Research Progress on the Biological Activities and Mechanisms of Pea Peptides

Pea, as an important legume crop conducive to food security and agricultural sustainability, is currently cultivated in more than 90 countries worldwide with an annual output of approximately 14 million tons [2]. The protein content of mature pea seeds is about 20%; pea protein is non-allergenic [3] and contains 8 essential amino acids for the human body, 7 of which are close to the recommended pattern values of FAO/WHO [4], showing great application potential in the food field. Many countries have used pea protein as a raw material or auxiliary material in products such as plant protein solid beverages, simulated meat, sausages, energy bars, cookies, and noodles [5].

The functionalization and high-value application of plant proteins are important measures to practice the “grand food concept” and achieve the strategic goals of “Healthy China 2030″. Preparing bioactive peptides from pea protein is an effective way to realize its high-value application. In recent years, pea peptides [6] have been confirmed to have various functional activities such as antioxidant, auxiliary blood pressure-lowering, auxiliary blood glucose-lowering, and intestinal flora regulation (see Figure 1), showing broad application prospects in the fields of health food and special medical food.

Research Review

This paper focuses on the in vivo and in vitro effects, related regulatory modes, and key signaling pathways of pea peptide activity, and summarizes the research status of the biological activities and mechanisms of pea peptides, aiming to provide a theoretical reference for the in-depth research, development and application of pea bioactive peptides.

Antioxidant Activity

In recent years, many scholars have carried out research on the antioxidant activity of pea protein hydrolysates. Zhang Shucheng et al. [7] found that the neutral protease hydrolysate of pea protein with a molecular weight less than 1,000 u had a superoxide anion radical scavenging rate of up to 79.3%, with a molecular weight concentrated at 200–800 u.

Zhao Dan et al. [8] found that the IC50 values of the alkaline protease and compound protease synergistic hydrolysate of pea protein with a molecular weight less than 1,000 u for scavenging DPPH and ABTS free radicals were 2.57 mg/mL and 1.32 mg/mL, respectively, with an ORAC value of 896.35 μmol TE/g. It could inhibit the auto-oxidation of linoleic acid to a certain extent within 7 days, and also confirmed that pea protein hydrolysate could inhibit the accumulation of ROS in HepG2 cells under oxidative stress.

Gu Hong et al. [9] found that the flavor protease hydrolysate of pea protein had DPPH and hydroxyl radical scavenging rates of 31.23% and 26.03%, respectively. Compared with the blank group, the serum SOD and CAT levels of mice in the pea protein hydrolysate gavage group were significantly increased, and the malondialdehyde (MDA) content was significantly decreased.

The above studies have shown that pea peptides can improve the activities of antioxidant enzyme systems such as CAT, SOD, and GSH-px, inhibit ROS accumulation, reduce MDA content, and decrease lipid peroxidation, thereby enhancing antioxidant defense capacity.

Antioxidant mechanism research is usually carried out from aspects such as scavenging reactive oxygen free radicals, chelating metal ions, inhibiting lipid peroxidation, and activating the body’s antioxidant defense system. As an important endogenous antioxidant regulatory signaling pathway in the body (see Figure 2), Keap1-Nrf2-ARE has also attracted much attention.

Li et al. [10] found that pea peptides could protect rat adrenal medullary pheochromocytoma cells (PC12) from lead-induced oxidative stress injury. Western blot confirmed that pea peptides reduced Keap1 expression and increased Nrf2 expression in oxidatively damaged cells, indicating that pea peptides are involved in the regulation of the Keap1-Nrf2 antioxidant pathway.

Existing studies have found that pea antioxidant peptides also have certain auxiliary effects in organic selenium supplementation [11], fatigue improvement [12], and chronic neurological diseases [13]. Therefore, exploring the effects of pea antioxidant peptides on oxidative stress in the body and their mechanisms is of great significance.

Auxiliary Blood Pressure-Lowering Activity

The blood pressure-lowering mechanism is shown in Figure 3. At present, clinically used blood pressure-regulating drugs include captopril, losartan, aliskiren, etc. [14]. However, long-term use of the above drugs will cause side effects such as human metabolic disorders and liver and kidney damage. Peptides derived from food proteins with blood pressure-lowering effects are considered safer substitutes for blood pressure-lowering drugs [15]. In recent years, ACE inhibitory peptides or ACE2 up-regulatory peptides have been discovered from pea protein hydrolysates, which play a positive role in maintaining healthy blood pressure levels.

Li et al. [16] verified that the thermolysin hydrolysate of pea protein (PPH-3) with a molecular weight less than 3,000 u had a blood pressure-lowering effect through spontaneously hypertensive rats (SHR) and hypertensive human subjects. After gavage of PPH-3 to SHR at a dose of 200 mg/kg body weight, it was found that the systolic blood pressure could be reduced, reaching a maximum reduction of 19 mmHg at 4 hours, and it was confirmed that the expression of Ang Ⅱ mRNA in the plasma of SHR in the PPH-3 gavage group was reduced by 45%; in the human trial, the systolic blood pressure of the group taking PPH-3 was reduced by 5 and 6 mmHg in the 2nd and 3rd weeks compared with the non-taking group, respectively.

Pea peptides can also assist in lowering blood pressure by up-regulating ACE2. Wang et al. [17] found that the pea-derived peptide LRW could reduce the production of superoxide induced by Ang II in vascular smooth muscle cells (VSMCs). LRW up-regulated the expression of ACE2 and its related receptor MasR, and could achieve auxiliary blood pressure-lowering effects by regulating the ACE2-Ang1-7-MasR pathway.

Auxiliary Blood Glucose-Lowering Activity

Diabetes is a chronic metabolic disease divided into type Ⅰ and type Ⅱ. Type Ⅰ diabetes is an autoimmune disease; type Ⅱ diabetes is caused by insufficient insulin supply or the inability of insulin to exert normal physiological effects in target cells, leading to disorders of sugar, protein and fat metabolism in the body [18]. Food-derived blood glucose-lowering peptides show potential for the adjuvant treatment of type Ⅱ diabetes, among which blood glucose-lowering peptides derived from pea protein exhibit potential auxiliary blood glucose-lowering effects. Their mechanisms of action mainly include alleviating insulin resistance, inhibiting digestive enzyme activity and inhibiting dipeptidyl peptidase-Ⅳ (DPP-Ⅳ) activity.

1.Alleviating insulin resistance: Insulin resistance occurs when signal conduction disorders occur during the binding of insulin to the insulin receptor (InsR) [19]. Cui Xinyue et al. [20] treated insulin-induced HepG2 cells with commercial pea peptides, and the expression level of InsR was increased by 1.19–1.34 times, and the glucose consumption was increased by 1.31–1.68 times, which had a certain alleviating effect on the formation of insulin resistance in HepG2 cells. Wang Sai et al. [21] gavaged type Ⅱ diabetic mice with pea oligopeptides isolated from the neutral protease hydrolysate of pea protein. The blood glucose of mice in the high-dose (500 mg/kg body weight) gavage group was reduced by 23.97% compared with the model group, the body weight increased by 12.24%, the expression of PI3K and AKT in the liver was increased by 278.49% and 21.78%, respectively, and liver cell damage was reduced.

2.Inhibiting digestive enzyme activity: Pea peptides can reduce the absorption of glucose and energy in the body by inhibiting the activities of digestive enzymes such as α-amylase, α-glucosidase and pancreatic lipase. Awosika et al. [22] used alkaline protease, pepsin, trypsin and chymotrypsin to hydrolyze pea protein respectively, and the obtained 4 hydrolysates could inhibit the activities of α-amylase, α-glucosidase and pancreatic lipase.

3.Inhibiting DPP-Ⅳ activity: DPP-Ⅳ is a cell surface serine protease that can decomppose a variety of active peptides, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP); DPP-Ⅳ inhibitors can increase the levels of endogenous GLP-1 and GIP, promote the release of insulin from islet β cells, and inhibit the secretion of glucagon from islet α cells, thereby increasing insulin levels and lowering blood glucose [23].

Wei et al. [24] evaluated the effects of pea oligopeptides obtained by sequential double-enzyme hydrolysis of pea protein with alkaline protease and neutral protease on the blood glucose and lipid status of type Ⅱ diabetic mice induced by high-fat diet and streptozotocin, and found that 4 pea oligopeptides ALP, LLP, VLP and SP with proline residues at the C-terminus could all inhibit DPP-Ⅳ activity, and significantly reduce the blood glucose, lipid content and liver fat deposition levels of diabetic model mice; pea oligopeptides could also improve glucose tolerance, promote glycogen synthesis, and protect the renal cortical tissues of the liver and kidneys in diabetic model mice.

Antibacterial Activity

Compared with antibiotics, plant antibacterial peptides have the advantage of not easily inducing drug resistance against bacteria, fungi, viruses, etc. [25].

Zhang Qiuping [26] hydrolyzed pea protein with papain and purified to obtain antibacterial peptides with a molecular weight range of 1.9–3.7 ku. At a mass concentration of 10 mg/mL, the antibacterial effect against Staphylococcus aureus was equivalent to that of 10.72 U/mL kanamycin sulfate and 2.08 U/mL gentamicin, with heat resistance, and the best antibacterial effect at pH 6.0.

Immunomodulatory Activity

Pea peptides have a certain improvement effect on the immune function of cyclophosphamide-induced immunosuppressed mice. Zhang Minjia et al. [27] gavaged commercial pea peptides to cyclophosphamide-induced immunosuppressed mice, and found that compared with the model group, there was no statistical difference in body weight in the pea peptide gavage group, the number of peripheral blood white blood cells was significantly increased to 3.56×10⁹/L; the percentage of CD3+ T lymphocytes was significantly increased to 26.07%; the percentages of CD4+ and CD8+ T lymphocytes were improved but not significantly different; the contents of immunoglobulins IgG and IgM related to humoral immune function were significantly increased; the concentration of cytokine IFN-γ was significantly decreased; the number of bone marrow nucleated cells was 48.83×10⁵/mL, and the DNA content was 0.015 μg/μL, both of which were significantly decreased.

Pea peptides can improve the immune function of cyclophosphamide-induced immunosuppressed mice by improving the development of immune organs, myelosuppression, immunoglobulin content, T lymphocyte subset percentage and cytokine levels.

Antitumor Activity

Pan Fen [28] found that the alkaline protease hydrolysate (APPH) and trypsin hydrolysate (TPPH) of pea protein could inhibit the growth of 4 common cancer cell lines, including human liver cancer cell line, human breast cancer cell line, human gastric cancer cell line and human lung cancer cell line, among which APPH had a maximum inhibition rate of more than 60% on the 4 cancer cell lines.

Intestinal Flora Regulation Activity

Intestinal flora imbalance is a phenomenon in which the symbiotic relationship formed by intestinal flora and the body’s interdependence and restriction is destroyed.

Pan Fen et al. [29] studied the effect of alkaline protease hydrolysate of pea protein on the growth of 17 common probiotics, and found that adding 4 mg/mL hydrolysate could significantly improve the growth of Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus acidophilus and Bifidobacterium in MRS medium, with the bacterial density increased by 10.78%–49.34% and the number of viable bacteria increased by 1–3 orders of magnitude, indicating that the alkaline protease hydrolysate of pea protein can significantly promote the growth of probiotics and improve the survival rate of probiotics.

Zhu Yan et al. [30] found that a commercial fermented pea protein peptide had a good effect on regulating intestinal flora. A mouse model of intestinal flora disorder was established by intraperitoneal injection of lincomycin hydrochloride. After gavage of pea protein peptide, the intestinal flora structure and colon pathological changes of the model mice were improved to varying degrees. When the gavage dose was 1.6 g/kg body weight, the flora diversity, abundance and composition of dominant species in the model mice were very similar to those in normal mice, indicating that pea peptides have a certain regulatory effect on intestinal flora.

Summary and Discussion

The exploration of the biological activities and mechanisms of pea peptides is of great significance for the research and development of pea peptide functional foods. This paper summarizes the research status of the biological activities and mechanisms of pea peptides in recent years, focusing on the antioxidant, auxiliary blood pressure-lowering and blood glucose-lowering activities and their mechanisms. At the same time, pea peptides also show positive effects in antibacterial, immunomodulatory, antitumor, intestinal flora regulation and probiotic growth promotion.

However, the current research on the mechanisms of biological activities of pea peptides needs to focus on molecular targets, structure-activity relationship, safety and in vivo digestion and absorption:

1. Aiming at the unclear mechanism of biological activity, it is possible to further determine the molecular targets of pea peptides exerting their biological activities in chemical systems, cell models, animal models and human trials;
2. Aiming at the unclear relationship between the functional structure and biological activity of pea peptides, it is possible to deeply explore the structure-activity relationship through bioinformatics and computer-aided molecular models to evaluate structural characteristics, active sites and modes of action;
3. Aiming at the unclear safety and in vivo digestion and absorption mechanism of pea peptides, it is possible to carry out in-depth research on the cytotoxicity, digestion stability, absorption mode and transport mechanism of pea peptides by using in vitro simulated gastrointestinal digestion and cell and animal models;
4. The mechanisms of various biological activities of pea peptides are correlated. It is possible to explore the correlation mechanism by evaluating the basis of activity to explore the dose-effect relationship and finding key signaling molecules related to the regulation of related signaling pathways.

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