, R.A. 2013. Antioxidant and func tional properties of protein hydrolysates from pink perch (Nemipterus japonicus) muscle. Journal of Food Science and Technology 50(5): 972-978. Ogunwa, T.H., Adeyelu, T.T., Fasimoye, R.Y., Oyewale, M.B., Ademoye, T.A., Ilesanmi, O.C., Awe, O.B., Ajiboye, S.A., Oloye, O.B. & Sholanke, D.R. 2016 Phytochemical evaluation and in vitro antioxidant status of Clerodendrum volubile (an indigenous medicinal plant). Pakistan Journal of Pharmaceutical Research 2(2):77-88 Olusola, A.O. & Ekun, O.E. 2019. Alpha-amylase – inhibitory properties and in vitro antioxidant potentials of cowpea seed protein hy drolysates. American Associ ati on for Science and Technology Communications 6(1): 1-12. Olusola, A.O., Ekun, O.E., David, T.I., Olorunfemi, O.E., & Oyewale, M.B. 2018. Moringa oleifera seed protein hydrolysates: Kinetics of α-amylase inhibition and antioxidant potentials. Global Advanced Research Journal of Medicine and Medical Sciences;7(9):190-201. Pham-Huy, L.A., He, H., & Pham-Huy, C. 2008. Free Radicals, Antioxidants in Disease and Health. Int. J. Biomed Sci. 4(2):89-96 Pohanka M. 2013. "Alzheimer´s disease and oxidative stress: a review". Current Medicinal Chemistry 21 (3): 356–364. Qadri, R. & Mahmood A. 2005. Ultra-Structural Studies on Root Nodules of Albizia Lebbeck (L.) Benth. Pak. J. Bot. 37(4): 815-
821. 
 Ramond, A., Godin-Ribuot, D., Ribuot, C., Totoson, P., Koritc hneva, I., Cachot, S. Levy, P. & Joyeux-Faure, M. 2011. "Oxidative stress mediates cardiac infarction aggravation induced by intermittent hypoxia.". Fundamental Clinical Pharmacology 27 (3): 252– 261. Razali, A.N., Amin, A.M & Sarbon, N.M. 2015. Antioxidant activity and functional properties of fractionated cobia skin gelatin hydrolysate at different molecular weight. International Food Research Journal 22(2): 651-660 Siow, H.L. & Gan, C.Y. 2016. Extraction, identification, and structure-activity relationship of antioxidative and alpha-amylase inhibitory peptides from c umin seeds (Cuminum cyminum). Journal of Functional Foods 22:1–12. http://www.eurekaselect.com/115342/article International Journal of Applied Biology, 6(2), 2022 114 Udenigwe, C.C. & Aluko, R.E. 2011 Chemometric analysis of the amino acid requirements of antioxidant food protein hydrolysates. International Journal of Molecular Sciences, 12, 3148–3161. Uma B., Prabhakar K., Rajendran S., Sarayu L.Y. 2009. Antimicrobial Activity of Albizzia Lebbeck Benth against Infectious Diarrhoea. The Internet Journal of Microbiol ogy 7:1-6. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M & Telser J. 2007. "Free radicals and antioxidants in normal physiological functions and human disease". International Journal of Biochemistry & Cell Biology 39 (1): 44–84. Verma, S.C., Vashishth, E., Singh, R., Kumari, A., Meena, A.K., Pant. P., Bhuyan, G.C & Padhi M.M. 2103 A Review on Parts of Albizia lebbeck (L.) Benth. Used as Ayurvedic Drugs Research J. Pharm. and Tech 6(11):1235-1241 Voet D, Voet, J.G & Pratt, C.W. 2016. Fundamentals of Biochemistry. 5th edition. Wiley. United States. Wu, H.C., Chen, H.M. & Shiau, C.Y. 2011. Free a mino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus). Food Research International 36:949-957 Xie, Z., Huang, J., Xu, X. & Jin Z. 2008. Antioxidant activity of peptides isolated from alfalfa leaf protein hydrolysate. Food Chemistry 111, 370–376. Zia-Ul-Haq, M., Ahmad, S., Qayum, M. & Ercişli, S. 2013. Compositional studies and antioxidant potential of Albizia lebbeck (L.) Benth. Pods and seeds. Turkish Journal of Biology
37: 25-32.