Effect of arginine : lysine ratio in free amino acid and protein form on L-NAME induced hypertension in hypercholesterolemic Wistar rats

Abstract

Arginine plays an important role in cardiovascular diseases, especially as a nitric oxide precursor leading to vasodilation. The present study focuses, on the effect of the arginine/lysine (Arg : Lys) ratio in the protein form and free amino acid form on hypertension in hypercholesterolemic Wistar rats. Hypertension was induced by administration of L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) orally for 16 weeks in high cholesterol fed Wistar rats. Arginine : lysine ratio is high in Moringa seed protein isolate (MPI) compared to other oilseed proteins. After inducting, the treatment groups were supplemented with diets containing Moringa seed protein isolate (MPI-LN) and free amino acids (FAA-LN) for 6 weeks. A decrease in systolic blood pressure (SBP) was observed after treatment. The MPI-LN and FAA-LN exhibited 126.6 ± 2.2 mmHg and 127.5 ± 0.2 mmHg of SBP respectively, which is less compared to the hypertensive rats (146.7 ± 4.5 mmHg). The hypertensive markers such as angiotensin-I converting enzyme (ACE) and nitrate levels in kidney were analysed. The MPI-LN and FAA-LN exhibited 10.30 ± 3.7 nmoles per mg per min and 37.56 ± 3.7 nmoles per mg per min ACE activity respectively. In both treatment groups, a 50% decrease in ACE activity was observed compared to hypertensive rats. Increase (30%) in nitrate levels was observed in MPI-LN (0.47 ± 0.04 μM g⁻¹) and FAA-LN (0.4 ± 0.06 μM g⁻¹) compared to hypertensive rats. From the results, it is evident that Arg : Lys has a role in hypertension regulation. Lipid profiles of blood plasma showed a decrease in plasma triacylglyceride and liver triacyglyceride levels in the treatment groups. The Arg : Lys ratio in both the protein form (MPI) and free amino acid form strongly affects the metabolic pathways of hypertension with moderate effect on hypercholesterolemia.

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1. Introduction

Hypertension is a cardiovascular disease, which is affecting around 1.5 billion people annually around the world and approximately 26% among them are the adult population worldwide.¹ Angiotensin I-Converting Enzyme (EC-3.4.15.1) (ACE) is a molecular marker of hypertension that converts the decapeptide angiotensin I to octapeptide angiotensin II, a vasoconstrictor that acts at both AT₁ and AT₂ receptors to stimulate the production of nitric oxide (NO).² L-Arginine is a precursor molecule for the synthesis of NO by the action of nitric oxide synthase (NOS), leading to relaxation of blood vessels and improving blood flow in endothelial cells.³ From the human clinical studies it is evident that treatment of patients with free form of L-arginine have resulted in the endothelium-dependent vascular dilation.⁴

L-Arginine is an imperative source of energy for cellular energy and muscular contraction, it is necessary for hepatic synthesis of creatine which is taken up by muscle where it phosphorylates to form phosphocreatine.⁵ Arginine : lysine (Arg : Lys) ratio plays a significant role as both the amino acids arginine and lysine compete each other for the same co-transporter molecule that transports the amino acids into the intestine based on the requirement.⁶ Studies have reported that along with antagonism, high lysine intake enhances arginine catabolism by inducing kidney arginase thereby depleting the arginine levels.⁵ L-Lysine has been reported to be hypercholesterolemic⁷ and an acute oral dose of L-Arg was found to increase flow-mediated dilation in patients with hypercholesterolemia.⁸

The cumulative effect of hypertension and hypercholesterolemia leads to additive impairment of the NO-dependent endothelial function.⁹ Many researchers have studied the blood pressure lowering effect as well as cholesterol lowering effect of dietary proteins, based on their amino acid profiles, especially arginine : lysine (Arg : Lys) ratio in hypertension, atherosclerosis and hypercholesterolemia conditions.¹⁰ The arginine-rich food sources should have the high Arg : Lys ratio levels to exert its affect on cardiovascular diseases.¹¹ Lysine rich animal proteins are known to be hypercholesterolemic and the different effects of plant and animal proteins on serum lipids appear to be primarily caused by differences in their amino acid composition.^6,12 Plant proteins with high Arg : Lys ratio are known to reduce cholesterol levels in hypercholesterolemic conditions.¹¹

Hypocholesterolemia was observed by supplementing diet with plant protein source like sesame seed globulin having high Arg : Lys ratio, compared to animal protein source like casein with low Arg : Lys ratio.^12,13 The difference in effects of plant and animal proteins on serum lipids appear to be primarily due to differences in their amino acid composition.^14,15 A study also suggests that the impact of dietary protein on lipid metabolism is source-dependent.¹⁶ Studies in rats have demonstrated that soy protein isolate (SPI) intake could modulate lipid and energy metabolism, including the synthesis and degradation of cholesterol.¹⁷ The lowering of serum and hepatic cholesterol levels was reported in rats fed with soy protein diet compared to those fed with a casein diet.¹⁸

The basic sources of dietary arginine are nuts and legumes where most grains have limited amount of arginine.¹¹ Moringa oleifera a tropical plant belongs to the family of Moringaceae. In the present study, along with Moringa various other oilseeds were screened for proteins with high arginine content for preparation of protein isolate. Oil seed proteins are the rich source of biologically active peptides. Peptides with biological activity could be produced from food in several ways. Hydrolysis of proteins using proteolytic enzymes produces peptides of various lengths and free aminoacids that exhibit biological activities. There are many reports on ACE inhibitory peptides that have been isolated from enzymatic digests of various food materials, including rape seed,¹⁹ sunflower,²⁰ peanut²¹ and flax seed.²²

In the present study Moringa seed protein isolate was prepared and subjected to in vitro enzymatic hydrolysis using commercially available food grade proteases and by human gastrointestinal juices (ex vivo) to study the digestibility of the MPI. The protein was examined for its hypotensive and hypocholesterolemic effect by in vivo methods. Many hypocholesterolemic plant proteins that are resistant to gastrointestinal digestion are known to bind bile acids/cholesterol and exhibit the said effect. Although the role of Arg : Lys ratio in hypercholesterolemia was known but the effective concentration ratio to show hypotensive and hypocholesterolemic effect is not known. Here, a comparative study of Arg : Lys ratio has been carried out in free form of aminoacids and in protein form. The present work focuses on the impact of dietary aminoacid/MPI with high Arg : Lys ratio and its influence on hypercholesterolemia and hypertension in rat models.

2. Materials and methodology

2.1. Materials

The Moringa oleifera seeds (Bhagya var.) and other oil seeds like groundnut (TMV-1), sesame seeds were obtained from University of Agricultural Sciences, Dharwad, Karnataka (India). All the solvents used were of analytical grade (AR), procured from Rankem fine chemicals Pvt. Ltd. (Bengaluru, India). Hippuryl-L-histidyl-L-leucine (HHL), L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) was purchased from Sigma-Aldrich (Bengaluru, India). HPLC grade chemicals and dimethyl sulphoxide (DMSO) was obtained from Merck India Ltd. (Bengaluru, India). All other chemicals used in the diet preparation were of analytical grade, obtained from Himedia, (Mumbai, India). Casein protein was procured from Nimesh Corporation (NewDelhi, India). All experiments were performed in compliance with the relevant laws and institutional guidelines, and also the institutional committee(s) have approved the experiments. The human gastric and duodenal juices were kind gift from Prof. G. E. Vegarud (Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Norway), which were aspirated from healthy volunteers and the aspiration of human contents were approved by the Norwegian Ethics Committee.

2.2. Preparation of Moringa oleifera seeds protein isolate (MPI)

Moringa seeds were dehulled, pulverized, defatted and powdered for extraction of protein. Defatted Moringa seed meal was dispersed in 10% (w/v) NaCl in 1 : 20 ratio and the slurry was stirred for 2 h at room temperature. The slurry was centrifuged at 9000 rpm for 15 min to remove insoluble material. The pH of the supernatant was adjusted to 4 and kept at cold conditions (4–8 °C) for 4 h to precipitate the protein. The protein fraction was collected by centrifugation at 9000 rpm for 15 min. The precipitated protein was dispersed in 5-fold water and again centrifuged (9000 rpm for 15 min) to obtain the protein precipitate (residue II) and the supernatant. The pH of the supernatant was adjusted to 8 and kept at cold condition (4–8 °C) for 4 h to precipitate the protein. The protein fraction (residue III) was collected by centrifugation at 9000 rpm for 15 min. The residue II and residue III were mixed and lyophilized in lyophilizer (Virtis, India) to get the protein isolate. The protein content was estimated by Kjeldahl's method according to Official Methods of Analysis (15^th edn). Washington, DC, USA: Association of Official Analytical Chemists²³ and the isolate was subjected to aminoacid analysis.

2.3. Amino acid analysis

A known amount of protein was hydrolyzed in 6 N HCl (distilled acid with 0.1% phenol) for 24 h under vacuum at 110 °C. Amino acid analysis was performed by pre-column derivatization with PITC (phenyl thiocarbamoyl). The phenyl thiocarbamoyl amino acids were analyzed by reverse phase high performance liquid chromatography (RP-HPLC).²⁴

2.4. Digestibility of MPI using commercial enzymes

The Moringa protein isolate was dispersed in distilled water (5%, w/v) and the pH was adjusted with 0.5 M NaOH/HCl. The digestibility was studied by adding alcalase (at pH 7.5, 50 °C), trypsin (at pH 7.5, 37 °C), pepsin (at pH 2, 37 °C), ficin (at pH 6.5, 45 °C) and pancreatin (at pH 7.5, 37 °C) at 1 : 100 ratio (w/w, protein basis) to the protein solution and hydrolysed for 5 h. Aliquots at different time intervals were collected to determine the degree of hydrolysis. The hydrolysates were subjected to aminoacid analysis.

2.5. Ex vivo digestibility of MPI using human gastro-duodenal enzymes

To 5 ml of 2.5% MPI prepared in water at pH 2, 2.5 ml of human gastric juice (HGJ) was added and the pH was checked and readjusted to pH 2. After 1 h incubation at 37 °C, the pH of the hydrolysate was adjusted to pH 6 to mimic in vivo conditions and 2.5 ml of human duodenal juice (HDJ) was added. Then, pH was adjusted to 7.5 and incubated for 2 h. At the end of 2 h the reaction was stopped by freezing the reaction and was used for further analysis.^25,26

2.6. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was carried out for the visualization of the protein bands of the digested samples. The assay was performed on 5% stacking and 12% separating gels, according to the protocol as described by Laemmli.²⁷ The protein bands were stained with Coomassie Brilliant Blue R-250 to visualize the separated proteins and released fragments.

2.7. Degree of hydrolysis (DH)

1 ml aliquot of the hydrolysate withdrawn at different time intervals was added to 1 ml of 5% trichloroacetic acid (TCA). The mixture was centrifuged at 8000 rpm for 15 min. The protein content in TCA-soluble fraction and the hydrolysate (without the addition of TCA) were determined by Lowry's method²⁸ and the DH value, expressed as a percentage, was calculated as the ratio of TCA-soluble protein to total protein in the hydrolysate.

2.8. Animal studies on hypertension

The experiments were performed in compliance with the relevant laws of CPCSEA and institutional guidelines, and approved by the IAEC (IAEC No. 222/12) the institutional committee(s). Twelve-week-old male Wistar rats (Rattus norvegicus), weighing 180 to 200 g, were obtained from the Animal House facility, CSIR-Central Food Technological Research Institute, Mysuru, India. The rats were housed individually at 25 ± 3 °C and 12 h light/dark cycle basis. Rats were acclimatized to these conditions for 2 weeks with water and AIN-93M diet ad libitum. After acclimatization, 4 rats in each group were placed as per Table 1 and were fed with the control diet (basal AIN-93M diet) and the high cholesterol diet (HCD) (with 1.0% cholesterol, 0.125% bile acid mixture and 10% fat as groundnut oil) for 16 weeks to induce hypercholesterolemia. After induction, the animals were randomized based on the systolic blood pressure to the groups as per Table 2. Three groups of HCD rats were administrated with Nω-nitro-L-arginine methyl ester hydrochlorides (L-NAME) orally 50 mg kg⁻¹ (ref. 29) (w/w, body weight) per day to induce hypertension and the treatment groups were supplemented with Moringa protein isolate (MPI) and free amino acids (FAA) with Arg : Lys ratio of 5 : 1 (w/w) and 5 : 1 (w/w) respectively for 6 weeks as shown in Table 1.

Table 1 Grouping of animals in hypertension induced in hypercholesterolemic models

Ingredients	Groups
	Control (g kg^−1)	HCD (g kg^−1)	LN (g kg^−1)	FAA-LN (g kg^−1)	MPI-LN (g kg^−1)
Corn starch	600	600	600	535	600
Cellulose	50	50	50	50	50
Casein protein	200	200	200	200	20
Moringa seed protein	—	—	—	—	180
Groundnut oil	100	100	100	100	100
Mineral mix	35	35	35	35	35
Vitamin mix	10	10	10	10	10
DL-Methionine	0.3	0.3	0.3	0.3	0.3
Sodium cholate	20	20	20	20	20
L-Arginine	—	—	—	65	—
Cholesterol	—	10	10	10	10
Bile acids	—	1.25	1.25	1.25	1.25
Arg : Lys ratio	0.37 : 1	0.37 : 1	0.37 : 1	5 : 1	5 : 1

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Table 2 Composition of experimental diets (g kg⁻¹)

Groups names	Groups label	Composition
Control	Control	Diet with 20% casein protein
High cholesterol diet (HCD) control	HCD	Diet with 20% casein protein with 1% cholesterol
HCD + L-NAME (LN) control	LN	Diet with 20% casein protein with 1% cholesterol + 50 mg kg^−1 BW of L-NAME
HCD + L-NAME + free amino acids (FAA)	FAA-LN	Diet with 20% casein protein + 1% cholesterol diet + 50 mg kg^−1 BW of L-NAME + 6.5% L-arginine
HCD + L-NAME + Moringa seed protein (MPI)	MPI-LN	Diet with 18% Moringa seed protein isolate (MPI) + 2% casein + 1% cholesterol diet + 50 mg kg^−1 BW of L-NAME

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2.9. Measurement of blood pressure

The Wistar rats were trained for 2 weeks during acclimatization for systolic blood pressure measurement by the tail-cuff method by placing the rats into restrainers with an electro sphygmomanometer³⁰ (Model 179, Blood Pressure Analyzer IITC, Woodland Hills, CA, USA). The experimental design was to induce hypertension through hypercholesterolemia in rats fed with cholesterol (16 weeks) followed by L-NAME (6 weeks). Blood pressure was measured thrice a week during experimental period and the blood pressure was calculated as the average of the four values for each animal in a group.

2.10. Blood and tissue sampling

Samples were collected at the end of the study by subjecting the animals to anesthesia followed by euthanasia. Blood was collected in 300 mM sodium citrate and plasma was separated for further analysis. Similarly, liver and kidney were homogenized in 400 mM Tris buffer (pH 7.2) containing protease inhibitors. Lipid profiles such as total cholesterol (TC), triglycerides (TAG) and phospholipids were measured in the plasma by the manufacturer's protocol (Agapee Diagnostic Kits, Kerala) and in liver tissue by chemical method. Liver damage markers like aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) were assessed in the plasma samples by the manufacturer's protocol (Agapee Diagnostic Kits, Kerala). Kidney tissues were assayed for molecular markers like Angiotensin-I Converting Enzyme (ACE) by Cushman and Cheung method,^32,33 nitric oxide (NO) by the modified Griess reaction method.³¹

2.11. Western blot analysis of hypertensive markers

Frozen kidney samples were homogenized in RIPA buffer containing 10 mM Tris–HCl (pH 6.8), 2% SDS, proteinase inhibitors (Sigma), and then centrifuged at 12 000 rpm for 20 min at 20 °C. Supernatants were collected, and the protein concentration was determined by Lowry's assay. 50 μg of total protein was loaded on 10% SDS-PAGE, separated and electroblotted onto PVDF transfer membrane (GILA laboratories). The following antibodies were used: rat polyclonal antibody against AT1R (sc-1173-G) (Santacruz), ACE 1 (ab134709), and polyclonal rabbit antibody against β-actin (ab8227) (Abcam). HRP-conjugated secondary antibody (sc-2030) was obtained from Santa Cruz Biotechnology. Immunodetection was accomplished with enhanced chemiluminescence by using Western Blotting Luminol Reagent (Bio-Rad Laboratories). Band intensities of analyzed proteins were quantified and normalized to the intensity of actin band, with control value assumed as 1. Quantification of western blots was performed using Genesys V1.4.0.0 (Syngene). The results were expressed in arbitrary units (A.U.), with one unit being the mean of corresponding protein (AT1R and ACE 1) level determined in the control group. Equal loading of the protein in each lane was verified by Pounceau staining.

2.12. Statistical analysis

All the results were expressed as means ± standard deviation. All the values were plotted using the Origin 6.1 software. One way analysis of variance (ANOVA) was employed to identify significant differences (p < 0.05) between data sets using Origin 6.1.

3. Results and discussion

3.1. Moringa seed protein isolate (MPI)

Moringa olifera (var. Bhagya) seeds dehulled, flaked and defatted were used for further study. The total protein content of the defatted Moringa seed flour was 56.46 ± 2.0 g/100 g sample. Our study showed that the protein extraction was dependent on pH, salt and concentration of the sample. Maximum extractability of Moringa seed protein in water is at acidic pH (Fig. 1). Beyond pH 4 towards alkaline pH, the protein extractability is very less. In presence of sodium chloride (NaCl) salt (10% w/v) extractability of Moringa seed protein was studied at different pH conditions (Fig. 1). The extractability improved in presence of NaCl salt which was 3-fold higher compared to water. The maximum extractability was observed at pH 6, with minimum extractability at pH 4 and pH 8 (Fig. 1). The minimum extractability at pH 4 and pH 8 were found to be the two isoelectric points of the proteins. Moringa seed protein with multiple isoelectric points has been reported earlier.^34,35 The Moringa seed protein isolate (MPI) preparation was optimized based on the solubility of MPI at different pH in NaCl solution. The method for the preparation of MPI was optimized by isoelectric precipitation in the presence of NaCl by varying the pH as shown in Fig. 2. The MPI prepared had a protein content of 85%. The proximate composition of MPI was performed and data shown in ESI (Table S1†). The protein isolate preparation process was optimized to remove the presence of hydrocolloids such as jellose/galactomannans, presence of which otherwise interfere in protein isolate preparation, as they are soluble in both water and salt solutions. Similar method for separation of hydrocolloid using salt has been reported in fenugreek seed.³⁶ Separation of tamarind jellose at high temperature and precipitating by using ethanol at various pH conditions has been reported earlier.³⁷

Fig. 1 Effect of pH (-●-) and sodium chloride (NaCl) (-■-) concentration on percent extractability of Moringa seed protein.

Fig. 2 Schematic representation showing the preparation of protein isolate from Moringa seed.

3.2. Digestibility by in vitro and ex vivo method

MPI was hydrolysed by different commercial food grade proteases viz., pepsin, pancreatin, alcalase, trypsin and ficin for about 5 h at their optimised condition of pH and temperature. Hydrolysis of MPI at 120^th min by enzymes such as pepsin showed 49.1 ± 2.8% DH, pancreatin showed 45.7 ± 1% DH, trypsin showed 38.8 ± 2.2% DH, alcalase with 56.2 ± 1.4% DH and ficin with 28.5 ± 3.9% DH. Thus, the pepsin, trypsin and pancreatin showed gradual increase of DH exponentially till 300 min in all enzymes except in alcalase, where after 60^th min DH remained constant (Fig. 3A). Thus, by in vitro method of digestion, both the enzymes pepsin and alcalase exhibited the highest digestibility and DH compared to the other enzymes at the end of 5 h of digestion. The aminoacid composition of all the hydrolysates showed arg : lys ratio of 3 : 1, data shown in ESI (Table S2†). The minimal inhibitory activities (IC₅₀) of the different hydrolysates is shown in Table S3.†

Fig. 3 (A) Degree of hydrolysis of Moringa seed protein isolate after digestion by different proteases. (B) SDS-PAGE pattern of protein bands of Moringa seed protein isolate (MPI) sequentially digested with human gastric and duodenal enzymes. Lane 1,7 – MPI hydrolyzed with gastric juice followed by duodenal juice, Lanes 2,6 – MPI hydrolysed with gastric juice, Lanes 3,5 – unhydrolysed MPI, Lane 4 – molecular weight markers, Lane 8 – human duodenal juice and Lane 9 – human gastric juice.

Pepsin preferentially cleaves at Phe, Tyr, Trp and Leu in position P1.³⁸ The gastro-duodenal juice digestion of MPI showed better digestibility as shown in electrophoretic profile (Fig. 3B). The control MPI exhibits protein bands in the molecular weight range of 45 to 21 and at ≤14 kDa. The electrophoretic protein profile shows the lower molecular weight protein (≤14 kDa) remains resistant and higher molecular proteins (45 to 21 kDa) get digested by gastro-duodenal juice (Fig. 3B). Not much work on Moringa seed protein purification and characterization has been carried out. Ghebremichael et al.³⁵ reported a cationic protein from Moringa seed with the molecular mass less than 6.5 kDa. Similar reports have shown water soluble Moringa lectin, with a polypeptide band of 26.5 kDa.³⁹ A protein dimer having haemagglutinin activity with a molecular mass of 14 kDa and subunits (7.1 kDa) has also been reported.⁴⁰ The MPI hydrolysed by (pepsin, trypsin) in vitro method and (duodenal juice, gastric juice) ex vivo method both exhibited similar trend of digestibility. The in vitro enzymatic processing facilitates the easy digestion of resistant plant storage protein by gastro-intestinal enzymes, but the enzymatic processing of MPI was not necessary, as the MPI can be directly digested by gastro-duodenal juice (in vivo) when supplemented to rats.

A variety of ACE inhibitors are derived from various plant and animal protein sources. Structure activity correlation among different peptide inhibitors indicate that ACE prefers substrates or competitive inhibitors mainly with hydrophobicity (aromatic and branched chain aminoacids), different amino acid sequences, side chain length,⁴¹ branched amino acids at N-terminus of peptides and bulkier aminoacids at the C-terminal.⁴²

3.3. Amino acid composition

Oil seed proteins with high arginine content were screened based on their amino acid profile. The total aminoacid composition of seed protein isolates was prepared from Moringa, sesame and groundnut (Table 3). The arginine : lysine (Arg : Lys) ratios of Moringa, sesame and groundnut protein isolates were 8.7, 4.8 and 2.3 respectively. Among the three arginine rich proteins, Moringa protein isolate (MPI) exhibited high Arg : Lys ratio. The essential and non-essential amino acid content of MPI was compared with that of casein (Table 4). The MPI and Moringa protein flour (MPF) exhibit 8.7 : 1 and 6 : 1 Arg : Lys ratios respectively, which indicated that MPI had significantly high Arg : Lys ratio compared to MPF (data not shown). The MPI with 85% protein has 15% arginine and 1.7% lysine (Table 3), whereas MPF with 56.5% protein showed 18% arginine and 7.8% lysine.

Table 3 Total aminoacid composition of protein isolates prepared from oil seeds

Aminoacids	Moringa (g%)	Sesame (g%)	Groundnut (g%)
Aspartate	8.08 ± 0.12	9.2 ± 0.09	20.65 ± 0.13
Glutamate	20.87 ± 0.31	22.8 ± 0.22	17.73 ± 0.06
Serine	4.35 ± 0.06	4.3 ± 0.04	6.18 ± 0.08
Glycine	7.51 ± 0.11	5.1 ± 0.05	5.24 ± 0.02
Histidine	1.26 ± 0.01	2.7 ± 0.02	1.14 ± 0.00
Arginine	15.25 ± 0.22	12.5 ± 0.12	11.11 ± 0.08
Threonine	3.69 ± 0.05	3.3 ± 0.03	3.18 ± 0.10
Alanine	6.21 ± 0.09	4.5 ± 0.05	5.41 ± 0.02
Proline	4.42 ± 0.06	4.7 ± 0.05	3.01 ± 0.07
Tyrosine	0.96 ± 0.01	2.1 ± 0.02	2.42 ± 0.04
Valine	5.73 ± 0.09	4.1 ± 0.04	5.51 ± 0.14
Methionine	2.01 ± 0.03	3.9 ± 0.04	0.69 ± 0.47
Cysteine	2.9 ± 0.04	3.3 ± 0.03	0.52 ± 0.13
Isoleucine	3.19 ± 0.05	4.9 ± 0.05	2.89 ± 0.01
Leucine	7.29 ± 0.11	7.5 ± 0.08	7.05 ± 0.01
Phenylalanine	4.52 ±± 0.06	2.1 ± 0.02	4.77 ± 0.06
Lysine	1.76 ± 0.03	2.6 ± 0.06	2.88 ± 0.09
Arg : Lys ratio	8.7 : 1	4.8 : 1	3.86 : 1

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Table 4 Essential aminoacid (EAA) and non-essential aminoacid (EAA) contents in Moringa protein isolate and casein protein (g per 100 g protein)

EAA	Moringa (g%)	Casein (g%)	NEAA	Moringa (g%)	Casein (g%)
Threonine	3.69	3.57	Aspartate	8.08	6.19
Valine	5.73	3.51	Glutamate	20.87	23.39
Methionine	2.01	8.16	Serine	4.35	4.67
Isoleucine	3.19	4.51	Glycine	7.51	1.73
Leucine	7.29	9.77	Histidine	1.26	2.52
Phenylalanine	4.52	4.66	Arginine	15.25	2.68
Lysine	1.76	7.16	Alanine	6.21	2.69
			Proline	4.42	10.91
			Tyrosine	0.96	1.00
			Cysteine	2.9	2.88

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The high Arg : Lys ratios is crucial because, the amino acids arginine and lysine compete for the same transporter, there by influencing each other absorption in the instestine.⁶ Also high ratio of arginine and lysine is essential because high arginine is responsible for hypotensive effect.¹³ As per the literature, lysine has been reported to be hypercholesterolemic.^7,43,44 The arginine content is found highest in globulin proteins belonging to most of the seed storage proteins. Plant proteins have higher absolute amounts of dispensable amino acids such as arginine, glycine and alanine whereas animal proteins have the higher amounts of indispensable amino acids such as lysine. Plant proteins (high arginine content) are known to be hypocholesterolemic compared to animal proteins (high lysine content) that are known to be hypercholesterolemic. As per the Table 4, MPI compared to casein has 23 fold high Arg : Lys ratio content. The amino acid data showed that MPI is rich in glutamic acid (20.9%) and arginine (15.3%) which is in corroboration with earlier report.⁴⁵ The essential amino acid content in MPI is at par with casein except for lysine and methionine. A study reported that Moringa showed high contents of glutamine, arginine and proline as well as other residues.⁴⁵

3.4. Effect on systolic blood pressure

This experiment was designed to study the efficacy of Arg : Lys aminoacid on hypertension induced by L-NAME in hypercholesterolemic Wistar rats. The hypertensive activities were indicated by the increased systolic blood pressure (SBP) from 114 ± 2.5 mmHg in control group to 160 ± 1.8 mmHg in high cholesterol diet fed (HCD) group from 0 to 16^th week. Subsequently, L-NAME an analogue of arginine was administered to HCD group rats to induce hypertension. Our observations showed that L-NAME + high cholesterol diet (LN) rats exhibited increased SBP of 146.7 ± 4.5 mmHg. The (FAA-LN) group fed with L-NAME + high cholesterol diet + free aminoacid with Arg : Lys ratio of 5 : 1 had 127.5 ± 0.2 mmHg SBP and the (MPI-LN) group fed with L-NAME + high cholesterol diet + Moringa seed protein isolate with Arg : Lys ratio of 5 : 1 had 126.6 ± 2.2 mmHg SBP.

Our study reported significant decrease in SBP in hypertensive rats in MPI-LN group (20.1 mm Hg) (p < 0.05) and FAA-LN group (19.2 mmHg) (p < 0.05) (Fig. 4A). Both the free form and protein form of Arg : Lys ratios show hypotensive effect within same time period (6 weeks) (Fig. 4B). Other studies have reported that the hydrolysates of pea,⁴⁶ soy,⁴⁷ apricot⁴⁸ exhibit SBP lowering effect by 10 mm Hg on oral gavage feeding which lasted for about 2 h. As per the literature, arginine rich peptide product showed greater hypotensive effect (at 2 h) than those of the flaxseed protein isolate (at 6 h) on treatment.⁴⁹

Fig. 4 (A) Systolic blood pressure (SBP, mmHg) measured in L-NAME induced hypertensive rats. (B) SBP values at different intervals were compiled. Different from control, (p < 0.05), indicates significant difference among the groups depicted by alphabets (a, b, c). Values are expressed as means ± standard deviation.

3.5. Effect on hypertensive markers in kidney

Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) was used as a nitric oxide synthase (NOS) inhibitor that induces hypertension and cardiovascular remodeling in which ACE activity and nitric oxide (NO) levels were effected.⁵⁰ Many studies have reported that chronic blockade of NO synthesis by NOS inhibitor like L-NAME causes endothelial dysfunction, a significant increase in blood pressure and further pathological injuries to the cardiovascular system and kidneys, which may lead to aggravation of hypertension.⁵¹ In our study, kidney angiotensin I converting enzyme (ACE) exhibited 154.5 ± 6.2 nmole per mg per min and 92.04 ± 5.5 nmole per mg per min activity in high cholesterol fed (HCD) group and L-NAME hypertensive (LN) rats respectively. Whereas, the effect of free form of aminoacid (FAA-LN) and protein form (MPI-LN) showed significant decrease in ACE activity with 37.56 ± 3.7 nmoles per mg per min and 10.30 ± 3.7 nmoles per mg per min respectively (Fig. 5A) (p < 0.05). This hypotensive effect is confirmed by western blot method, ACE levels were significantly down-regulated in Moringa seed protein isolate treatment (MPI LN) and AT1 levels were significantly down-regulated by free aminoacid (FAA LN) as shown in Fig. 6A and B. Another study reported earlier the kidney ACE activities (0.5–0.65 μmole per mg) remained unchanged for 12 weeks after treatment.⁵²

Fig. 5 Kidney tissue exhibiting the (A) angiotensin I converting enzyme (ACE) activity (p < 0.05), (B) nitrate levels (p < 0.05) in hypertensive Wistar rats where L-NAME is used as negative control of hypertensive markers. Control diet, HCD: high cholesterol diet, LN: high cholesterol diet + L-NAME, FAA-LN: high cholesterol diet + L-NAME + free Arg : Lys aminoacid, MPI-LN: high cholesterol diet + L-NAME + Moringa seed protein isolate. Protein expression of ACE and AT1R values were normalized with β-actin. Values are expressed as means ± standard deviation (p < 0.05).

Fig. 6 Relative intensity of (A) angiotensin I converting enzyme (ACE) and (B) angiotensin II type I receptor (AT1R), in lung tissue of Wistar rats where L-NAME is used as negative control of hypertensive markers. Control diet, HCD: high cholesterol diet, LN: high cholesterol diet + L-NAME, FAA-LN: high cholesterol diet + L-NAME + free Arg : Lys aminoacid, MPI-LN: high cholesterol diet + L-NAME + Moringa seed protein isolate. Protein expression of ACE and AT1R values were normalized with β-actin. Values are expressed as means ± standard deviation (p < 0.05).

The plasma parameters were analyzed for blood urea nitrogen (BUN), creatinine and albumin content that did not show significant change in hypertensive and control group rats. The kidney damage markers like protein carbonyl and reactive oxygen species (ROS) levels analysed in kidney showed no significant change in hypertensive rats compared to control rats (Table S4†).

The free form arginine supplemented to L-NAME fed rats showed decrease in ACE levels as well as systolic blood pressure (SBP) values with increase in nitric oxide synthase (NOS) activity. There was a significant increase in nitrate levels indicating vasodilation process involved in hypotension in both MPI-LN and FAA-LN groups. The nitrate levels in control group was 0.56 ± 0.03 μM g⁻¹, HCD and LN groups showed lowest levels among the group with 0.35 ± 0.03 μM g⁻¹ and 0.31 ± 0.02 μM g⁻¹ respectively. In case of treated groups the FAA-LN and MPI-LN groups showed moderate increase in the levels of nitrate with 0.4 ± 0.06 μM g⁻¹ and 0.47 ± 0.04 μM g⁻¹ respectively compared to hypertensive groups (Fig. 5B). Both the treatment groups showed decrease in the ACE activity indicating the antihypertensive activity of the peptides from Moringa protein. The action of MPI may be due to the formation of peptides by gastro-intestinal digestion in rat. Flax seed protein hydrolysate rich in arginine showed renin and ACE inhibitory effect by in vitro method which also showed reduction in the blood pressure in spontaneous hypertensive rats.²² Rice protein hydrolysed by alcalase showed ACE inhibitory activity with significant decrease in systolic blood pressure.⁵³

3.6. Effect on hypercholesterolemia

In the present study, total cholesterol (TC) and triglyceride (TAG) levels increased significantly in HCD group after 13 weeks of intervention. The blood plasma showed TAG value 78.2 ± 7.3 mg dL⁻¹ and 42.6 ± 6.9 mg dL⁻¹ in HCD and LN groups, where as FAA-LN (93.2 ± 9.9 mg dL⁻¹) and MPI-LN groups (62.8 ± 1.2 mg dL⁻¹) did not show any significant change in TAG after treatment (Table 5). Liver TAG levels decreased in FAA-LN group (21.6 ± 4.7 mg g⁻¹) and MPI-LN group (20.9 ± 3.9 mg g⁻¹) compared to LN group (27.9 ± 2.0 mg g⁻¹).

Table 5 Estimation of biochemical parameters like total cholesterol (TC), triglycerides (TAG), in blood plasma and liver TC, TAG, phospholipids (PC). Liver damage enzyme markers like alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), in blood plasma. Control, HCD: high cholesterol diet fed, LN: high cholesterol diet + L-NAME, FAA-LN: high cholesterol diet + L-NAME + free Arg : Lys aminoacid, MPI-LN: high cholesterol diet + L-NAME + Moringa seed protein isolate. Values are expressed as mean ± standard deviation (p < 0.05)

Parameters	Nor Con	HCD Con	LN Con	FAA-LN	MPI-LN
Plasma TAG (mg dL^−1)	67.2 ± 8.6	78.2 ± 7.3	42.6 ± 6.9	93.2 ± 9.9	62.8 ± 1.2
Plasma TC (mg dL^−1)	47.3 ± 8.9	93.8 ± 2.2	114.6 ± 4.5	140.7 ± 9.9	120.0 ± 17.5
Liver TAG (mg g^−1)	1.2 ± 0.3	18.6 ± 3.0	27.9 ± 2.0	21.6 ± 4.7	20.9 ± 3.9
Liver TC (mg g^−1)	3.0 ± 0.3	26.1 ± 4.1	37.4 ± 2.9	44.6 ± 5.6	44.6 ± 8.6
Liver PL (mg g^−1)	4.2 ± 0.9	3.5 ± 0.4	2.6 ± 0.4	6.3 ± 0.7	6.3 ± 1.3
ALP (U L^−1)	366.4 ± 14.7	380.8 ± 9.2	346.8 ± 1.4	389.4 ± 48.5	293.6 ± 2.9
AST (U L^−1)	167.2 ± 11.1	253.0 ± 23.3	195.8 ± 13.7	148.3 ± 39.7	160.10 ± 39.7
ALT (U L^−1)	96.9 ± 17.5	111.5 ± 14.7	104.1 ± 13.3	118.0 ± 9.8	110.71 ± 12.8

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The TC levels in blood plasma in hypertensive rats were not significantly affected (Table 5). The total cholesterol in liver of HCD showed 26.1 ± 4.1 mg g⁻¹ and the LN showed 37.4 ± 2.9 mg g⁻¹, with no significant change in the treatment groups (Table 5). There was no change observed in liver phospholipids, liver injury markers like AST, ALT and ALP in blood plasma (Table 3). The body weight of the rats and food ingested by rats was monitored throughout the experiment, these parameters did not show any effect in hypertensive rats shown in (Table 6). The affect of diet on the weight changes in all groups as well as the animal appetite were monitored, since increased food intake might affect all parameters involved in controlling hypertension (Table 6). The weight of heart, kidneys, lungs and liver have been tabulated (Table 7).

Table 6 Body weight and diet weight of experimental hypertensive rats. Control, HCD: high cholesterol diet fed, LN: high cholesterol diet + L-NAME, FAA-LN: high cholesterol diet + L-NAME + free Arg : Lys aminoacid, MPI-LN: high cholesterol diet + L-NAME + Moringa seed protein isolate. Values are expressed as mean ± standard deviation (p < 0.05)

	Samples	Control	HCD	LN	LN FAA	LN MPI
Body weight (g)	Week 1	214 ± 21	224 ± 16	—	—	—
	Week 14	285 ± 23	294 ± 26	307 ± 21	305 ± 15	322 ± 32
	Week 20	308 ± 11	305 ± 15	317 ± 22	317 ± 19	321 ± 18
Diet intake (g)	Week 1	11 ± 2	11 ± 2	—	—	—
	Week 14	15 ± 1	15 ± 1	16 ± 1	14 ± 2	15 ± 3
	Week 20	14 ± 1	14 ± 1	13 ± 1	16 ± 2	13 ± 1

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Table 7 Major organ weights of all the groups of hypertensive rats

Samples	Control	LN	HCD	LN FAA	LN MPI
Kidneys (g)	1.4 ± 0.6	1.0 ± 0.1	1.4 ± 0.3	1.0 ± 0.1	1.1 ± 0.2
Liver (g)	9.1 ± 1.2	11.2 ± 1.3	11.9 ± 2.2	10.8 ± 0.5	11.1 ± 0.7
Lungs (g)	1.4 ± 0.1	1.6 ± 0.4	1.3 ± 0.3	1.3 ± 0.3	1.1 ± 0.1
Heart (g)	0.65 ± 0.1	0.63 ± 0.1	0.66 ± 0.2	0.64 ± 0.1	0.68 ± 0.2

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Literature shows Sprague-Dawley rats fed with high Lys : Arg ratio (1.58 : 1) diet exhibited high levels of total and HDL-cholesterol compared to rats fed with low Lys : Arg ratio (0.36 : 1) diet which exhibited low total and HDL-cholesterol levels.⁵⁴ The diet supplemented with low Arg : Lys ratio (0.17 : 1) showed maximum elevation of serum cholesterol, whereas Arg : Lys ratio of 0.48 : 1 caused moderate antihypercholesterolemic effect.⁵⁵ Addition of moderate amount of Arg to the hypercholesterolemic diets having Lys or Met aminoacids did not alter the total, LDL, VLDL and HDL cholesterol and had no effect on serum TAG levels.⁵⁶ An inhibition of NO synthesis causes hyperlipidemia and fat accretion in rats, whereas dietary arginine supplementation reduces fat mass in diabetic fatty rats.⁵⁷ Our observations showed hypocholesterolemic and hypotensive effect after MPI and the free aminoacid treatment. We report MPI treatment significantly decreases liver TAG levels exhibiting hypocholesterolemic effect whereas decrease in kidney ACE levels and increased nitrate levels showed hypotensive effect.

4. Conclusion

Screening for arginine-rich seed protein was carried out and protein isolate (MPI) from Moringa seed was prepared by iso-electric precipitation method having two iso-electric points. The in vitro enzymatic hydrolysis of MPI with Arg : Lys ratio of 8.7 : 1, exhibited highest protein digestibility in pepsin even in the ex vivo digestion using human gastro-duodenal juice. Hypertension was induced by high cholesterol diet as well as L-NAME intubation in Wistar rats in vivo model. The Arg : Lys ratio of MPI with 8.7 : 1 was adjusted to 5 : 1 by supplementing with casein protein. The hypotensive activity of Arg : Lys ratio observed may be due to overall effect on both renin–angiotensin and nitric oxide–cGMP mechanistic pathways of hypertension involving vasodilatory function. Our observations showed that there was moderate hypocholesterolemic effect of MPI and FAA. From the enzymatic hydrolysis of arginine rich protein (MPI), the peptides were released having potential hypotensive effect like ACE inhibitory activity. Hence, from our study we conclude, the free form of arginine and protein form both execute hypotensive effect and hypocholesterolemic effect. The MPI with therapeutic properties can be promoted as a functional food ingredient.

Acknowledgements

We thank our Director, CSIR-CFTRI, Mysuru for his constant support and providing the facility to carry out the work in the institute. The authors are thankful to Prof. G. E. Vegarud, Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Norway for providing help in ex vivo digestion experiments. This research was funded and supported by five-year plan project (WELFO), CSIR, New Delhi.

References

1. P. M. Kearney, M. Whelton, K. Reynolds, P. Muntner, P. K. Whelton and J. He, Global burden of hypertension: analysis of worldwide data, Lancet, 2005, 365, 217–223.
2. W. Chai, W. Wang, J. Liu, E. J. Barrett, R. M. Carey, W. Cao and Z. Liu, Angiotensin II type 1 and type 2 receptors regulate basal skeletal muscle microvascular volume and glucose use, Hypertension, 2010, 55, 523–530.
3. B. Murray, R. J. FitzGerald, H. Miesel and D. J. Walsh, Nutraceutical Proteins and Peptides in Health and Disease, ed. F. S. Oshinori Mine, CRC Press, 2005.
4. K. S. Heffernan, C. A. Fahs, S. M. Ranadive and E. A. Patvardhan, Review Article: L-Arginine as a Nutritional Prophylaxis Against Vascular Endothelial Dysfunction With Aging, J. Cardiovasc. Pharmacol. Ther., 2010, 15, 17–23.
5. M. G. Di Pasquale, Amino Acids and Proteins for the Athlete: The Anabolic Edge, CRC Press, New York, 2nd edn, 2007.
6. H. Azuma, J. Sato, H. Hamasaki, A. Sugimoto, E. Isotani and S. Obayashi, Accumulation of endogenous inhibitors for nitric oxide synthesis and decreased content of L-arginine in regenerated endothelial cells, Br. J. Pharmacol., 1995, 115, 1001–1004.
7. P. Hevia, F. W. Kari, E. A. Ulman and W. J. Visek, Serum and liver lipids in growing rats fed casein with L-lysine, J. Nutr., 1980, 110, 1224–1230.
8. P. Clarkson, M. R. Adams, A. J. Powe, A. E. Donald, R. McCredie, J. Robinson, S. N. McCarthy, A. Keech, D. S. Celermajer and J. E. Deanfield, Oral L-arginine improves endothelium-dependent dilation in hypercholesterolemic young adults, J. Clin. Invest., 1996, 97, 1989–1994.
9. V. Vermeirssen, J. Van Camp and W. Verstraete, Bioavailability of angiotensin I converting enzyme inhibitory peptides, Br. J. Nutr., 2004, 92, 357–366.
10. A. R. Tovar, F. Murguía, C. Cruz, R. Hernández-Pando, C. A. Aguilar-Salinas, J. Pedraza-Chaverri, R. Correa-Rotter and N. Torres, A soy protein diet alters hepatic lipid metabolism gene expression and reduces serum lipids and renal fibrogenic cytokines in rats with chronic nephrotic syndrome, J. Nutr., 2002, 132, 2562–2569.
11. S. Vega-López, N. R. Matthan, L. M. Ausman, S. V Harding, T. C. Rideout, M. Ai, S. Otokozawa, A. Freed, J. T. Kuvin, P. J. Jones, E. J. Schaefer and A. H. Lichtenstein, Altering dietary lysine : arginine ratio has little effect on cardiovascular risk factors and vascular reactivity in moderately hypercholesterolemic adults, Atherosclerosis, 2010, 210, 555–562.
12. T. Rajamohan and P. A. Kurup, Antiatherogenic effect of a low lysine : arginine ratio of protein involves alteration in the aortic glycosaminoglycans and glycoproteins, J. Biosci., 1990, 15, 305–311.
13. T. Rajamohan and P. A. Kurup, Lysine : arginine ratio of a protein influences cholesterol metabolism. Part 1 – studies on sesame protein having low lysine : arginine ratio, Indian J. Exp. Biol., 1997, 35, 1218–1223.
14. V. R. Young, Soy protein in relation to human protein and amino acid nutrition, J. Am. Diet. Assoc., 1991, 91, 828–835.
15. A. Sánchez, D. A. Rubano, G. W. Shavlik, R. Hubbard and M. C. Horning, Cholesterolemic effects of the lysine/arginine ratio in rabbits after initial early growth, Arch. Latinoam. Nutr., 1988, 38, 229–238.
16. L. L. Ma, G. Y. Ji and Z. Q. Jiang, Influence of Dietary Amino Acid Profile on Serum Lipids in Hypercholesterolemic Chinese Adults, J. Nutr. Food Sci., 2014, 4, 1–6.
17. N. Tachibana, I. Matsumoto, K. Fukui, S. Arai, H. Kato, K. Abe and K. Takamatsu, Intake of soy protein isolate alters hepatic gene expression in rats, J. Agric. Food Chem., 2005, 53, 4253–4257.
18. A. R. Tovar, I. Torre-Villalvazo, M. Ochoa, A. L. Elías, V. Ortíz, C. A. Aguilar-Salinas and N. Torres, Soy protein reduces hepatic lipotoxicity in hyperinsulinemic obese Zucker fa/fa rats, J. Lipid Res., 2005, 46, 1823–1832.
19. E. D. Marczak, H. Usui, H. Fujita, Y. Yang, M. Yokoo, A. W. Lipkowski and M. Yoshikawa, New antihypertensive peptides isolated from rapeseed, Peptides, 2003, 24, 791–798.
20. C. Megías, M. del Mar Yust, J. Pedroche, H. Lquari, J. Girón-Calle, M. Alaiz, F. Millán and J. Vioque, Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates, J. Agric. Food Chem., 2004, 52, 1928–1932.
21. S. N. Jamdar, V. Rajalakshmi, M. D. Pednekar, F. Juan, V. Yardi and A. Sharma, Influence of degree of hydrolysis on functional properties, antioxidant activity and ACE inhibitory activity of peanut protein hydrolysate, Food Chem., 2010, 121, 178–184.
22. C. C. Udenigwe and R. E. Aluko, Multifunctional Cationic Peptide Fractions from Flaxseed Protein Hydrolysates, Plant Foods Hum. Nutr., 2012, 67, 1–9.
23. Anonymous, in Official Methods of Association of Official Analytical chemists (AOAC), AoAc Inc.; Suite 4000, Virginia, USA, 15th edn, 1990.
24. B. A. Bidlingmeyer, S. A. Cohen and T. L. Tarvin, Rapid analysis of amino acids using pre-column derivatization, J. Chromatogr., 1984, 336, 93–104.
25. A. Tapal, G. E. Vegarud, A. Sreedhara, P. Hegde, S. Inamdar and P. K. Tiku, In vitro human gastro-intestinal enzyme digestibility of globulin isolate from oil palm (Elaeis guineensis var. tenera) kernel meal and the bioactivity of the digest, RSC Adv., 2016, 6(24), 20219–20229.
26. J. Borawska, M. Darewicz, G. E. Vegarud, A. Iwaniak and P. Minkiewicz, Ex vivo digestion of carp muscle tissue – ACE inhibitory and antioxidant activities of the obtained hydrolysates, Food Funct., 2015, 211–218.
27. U. K. Laemmli, Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4, Nature, 1970, 227, 680–685.
28. O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 1951, 193, 265–275.
29. B. Piotrkowski, V. Calabró, M. Galleano and C. G. Fraga, (−)-Epicatechin prevents alterations in the metabolism of superoxide anion and nitric oxide in the hearts of L-NAME-treated rats, Food Funct., 2015, 155–161.
30. R. Senthilkumar and R. Veerappan, Chrysin enhances antioxidants and oxidative stress in L-NAME-induced hypertensive rats, Int. J. Nutr., Pharmacol., Neurol. Dis., 2015, 5, 20.
31. K. M. Miranda, M. G. Espey and D. A. Wink, A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite, Nitric Oxide, 2001, 5, 62–71.
32. V. S. Vallabha and P. K. Tiku, Antihypertensive Peptides Derived from Soy Protein by Fermentation, Int. J. Pept. Res. Ther., 2013, 1–8.
33. D. W. Cushman and H. S. Cheung, Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung, Biochem. Pharmacol., 1971, 20, 1637–1648.
34. A. Ndabigengesere, K. S. Narasiah and B. G. Talbot, Active agents and mechanism of coagulation of turbid waters using Moringa oleifera, Water Res., 1995, 29, 703–710.
35. K. A. Ghebremichael, K. R. Gunaratna, H. Henriksson, H. Brumer and G. Dalhammar, A simple purification and activity assay of the coagulant protein from Moringa oleifera seed, Water Res., 2005, 39, 2338–2344.
36. E. G. Mhlanga, Extraction of the hydrocolloids from fenugreek seed (Trigonella foenum graecum), WO 2012031592 A1, March 15, 2012.
37. S. Sukhawanli and P. Thamakorn, Extraction of tamarind seed jellose under different conditions and their rheological properties, Food and Applied Bioscience Journal, 2014, 2(1), 61–68.
38. B. Keil, Specificity of Proteolysis, Springer, Berlin, Heidelberg, 1992.
39. A. F. S. Santos, L. A. Luz, A. C. C. Argolo, J. A. Teixeira, P. M. G. Paiva and L. C. B. B. Coelho, Isolation of a seed coagulant Moringa oleifera lectin, Process Biochem., 2009, 44, 504–508.
40. U. V. Katre, C. G. Suresh, M. I. Khan and S. M. Gaikwad, Structure–activity relationship of a hemagglutinin from Moringa oleifera seeds, Int. J. Biol. Macromol., 2008, 42, 203–207.
41. A. Hugo Pripp, T. Isaksson, L. Stepaniak and T. Sorhaug, Quantitative structure–activity relationship modelling of ACE-inhibitory peptides derived from milk proteins, Eur. Food Res. Technol., 2004, 219, 579–583.
42. M. A. Ondetti and D. W. Cushman, Enzymes of the renin–angiotensin system and their inhibitors, Annu. Rev. Biochem., 1982, 51, 283–308.
43. D. H. Baker, Lysine, Arginine, and Related Amino Acids: An Introduction to the 6^th Amino Acid Assessment Workshop, J. Nutr., 2007, 137, 1599S–1601S.
44. J. D. Jones, R. Wolters and P. C. Burnett, Lysine–Arginine-Electrolyte Relationships in the Rat, J. Nutr., 1964, 250, 171–188.
45. U. Gassenschmidt, K. D. Jany, B. Tauscher and H. Niebergall, Isolation and characterization of a flocculating protein from Moringa oleifera Lam., Biochim. Biophys. Acta, 1995, 1243, 477–481.
46. H. Li, N. Prairie, C. C. Udenigwe, A. P. Adebiyi, P. S. Tappia, H. M. Aukema, P. J. H. Jones and R. E. Aluko, Blood pressure lowering effect of a pea protein hydrolysate in hypertensive rats and humans, J. Agric. Food Chem., 2011, 59, 9854–9860.
47. T. Kodera and N. Nio, Identification of angiotensin I-converting enzyme inhibitory peptides from protein hydrolysates by a soybean protease and the antihypertensive effects of hydrolysates in spontaneously hypertensive model rats, J. Food Sci., 2006, 71, C164–C173.
48. C. Wang, J. Tian and Q. Wang, ACE inhibitory and antihypertensive properties of apricot almond meal hydrolysate, Eur. Food Res. Technol., 2011, 232, 549–556.
49. C. C. Udenigwe, A. P. Adebiyi, A. Doyen, H. Li, L. Bazinet and R. E. Aluko, Low molecular weight flaxseed protein-derived arginine-containing peptides reduced blood pressure of spontaneously hypertensive rats faster than amino acid form of arginine and native flaxseed protein, Food Chem., 2012, 132, 468–475.
50. H. Y. Yang, S. C. Yang, S. T. Chen and J. R. Chen, Soy protein hydrolysate ameliorates cardiovascular remodeling in rats with L-NAME-induced hypertension, J. Nutr. Biochem., 2008, 19, 833–839.
51. M. L. Graciano, R. d. C. Cavaglieri, H. Dellê, W. V. Dominguez, D. E. Casarini, D. M. A. C. Malheiros and I. L. Noronha, J. Am. Soc. Nephrol., 2004, 15, 1805–1815.
52. A. M. Sharifi, N. Akbarloo and R. Darabi, Investigation of local ACE activity and structural alterations during development of L-NAME-induced hypertension, Pharmacol. Res., 2005, 52, 438–444.
53. G.-H. Li, M.-R. Qu, J.-Z. Wan and J.-M. You, Antihypertensive effect of rice protein hydrolysate with in vitro angiotensin I-converting enzyme inhibitory activity in spontaneously hypertensive rats, Asia Pac. J. Clin. Nutr., 2007, 16(1), 275–280.
54. M. S. Park and G. U. Liepa, Effects of dietary protein and amino acids on the metabolism of cholesterol-carrying lipoproteins in rats, J. Nutr., 1982, 112, 1892–1898.
55. K. Weisse, C. Brandsch, B. Zernsdorf, G. S. Nkengfack Nembongwe, K. Hofmann, K. Eder and G. I. Stangl, Lupin protein compared to casein lowers the LDL cholesterol : HDL cholesterol-ratio of hypercholesterolemic adults, Eur. J. Nutr., 2010, 49, 65–71.
56. S. Nagaoka, K. Miwa, M. Eto, Y. Kuzuya, G. Hori and K. Yamamoto, Soy protein peptic hydrolysate with bound phospholipids decreases micellar solubility and cholesterol absorption in rats and caco-2 cells, J. Nutr., 1999, 129, 1725–1730.
57. W. S. Jobgen, S. K. Fried, W. J. Fu, C. J. Meininger and G. Wu, Regulatory role for the arginine–nitric oxide pathway in metabolism of energy substrates, J. Nutr. Biochem., 2006, 17, 571–588.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra13632j

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