Legumes are edible seeds of plants belonging to the Leguminosae family and are considered the third-biggest flowering plant family, with 946 genera and 24,505 species [1]. Legumes, such as soybeans, peas, chickpeas, lentils, beans, and peanuts, are considered a rich source of protein (30–35%), and are an inexpensive and popular food for many people [2]. Legumes are widely grown and consumed worldwide because they play an essential role in the human diet. Their rich sources of protein, minerals, carbohydrates, and vitamins are related to the prevention of chronic diseases due to their contents of bioactive peptides [3]. Therefore, legume protein hydrolysates have been widely studied to produce peptides with biological properties such as antihypertensive, antioxidant, antiproliferative, anticoagulant, anti-inflammatory, anticancer, hypoglycemic, calcium-binding, immunomodulatory, and anti-obesity ^[4][5][6][4,5,6].

The production and quality of protein from legumes are impacted by extraction techniques [7]. One of the common processes, referred to as isoelectric precipitation, that is used to extract isolated protein from legumes depends on the solubility. The pH ranges of alkaline and acid lead to high solubility, while the isoelectric point (around pH 4–5) exhibits low solubility [8]. Because alkaline solutions can accelerate the dissolution of protein from legumes, several legumes are employed to extract protein using alkaline solutions (NaOH) [7], such as lupine [9], chickpea [10], black bean [11], soy [12], and pigeon pea [13]. In addition, Langton et al. [14] mentioned that the extraction conditions such as pH, choice of solvent, protein fractions and other compounds in protein isolate, and temperature can affect the characteristics of the protein extracted from legumes. In contrast, enzymatic hydrolysis increases the rate at which protein is extracted by hydrolyzing the protein, enhancing protein solubility, and rupturing the legume cell wall, which encourages the solvent to penetrate the tissue of the legumes, thereby increasing the protein dissolution [7]. Besides, enzymatic hydrolysis is the most popular method for generating hydrolysate and bioactive peptides from legumes ^[15][16][15,16]. Recently, enzymatic hydrolysis of the legume proteins has been reported as an effective, cheaper, and safe method to release bioactive peptides with specific health benefits, such as antioxidant properties and antihypertensive (inhibitors for the angiotensin-I converting enzyme (ACE) [17].

Bioactive peptides may be considered edible nutraceutical agents with health benefits related to disease treatment or prevention [18]. Throughout the past few decades, many studies have focused on the isolating and purifying of antioxidant and ACE-inhibiting peptides of legume protein hydrolysates such as lentils [19], black bean [20], mung bean [21], chickpea [22], soybean [23], and pigeon pea [24]. Synthetic ACE inhibitors have various adverse side effects, while synthetic antioxidants are banned due to health risks [25]. On the other hand, antihypertensive and antioxidant peptides derived from natural plant and animal sources such as tuna muscle, wheat, casein, soy, eggs, milk, azufrado beans, cheese, fish, meat, corn gluten, chickpea, peanut soybean, lupin, hemp, pea, lentil, Bambara, and common bean have potentially beneficial effects due to their high activity, low cost, easy absorption in the human body, and low molecular weight. In addition, they have little or no adverse side effects and strong antioxidant activities [26]. Wang et al. [27] stated that antioxidant peptides are shown to have ACE-inhibitory activity concurrently. The ACE-inhibitory and antioxidant peptides have been deeply related to their structure, sequencing, chain length, and amino acid composition [28]. In addition, Wang et al. [29] mentioned that the presence of hydrophobic amino acids (Cys, Pro, Leu, Met), aromatic amino acids (Trp, Phe, and Tyr), positively charged amino acids (Arg and Lys), and branched-chain aliphatic amino acids (Val, and Ile) is a typical characteristic of peptides with high ACE-inhibitory activities.

2. Legume Protein Isolate and Hydrolysate

The extraction of protein isolates and other components from legume seeds is currently performed using advanced legume extraction and fractionation technologies. Protein isolation can be done in two ways: dry processing for legume seeds, which preserves protein functionality, and wet processing for flours, which yields better protein purity ^[30][34]. Previous studies have reported on legume protein isolation using the acid-base extraction method, such as chickpea ^[31][35], black gram ^[32][36], pea ^[33][37], pinto beans ^[34][38], string beans ^[35][39], and lupin ^[36][40]. The legume protein isolation begins with extracting the protein in an alkaline solution, followed by isoelectric precipitation (IP), and finally, drying to get the final isolated protein ^[37][38][41,42]. Los et al. ^[39][43] extracted carioca bean protein before hydrolysis with NaOH (pH 8.0) and then precipitated it with HCl (pH 4.5). Legume protein hydrolysate can be produced using numerous methods, such as chemical hydrolysis, enzymatic hydrolysis, or microbial fermentation ^[34][40][38,44]. Enzymatic hydrolysis converts the protein molecules into peptides quickly for various sizes and free amino acids by breaking down certain peptide bonds of the parent protein using proteases with a positive impact on human health ^[41][45]. In this context, through the extraction of protein isolate, enzymatic hydrolysis, chemical and fermentation synthesis, separation, and purification (ultrafiltration membrane, gel filtration, reverse phase high performance liquid chromatography, and ion exchange chromatography), various legume sources can be converted into antioxidants, ACE-inhibitory, and anti-inflammatory peptides ^[2][23][42][2,23,31]. These peptides are regarded as a good source and an alternative food for use as antioxidants, antihypertensive, and anti-inflammatory peptides ^[43][44][45][46,47,48]. Moreover, antioxidant and ACE-inhibitory properties of legume protein hydrolysates are strongly influenced by hydrolysis, which changes the structural and composition characteristics such as molecular weight (MW) ^[11][46][47][11,49,50]. Putra et al. [13] mentioned that peptide fractions with MW below 1 kDa demonstrated the highest ACE-inhibitory activity among the other fractions derived from pigeon pea hydrolysates because of their capacity to bind Zn²⁺ and form Peptide- Zn²⁺ complex, which prevents ACE from using the Zn²⁺ ions that are already present as a cofactor. According to Chen et al. ^[48][51], antioxidant peptides were separated from the black bean soybean protein hydrolysate using ultrafiltration, gel filtration (GF), and reverse phase high performance liquid chromatography (RP-HPLC). The amino acid sequence of the sub-fraction (F2-c) peptide (455 Da) was identified as Leu-Val-Pro-Leu-Lys and Ile-Val-Pro-Leu-Lys and has the highest antioxidant of DPPH and HRSA (IC₅₀: 0.12 µM and 0.037 µM).

3. In Vitro and In Vivo Studies on the Antioxidant, ACE-Inhibitory, and Anti-Inflammatory Peptides from Legume Protein Hydrolysate

3.1. In-Vitro Study of Antioxidant, ACE-Inhibitory, and Anti-Inflammatory Peptides

3.1.1. Antioxidant Peptide

Reactive oxygen species “(ROS)” are highly m molecules produced by all aerobic cells that can be free radicals, such as hydroxyl radical(•OH), and superoxide radical(•O₂) or non-radicals, such as singlet oxygen(¹O₂) and hydrogen peroxide(H₂O₂) ^[49][52]. Free radicals are unavoidable metabolic by-products that are the result of oxidative stress which can damage proteins, cell membranes, phospholipids, and DNA, resulting in severe human diseases such as diabetes, coronary heart disease, hypertension, stroke, cancer, arteriosclerosis, and Alzheimer’s ^[50][51][53,54]. Therefore, antioxidants play a crucial role in preventing or delaying the autoxidation of food components and the human body by inhibiting oxidation reactions and producing free radicals ^[52][55]. Antioxidant peptides released during enzymatic hydrolysis act as free radical scavengers, metal inactivators, oxygen inhibitors, or peroxide to protect the body and food system from reactive oxygen species ^[53][56]. Antioxidants are classified into synthetic and natural categories ^[54][57]. Although synthetic antioxidants such as “butylated hydroxytoluene (BHT), propyl gallate (PG), butylated hydroxyanisole (BHA), and tert-butyl hydroquinone (TBHQ)” are efficient and relatively cheap, they have displayed some toxic and hazardous properties ^[55][58]. According to Lourenço et al. ^[56][59], several studies have been published showing a correlation between the long-term consumption of synthetic antioxidants and specific health problems such as digestive system disorders, skin allergies, and occasionally leading to increased risk of cancer. Additionally, in animal studies, BHT and BHA have already been shown to be responsible for adverse effects on carcinogenesis and the liver. Large dosages of synthetic antioxidants may cause premature senescence and damage the DNA. For this reason, studies have focused on searching for and developing antioxidant peptides from natural sources such as plants and animals ^[57][60]. These natural antioxidant peptides can be easily absorbed more readily and without adverse side effects compared to synthetic antioxidants. Furthermore, the primary mechanism of these antioxidant peptides is through the ability to inactivate intracellular reactive oxygen species (ROS), scavenge free radicals, reduce lipid peroxidation, and chelate transition metals, among other things, which have all been linked to their antioxidant activities ^[58][61]. Nevertheless, compared to synthetic antioxidants, some natural antioxidants have lower antioxidant activity, indicating that they need to be used in higher dosages and may result in unsafe dosages. Despite this, if they are taken within the limits of the law, natural antioxidants are a helpful alternative to synthetic ones ^[56][59]. Food-derived antioxidant peptides are considered natural antioxidant resources which possess acceptable nutritive value, minimal adverse side effects (safe), high efficiency, low molecular weight, high activity, low cost, and are easily absorbable to replace synthetic antioxidants for use in food ^[49][59][52,62]. Many studies have shown that legumes, such as chickpeas, pigeon peas, beans, soybeans, and lentils, can produce antioxidative peptides in vitro and positively impact human health when used as alternative antioxidants [3]. The antioxidant activity of peptides depends on molecular weight, amino acid composition and sequence, size, hydrophobicity, enzyme specificity, and degree of hydrolysis ^[20][43][20,46]. Figure 1 shows the main steps for preparing and identifying antioxidant peptides from natural protein sources. In this context, if an antioxidant compound prevents the generation of free alkyl radicals or disrupts the free radical chain’s propagation, the lipid oxidation’s chemical reaction rate is delayed or slowed. The use of singlet oxygen inhibitors, metal-chelating agents, and peroxide stabilizers caused this delay by donating hydrogen from antioxidants and metal-chelating agents ^[60][63]. Recent interest in antioxidant peptides derived from food proteins has grown owing to their prominent role in the prevention mechanisms of oxidative stresses linked to various diseases ^[61][64]. Consequently, different methods of in vitro antioxidant systems have been used to assess the antioxidant capacity of legume protein hydrolysates and peptides such as (DPPH) 2,2-diphenyl-1-picrylhydrazyl, (HRSA) hydroxyl radical scavenging activity, (ABTS) 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging, and (ORAC) oxygen radical absorbance capacity, etc. ^[62][63][65,66]. Lentil protein hydrolysate with an amino acid sequence Ala-Leu-Gly-Pro-Val-Met (587.31 Da) exhibited the highest DPPH (63%) and β-carotene-linoleate model system (73%), respectively ^[62][65]. Wali et al. ^[64][67] also mentioned that the identified antioxidant peptide of chickpea protein hydrolysate (NF2-4-1): Leu-Thr-Glu-IIe-IIe-Pro (685.41 Da) showed the highest DPPH and OH scavenging activities, IC₅₀: 0.24 mg/mL and 0.57 mg/mL, respectively. Peptides with low molecular weight have strong antioxidant activities than higher molecular weight peptides because they have a better chance to cross the intestinal barrier to exert antioxidant effects and favor increased interactions with the free radical ^[65][68]. The presence of hydrophobic (Pro, Val, Leu, Ala, Tyr, Trp, Met), basic (Arg, His), and aromatic (Trp, Phe, Tyr) amino acid residues in the peptide sequence can contribute to increasing antioxidant activities ^[38][42]. Phongthai and Rawdkuen ^[55][58] clarified that Val and Ile might be responsible for generating a suitable hydrophobic microenvironment for peptide molecules, while indole and pyrrolidine rings in Pro and Trp, respectively, could also act as hydrogen donors via their hydroxyl groups. In addition, the presence of positively-charged, acidic, and sulfur-containing amino acids improved the ability of antioxidant protein hydrolysates to scavenge free radicals ^[66][74]. For instance, Chunkao et al. [21] found that mung bean peptides having “Leu-Leu-Gly-Ile-Leu, Leu-Leu-Leu-Leu-Gly, Pro-Ala-Ile-Asp-Leu, and Pro-Ala-Ile-Asp-Leu and Ala-Ile-Val-Ile-Leu” amino acids sequences which had the highest DPPH (81.27%), FRAP (0.05 mM/mg), HRSA (EC₅₀: 0.09 mM) and SRSA (EC₅₀: 0.07 mM), respectively. Wali et al. ^[64][67] mentioned that different amino acid chains form with varying lengths and compositions. The varied forms play different roles in peptide function, such as a significant role in antioxidant activity. Furthermore, the presence of hydrophobic amino acids inside the peptides causes them to increase their hydrophobic solubility and leads to easier interaction among peptides and donation of protons to radical species ^[43][46]. From pinto bean protein hydrolysate, Ngoh and Gan ^[34][38] found six sequences for antioxidant peptides, and these peptides’ fraction is <3 kDa. These sequences are “Ala-Cys-Ser-Asn-His-Ser-Pro (1420.64 Da), Pro-Leu-Pro-Leu-His-Met-Leu-Pro (916.52 Da), and Pro-Pro-His-Met-Leu-Pro” (690.35 Da), which displayed the highest FRAP and ABTS, 81 mM, 42.2%, respectively. According to Li et al. ^[67][75], increased hydrophobic amino acid content and their location at the third position adjacent to the “C-terminus or the N-terminus” of the sequence were favorable to antioxidant activity. The His-containing peptide could donate protons and scavenge free radicals owing to the imidazole ring. In addition, due to the existence of the phenolic group, F (Phe) might serve as a hydrogen donor, while the SH group in C (Cys) could interact with radicals directly. Furthermore, amino acid residues such as cysteine, histidine, and methionine are essential to the radical scavenging activity of peptides that enhance antioxidant activities due to their unique structural characteristics. “Cysteine” donates sulfur hydrogen; the imidazole group in “histidine” has a proton-donation ability, while “methionine” is prone to oxidation of the methionine sulfoxide ^[34][38].

3.1.2. ACE-Inhibitory Peptide

Hypertension, a global issue affecting one-fourth of the world’s adults, is the leading cause of heart disease ^[68][76]. The two of the most powerful systems for maintaining blood pressure regulation in humans via the angiotensin-I converting enzyme (ACE) are the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS), as shown in Figure 2. Angiotensinogen is a substrate for renin to produce angiotensin (I), which is then converted into potent vasoconstrictor angiotensin (II) by ACE, causing raised blood pressure ^{[28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77]}[28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77]. In this context, angiotensin (II) raises oxidative stress when blood pressure is high because it interferes with several of the cell’s functions by increasing the creation of intracellular reactive oxygen species [78]. In contrast, ACE inhibition blocks the first step in the renin-angiotensin system (reduction of angiotensin II), resulting in the treatment of hypertension and enhancing the antioxidative defense system [3]. Inhibitors bind strongly to the ACE active site, contending for occupancy with angiotensin (I); as a result, ACE is unable to convert angiotensin (I) to angiotensin II [79]. Many anti-ACE drugs, such as ramparil, lisinopril, zestril, enalapril, captopril, etc., are commonly used as antihypertensive treatments; with adverse side effects, including taste disturbances, cough, angioedema, and skin rashes [80]. Consequently, natural compounds, especially from plant sources with ACE-inhibitory potential, seem to be of ongoing interest as alternatives to synthetic drugs [81]. Studies have shown that legumes contain beneficial nutrients, bioactive peptides, and high-quality proteins, which have been demonstrated as potent in vitro ACE inhibitors [82]. In bioactive environments, ACE inhibitors can reduce angiotensin (II) production and hence reduce hypertension; for instance, ACE inhibitors are produced from peptides derived from various legumes in vitro. Shi et al. [83] found that a peanut peptide with the amino acid sequence Lys-Leu-Tyr-Met-Arg-Pro and a molecular weight of 808.8 KDa had the highest ACE inhibiting activity at 85.77% (IC₅₀: 0.0052 mg/mL). According to Puspitojati et al. [84], ACE-inhibitory peptides usually contain hydrophobic amino acids (Val, Trp, Ile, Phe, Met, Tyr, and Ala) or (Pro) at the C terminal or positively charged amino acids (Lys and Arg). The differences in ACE-inhibitory activity can be attributed to a variety of factors, including the type of legumes, protein extraction technique, type of enzyme used for proteolysis, hydrolysis conditions (temperature, pH, substrate/enzyme ratio, time), and the analytical approach used to determine ACE-inhibitory activity [79]. Figure 1 shows the main steps for preparing and identifying ACE peptides from natural protein sources. Jakubczyk et al. [80] have determined that the peptide fraction III (3.5–7 kDa) of bean protein hydrolysate under conditions of 3 h and a temperature of 22 °C was “Ile-Asn-Glu-Gly-Ser-Leu-Leu-Pro-His” and “Phe-Val-Val-Ala-Glu-Gln-Ala-Gly-Asn-Glu-Glu-Gly-Phe-Glu”, which showed the highest ACE-inhibitory activity (IC₅₀: 0.20 μg/mL). Additionally, Sonklin et al. [15] reported that the ACE inhibition activity of the mung bean hydrolysate was highest in peptide fractions with low MW (<1 kDa, IC₅₀: 0.50 mg/mL) when compared to other peptide fractions. The high ACE inhibition activity was due to the lower MW peptides bound to active ACE sites more easily than higher MW peptides after ultrafiltration separation of the protein hydrolysate, resulting in increased ACE inhibition activity. Similarly, Gupta and Bhagyawant ^[85][88] found that C. arietinum produced by Alcalase enzyme at 60 min had the highest ACE-inhibitory activity (IC₅₀: 0.182 mg/mL) compared to flavourzyme at 100 min (IC₅₀: 0.365 mg/mL). The most increased ACE-inhibitory activity is because the peptides made by alcalase are resistant to gastrointestinal proteases, thus absorbed in the small intestine, suggesting that these peptides could be used in the food industry to help people with high blood pressure. In addition, Jakubczyk and Baraniak ^[33][37] also demonstrated that peptide fraction (F8B) of pea protein hydrolysate had the highest ACE-inhibitory activity (IC₅₀: 0.073 mg/mL) and was identified as these sequences “Gly-Gly-Ser-Gly-Asn-Tyr, Asp-Leu-Lys-Leu-Pro, Gly-Ser-Ser-Asp-Asn-Arg, and His-Asn-Thr-Pro-Ser-Arg”. In this context, the C-terminal amino hydrophobic or aromatic residues significantly impact ACE binding, with proline being the most preferred for high ACE-inhibitory activity; consequently, there is a relationship between ACE-inhibitory peptide activity and structure [23]. Moreover, Hanafi et al. [4] mentioned that the hydrophobicity of the C-terminal amino acids and three-dimensional chemical characteristics significantly impacted ACE-inhibitory activity, demonstrating that the amino acids with the highest volume and hydrophobicity have the strongest ACE-inhibitory activity. Certainly, ACE-inhibitory peptides produced from legume proteins have gained a lot of interest recently, and their ability to prevent hypertension in vitro and in vivo has been thoroughly investigated. Therefore, the use of natural components, such as bioactive legume peptides, to suppress ACE activity has been implemented in studies on cardiovascular problems, particularly hypertension [81].

3.1.3. Anti-Inflammatory Peptide

Inflammation is the immune system’s reaction to adverse stimuli, including infection, tissue damage, or toxic substances. It aims to eliminate pathogenic microorganisms or irritants and promote tissue repair ^[86][89]. Nitric oxide (NO) and prostaglandin E2 (PGE2) are pro-inflammatory mediators that are released by microglia in response to inflammatory stimuli through the activation of nuclear factor (NF)-B, which ordinarily activates a protective response in the central nervous system (CNS) to eliminate pathogens and infected cells ^[87][90]. There are two lines of defence. The first one is acute inflammation, which happens right after inflammatory response induction and temporarily activates cellular and molecular activities and interactions to stop the spread of infection or damage. The second line is chronic inflammation linked to a higher risk of developing chronic diseases and disorders, including type cancer, 2 diabetes, asthma, and inflammatory bowel disease ^[88][91]. Some of these diseases are related to inflammation, insulin resistance, and lipotoxicity caused by an excess of adipose tissue (obesity and overweight). Depending on the facts of each case, it is necessary to use drugs with side effects and unfavourable effects. Therefore, pro-inflammatory cytokine inhibitors have been evaluated as potential anti-inflammatory medication options. In contrast, the rise in inflammatory disorders has prompted the search for proteins and peptides with anti-inflammatory effects ^[89][92]. In this case, one technique is to research possible compounds, such as plant proteins, that have been studied as a source for bioactive peptides with anti-inflammatory activity agents ^[90][93]. Hydrolysates and peptides generated from legumes can have various biological effects, including anti-inflammatory, antioxidant, antihypertensive, anticancer, and immunomodulatory properties [6]. A few particular peptides, such as lunasin, a 43-amino acid chemopreventive peptide extracted from soybean, barley, wheat, rye, and triticale, have been identified as anti-inflammatory peptides from various plant protein hydrolysates ^[89][92]. Indrati et al. ^[91][94] mentioned that bioactive legume peptides, especially soybeans and Phaseolus vulgaris L., can control a variety of inflammatory indicators, including prostaglandin E2 (PGE2), nitric oxide (NO), induced nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), cytokines, and chemokines. Additionally, Montserrat-de la Paz et al. ^[92][95] reported that lupin protein hydrolysate hydrolyzed by alcalase reduced the levels of nitric oxide and reactive oxygen species in cells and crossed the Caco-2 monolayer of the human intestinal tract to exert anti-inflammatory activity in macrophages located in the basement region by production the pro-inflammatory cytokines and lowering mRNA levels in the vitro study. López-Barrios et al. ^[93][96] reported anti-inflammatory activity of Phaseolus vulgaris L. peptides was low molecular weight (MW) and contained three to eleven amino acids. Similarly, Garcia-Mora et al. ^[69][30] found that pinto beans produced by Alcalase enzyme at 120 min had a higher concentration of small peptides (<3 kDa) with anti-inflammatory activity (28–16%). Moreover, Cruz-Chamorro et al. ^[94][97] mentioned that the lupin protein hydrolysate (LPHs) decreased human peripheral blood mononuclear cells proliferation (PBMCs) and the levels of T helper cells (Th1, Th9 and Th17) pro-inflammatory cytokines without being cytotoxic. This improves the anti-inflammatory/pro-inflammatory cytokine balance and reduces T-cell inflammatory responses. High molecular weight peptide fraction (5–10 KDa) from soybean protein hydrolysate showed the best anti-inflammatory activity by reducing the secretion of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 cells ^[95][98].