Antioxidant properties, ACE/renin inhibitory activities of pigeon pea hydrolysates and effects on systolic blood pressure of spontaneously hypertensive rats

Abstract Legumes are rich sources of protein in human diet and their consumption has been associated with the prevention of chronic diseases attributable to their bioactive components. Pigeon pea (Cajanus cajan) is an underutilized legume with relatively high protein content (~24%). Protein hydrolysates were prepared from pea isolate by enzymatic hydrolysis using pepsin and pancreatin. Hydrolysates were evaluated for their amino acid composition, antioxidant properties, in vitro and in vivo antihypertensive properties. The hydrolysates had high hydrophobic amino acids, especially isoleucine, phenylalanine, and leucine. Pepsin‐pancreatin‐hydrolyzed pea protein (PPHPp) showed significantly higher ability to scavenge DPPH˙ while pancreatin‐hydrolyzed pea protein (PPHPa) had higher ˙OH, ABTS˙+ scavenging, Fe3+ reducing and linoleic acid peroxidation inhibition. PPHPp exhibited superior angiotensin‐converting enzyme inhibition (61.82%) while PPHPa showed higher renin inhibition (14.28%). PPHPp exhibited strong antihypertensive effect, showing an instantaneous systolic blood pressure lowering effect (−26.12 mmHg) within 2‐h post‐oral administration. Pigeon pea protein hydrolysate (especially from pancreatin digest) could therefore, be a promising source of bioactive peptides and potential ingredient for formulation of functional foods against oxidative stress and hypertension.

antioxidants from hydrolysates have been of major research interest being rich sources of peptides with physiological antioxidative effects and prevention of oxidative food degradation. They serve as alternatives to existing synthetic antioxidants (butylated hydroxylanisole, butylated hydroxytoluene) whose usage is associated with undesirable and adverse side effects.
The physiological blood pressure is regulated by the reninangiotensin system (RAS). Renin (produced in the kidney) converts angiotensinogen (in the liver) to a decapeptide, angiotensin I, which is biologically inactive, but activated by conversion to angiotensin II (a major vasoconstrictor) by angiotensin-converting enzyme (ACE).
Persistent high blood pressure results from uncontrolled release of angiotensin II, and is a controllable risk factor for cardiovascular diseases such as stroke and heart failure, which are the leading causes of death and disability worldwide (Jensen, Eysturskarö, Madetoja, & Eilertsen, 2014). Inhibition of enzymes in the RAS pathway, especially ACE, is regarded to be a potent therapeutic approach in the treatment of hypertension (Lin, Lv, & Li, 2012). ACE inhibition functions to block the first step in the renin-angiotensin system thereby interrupting the negative feedback effects of angiotensin II.
Synthetic ACE inhibitors (captopril, lisinopril, enalapril) extensively used as antihypertensive drugs are available on the market, but their use is associated with adverse effects and nontolerance in some patients. ACE inhibitory peptides can be derived from different plant sources and may function as potent alternatives to synthetic drugs as they have been adjudged to be safe and economical. Legumes Based on the demonstrated relationship between oxidative stress and high blood pressure, the use of a single agent with antioxidant and antihypertensive properties could provide an effective strategy for the prevention and treatment of hypertension. Pigeon pea is an underutilized legume in Nigeria, used as food and forage, a good source of protein (20-28%). It used to be a delight and delicacy about two decades ago but presently tending toward extinction owing to scanty information on its nutritional and nutraceutical potential compared to other common legumes (bambara, cowpea, African yam bean, etc.). Therefore, the objective of this study was to evaluate pigeon pea protein hydrolysates for antioxidative capacity, as well as in vitro and in vivo antihypertensive properties.

| Materials
Pigeon pea (Cajanus cajan) seed (TCc-AO/TB78-9) was obtained from the Gene bank of the International Institute of Tropical Agriculture, Ibadan, Oyo State, Nigeria. Reagents used were purchased from Sigma (St. Louis, MO, USA) and Fisher Scientific (Oakville, ON, Canada).

| Preparation of pigeon pea protein isolate (PPI)
Pigeon pea isolate was obtained by isoelectric precipitation.
Pigeon pea flour (90 g) was suspended in 1800 ml of double distilled water (DDQ), adjusted to pH 9.0 using 1 M NaOH and extracted for 1 h at 25°C on a stirrer plate. pH was adjusted to 4.5 using 1 M HCl solution. The precipitate was centrifuged (9000 g; 30 min; 4°C), washed with DDQ thrice, adjusted to pH 7.0, lyophilized and stored at −20°C.

| Enzymatic hydrolysis of PPI
PPI (5%, w/v) was hydrolyzed with pancreatin (pH 8.0, 37°C) and pepsin (pH 2.0, 37°C) for 4 h at enzyme to substrate ratio of 1:20 (w/w). Also, a sequential hydrolysis with pepsin followed by pancreatin for 2 h each was done. The pH was maintained during each hydrolysis using either 1 M NaOH or 1 M HCl as appropriate, while the temperature was maintained using a thermostat. The enzymes were inactivated after hydrolysis, by heating and holding at 85°C for 15 min. The cooled reaction mixtures were centrifuged (9000 g; 30 min; 4°C), supernatant lyophilized and stored at −20°C.

| Determination of amino acid composition
The amino acid profiles were determined using the HPLC Pico-Tag system according to the method previously described after samples were digested with 6 M HCl for 24 h (Bidlingmeyer, Cohen, & Tarvin, 1984). The cysteine and methionine contents were determined after performic acid oxidation and the tryptophan content was determined after alkaline hydrolysis.

| Determination of antioxidant properties
DPPH radical scavenging activity was determined by the method of by Girgih, Udenigwe, Li, Adebiyi, and Aluko (2011)  DPPH was dissolved in methanol to a final concentration of 100 μM.
One hundred microlitre (100 μl) aliquot of each sample was mixed with 100 μl of the DPPH radical solution in a 96-well plate to final concentrations of 0.5-4 mg/ml and incubated at room temperature in the dark for 30 min. The buffer was used in the blank assay while reduced glutathione (GSH) served as the positive control.
Absorbance was measured at 517 nm using a spectrophotometer and the percentage DPPH radical scavenging activity was determined using the following equation: Hydroxyl radical scavenging activity (HRSA) was also based on a method described by Girgih et al. (2011) with modifications. Samples, GSH and 1, 10-phenanthroline (3 mM) were each separately dissolved in 0.1 M phosphate buffer (pH 7.4) while FeSO 4 (3 mM) and 0.01% hydrogen peroxide were each separately dissolved in distilled water. An aliquot (50 μl) of sample 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 FeSO 4 . To initiate reaction in the wells, 50 μl of hydrogen peroxide (H 2 O 2 ) 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 hydroxyl radical scavenging activity was calculated as follows based on change in absorbance (ΔA): The ferric reducing antioxidant power (FRAP) of the protein was determined according to the modified method of Benzie and Strain (1996). Peptide or GSH was dissolved in 0.3 M acetate buffer (pH 6.6). The FRAP reagent was prepared by mixing 0.3 M acetate buffer with 10 mM TPTZ (2,4,6 tripyridyl-s-triazine) at pH 3.6 in 40 mM HCl and 20 mM FeCl 3 .6H 2 O pH 3.6, at the ratio of 5:1: ABTS radical scavenging activity (ARSA) was carried out according to a previously described method (Arts, Dallinga, Voss, Haenen, & Bast, 2004). Briefly, ABTS + was prepared by dissolving 7 mM ABTS and 2.45 mM potassium persulphate in phosphate buffered saline (PBS), pH 7.4 and allowing this to stand in the dark for 16 h to generate the ABTS radical cation (ABTS˙+). For the analysis, the ABTS˙+ stock was diluted using PBS buffer and equilibrated at 30°C to an absorbance of 0.7 (±0.02) at 734 nm using a Heliosk thermo spectrophotometer (Electron Corporation Helios Gamma, England). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxy lic acid) was dissolved in 80% ethanol. The antioxidant capacity was measured by mixing 200 μl of samples with 2 ml of ABTS + solution and the decline in absorbance was observed for 5 min. Appropriate blanks were run for each sample and the radical scavenging capacity was compared to that of Trolox (6.25-200 μM) and results were expressed as mM Trolox equivalent (TE) per gram of sample on protein equivalent basis. The percentage ABTS˙+ scavenged was calculated using the following equation:

| Determination of linoleic acid oxidation
Linoleic acid oxidation was determined using the method described by Girgih et al. (2011). Samples or GSH were each dissolved in 1.5 ml of 0.1 M phosphate buffer, pH 7.0. Each sample solution was added (final assay concentration of 0.5 mg/ml) 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 mixture was added to a clear bottom 96-well plate and the degree of color development was measured at 500 nm after 3 min incubation at room temperature.

| Determination of angiotensin I-converting enzyme (ACE) inhibitory activity
The inhibition of ACE was determined as described by Udenigwe, Lin, Hou, and Aluko (2009) ACE inhibition was calculated as follows:

| Determination of renin inhibitory activity
The ability of peptide samples to inhibit renin activity was determined according to a previously described method (Li & Aluko, 2010)

| Antihypertensive activity study using spontaneously hypertensive rats (SHRs)
Animal experiments were performed according to protocols ap-

| Statistical analysis
Data were generated in triplicates and subjected to Analysis of Variance (ANOVA) using Statistical Package for Social Sciences (SPSS) V. 17.0. The means were separated using Duncan Multiple Range Test (DMRT) at 95% confidence level.

| Amino acid composition of pigeon pea protein and hydrolysates
The amino acid composition of pigeon pea isolate and hydrolysate showed that glutamic acid, aspartic acid, lysine, leucine, arginine, and phenylalanine were predominant (Table 1) exhibited considerable content of the hydrophobic amino acids especially isoleucine (3.46-3.68 g/100 g), leucine (6.88-8.37 g/100 g), phenylalanine (6.80-8.58 g/100 g), glycine (3.12-3.63 g/100 g), alanine (3.82-4.04 g/100 g), and proline (4.83-5.06 g/100 g). AAAs are known to freely donate hydrogen atom to electron deficient free radicals hence neutralizing the radical as well as breaking the radical chain. The presence of AAA has also been reported to enhance antioxidant capacity of peptides (Girgih et al., 2014). Cysteine with sulfhydryl (SH) group can also donate hydrogen atom from the SH group. Hence, the presence of cysteine and tryptophan in the same peptide chain would make a significant contribution to the antioxidant activities of such peptide (Zou et al., 2016).
The presence of peptides with significant aromatic residues (tyrosine, phenylalanine, tryptophan) at C-terminal and basic residues (lysine, histidine, arginine) at the N-terminal have been suggested to have strong and competitive ACE inhibitory activity (Hwang & Ko, 2004). AAA also contribute as inhibitors of Angiotensin Converting Enzyme (ACE) by interacting between three subsites at the active site of ACE.

Renin inhibition
The result of the present study therefore, suggests that the presence of aromatic and hydrophobic amino acids in pigeon pea proteins is a valuable contributor to its antioxidative and antihypertensive properties.

| Antioxidant properties of pigeon pea protein hydrolysate
DPPH˙ is used to measure the ability of antioxidative compounds to donate electrons or hydrogen ion to free radicals to form a more stable compound. DPPH˙ receives hydrogen from antioxidant to form a stable diamagnetic molecule (yellow-colored diphenyl picrylhydrazyl), and the extent of discoloration of the radical from purple to yellow indicates the scavenging potential of the antioxidant compound with respect to its hydrogen-donating ability. The protein hydrolysates had better DPPH˙ scavenging activity than the intact protein (PPI), and thus indicates the release and exposure of peptides with enhanced radical scavenging activity from the isolate during enzymatic proteolysis (Figure 1a). However, activities of the protein isolate and hydrolysates (3.54-40.98%) were significantly lower than that of GSH an excellent free radical scavenger (Galano & Alvarez-Idaboy, 2011 However, results from this present study showed an independent relationship between hydrophobic character of the pigeon pea hydrolysates and ability to scavenge hydroxyl radical.
Results of ABTS˙+ scavenging activity is presented in Figure 1c.
There was no significant difference (  of this study corroborate the reports of Alashi et al. (2014) where ABTS˙+ scavenging activity of canola protein hydrolysate using pancreatin enzyme was more effective than those of pepsin.
Antioxidants donate hydrogen atom to electron deficient free radical and this electron donating ability can be evaluated using ferric reducing antioxidant power. The antioxidant activity of a peptide directly correlates to its ferric ion reducing ability. The ability of pigeon pea hydrolysates to reduce Fe 3+ ranged from 0.027 to 0.069 mmol Fe 2+ /mg, and was comparable to that of GSH (Figure 1d). There was no correlation between ferric reducing antioxidant power and ABTS˙+ scavenging activity as the type of radical and their mechanism of reaction differs. The ferric reducing antioxidant power shows the reducing potential of an antioxidant (peptide) when it reacts with Fe 3+ -TPTZ complex resulting in a deep blue colored compound (Fe 2+ -TPTZ). In this reaction, the peptide serves as a hydrogen atom donator to stabilize or neutralize the free radical.
Moreso, the reaction is conducted under acidic condition of pH 3.6 so as to iron solubility. On the other hand, ABTS˙+ scavenging activity was conducted at pH 7.4 and ABTS˙+ serves as the oxidant and produces a green colored compound in which the extent of decolorization of the color relates with the percentage inhibition of radical or its scavenging ability.

| Inhibition of linoleic acid oxidation
Lipid peroxidation is of serious concern in the food industry because of its contribution to the development of undesirable offflavors, odors, dark colors as well as toxic products. Antioxidants function by reducing the peroxyl radical formed during lipid peroxidation to hydro-peroxide in order to prevent the propagation of the radical chain. The ability of pigeon pea protein to inhibit lipid peroxidation was evaluated using a linoleic acid model system and the results are presented in Figure 2. The extent of peroxide formation in blank (without protein sample) increased rapidly within the first 2 days of incubation to reach its maximum absorbance (0.81) before declining. The rapid decline is supposedly due to the formation and decomposition of linoleic oxidation products such as hydroperoxides, which are unstable and gradually decomposes into secondary metabolites with the progression of incubation period. Regardless of the decline, the absorbances of the control reaction until the fifth day were still higher than values obtained in protein samples. The low inhibitory activity of reduced glutathione (GSH) has been attributed to the oxidation of GSH to glutathione sulphide (GSSG) after some days and due to the long incubation period (7 days), and its impossibility to regenerate the antioxidant form of the peptide (GSH) (Ajibola, Fashakin, Fagbemi, & Aluko, 2013). The peptides showed a good antioxidant property when compared to the control (blank) in inhibiting lipid peroxidation which is evident by the reduced absorbance or absorption intensity observed in samples with the polypeptide. PPI exhibited the weakest inhibitory activity after the second day of incubation.
PPHPa was the most active in suppressing lipid peroxidation for 6 days evident by almost linear curve revealing an almost complete prevention of lipid peroxidation. This can be attributed to its high histidine content which correlates to have high lipid peroxyl radical trapping ability (Erdmann, Cheung, & Schröder, 2008). The absorbances obtained in the present study are lower than those reported for rapeseed (Mákinen, Johannson, Vegarud, Pihlava, & Pihlanto, 2012) and pumpkin (Venuste et al., 2013) hydrolysates.

| In vitro ACE and renin inhibitory activities
Pigeon pea exhibited good ACE and renin inhibitory activity may be due to higher proline content. The first two ACE inhibitory peptides (VPP and IPP) isolated from fermented milk both contained proline, which has been suggested to enhance ACE inhibition (Nakamura, Yamamoto, Sakai, & Takano, 1995

| Antihypertensive effect of pigeon pea isolate and hydrolysates in spontaneously hypertensive rats (SHRs)
The effect of single oral administration of pigeon pea protein isolate and hydrolysate on systolic blood pressure (SBP) is shown in Figure 4. reported similar blood pressure lowering effect for a pentapeptide (IPAGV) from hemp seed 4 h after oral administration. Similar change (-36 mmHg) in SBP was reported for another pentapeptide (LTFPG) from yellow field pea obtained 2 h after oral administration (Aluko et al., 2015). The significant decrease in blood pressure lowering activities in spontaneously hypertensive rats (SHRs) after a single oral administration demonstrates the potential of pigeon pea hydrolysates as suitable functional foods ingredient and nutraceuticals for controlling blood pressure elevation.

| CON CLUS IONS
Oxidative stress constitutes an important pathophysiology in many chronic diseases, and can be controlled to reduce disease progression. The free radical scavenging, lipid peroxidation inhibitory, and ferric reducing activities of pigeon pea protein hydrolysates suggest they can attenuate tissue oxidative stress. Also, as inhibitors of ACE and renin, they can be further investigated as natural agents for managing hypertension, which is a strong risk factor for stroke. The multifunctional capacity of the pigeon pea peptides provides an opportunity to formulate functional foods and nutraceuticals with potential role in the prevention and treatment of chronic diseases.

ACK N OWLED G M ENTS
This work was supported by Tertiary Education Trust Fund (TETFund), Nigeria.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.