Inflammatory Peptide - an overview

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3.7 Anti-inflammatory peptides

Inflammation is the body's natural response to the infection or injury and it is either acute or chronic. Chronic inflammation is a major concern since it is associated with the various lifestyle diseases such as arthritis, type-2 diabetes, cardiovascular diseases, and inflammatory bowel disease (Serhan & Savill, 2005; Wang et al., 2011). The adverse side effects on prolonged consumption of drugs to treat the diseases is a major concern (Carter et al., 2014; Roubille et al., 2013). Hence, food-derived anti-inflammatory peptides is a choice to fight against chronic inflammation. Hydrophobicity and cationic character of peptides are responsible for the anti-inflammatory properties (Guha & Majumder, 2018). Most of the anti-inflammatory peptides have positively charged AA such as Arg and Lys. The anti-inflammatory peptides contains hydrophobic AA at the N-terminal and polar AA at the C-terminal. Hence, the presence of AA such as Val-His, Ile-Ala, Ile-Pro-Pro, Val-Pro-Pro, Ile-Arg-Trp, Ile-Gln-Trp, Phe-Leu-Val, Leu-Asp-Ala-Val-Asn-Arg, Val-Pro-Tyr, and Met-Met-Leu-Asp-Phe at the N-terminal of peptides showed an anti-inflammatory activities (Chakrabarti & Wu, 2015; Guha & Majumder, 2018; Kwak et al., 2016; Zhao et al., 2016). Bioactive peptides showed their anti-inflammatory property by inhibiting the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) or MAPK (mitogen-activated protein kinase) pathway, the two important pathways involved in chronic inflammation (Guha & Majumder, 2018). The milk derived peptides showed an improvement in chronic diseases because of their antioxidative and anti-inflammatory bioactivity (Bamdad et al., 2017; Chakrabarti et al., 2014). Trypsin hydrolyzed β-casein generates the anti-inflammatory peptides (Altmann et al., 2016). Similarly, corolase enzyme (bacterial endopeptidase enzyme preparation) digested casein showed an anti-inflammatory action on activated macrophages (Aihara et al., 2009). The casein derived multifunctional peptides, i.e., Ile-Pro-Pro and Val-Pro-Pro have shown an anti-inflammatory activity by controlling the activation of NF-κB pathway (Chakrabarti & Wu, 2015) besides improving atherogenesis in mice deficient in apolipoprotein-E (Nakamura et al., 2013). Casein derived peptide with sequence Gln-Glu-Pro-Val has an inhibitory action on nitric oxide release besides increasing the secretion of an anti-inflammatory mediators IL-4 and IL-10 (Jiehui et al., 2014).

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3.1 Anti-inflammatory peptides

Peptides are short polymers of amino acids linked by peptide bonds. They have the same peptide bonds as those in proteins, but are commonly shorter in length.

Peptides with anti-inflammatory properties are of great interest as drug delivery molecules. Several forms and origins of peptide make this drug class attractive for drug delivery. For example, depsipeptides and biooligomers found in microorganisms and marine invertebrates have been shown to have anti-inflammatory and anticancer potentialities (Ballard et al., 2002). Several key advantages are known such as (1) high specificity, (2) high activity, (3) little unspecific binding with untargeted molecular structure, (4) less accumulation in tissues, (5) lower toxicity, (6) minimization of peptide intermolecular binding, and (7) unlimited potential for synthetic peptides. Low oral bioavailability requires intravenous or local injection, as peptides (used as a simple molecule alone) are difficult to deliver across biological membranes, are rapidly cleared from the body and nonstable. Anti-inflammatory peptides such as the somatostatin analogs octreotide, lanreotide, and vapreotide are now clinically available to treat GI tumors (Froidevaux and Eberle, 2002).

Once coupled to drug delivery nanocarriers (NPs, liposomes, etc.), anti-inflammatory peptides have tremendous results as the peptide has a high activity and specificity. In one recent study (Laroui et al., 2010a), an anti-inflammatory peptide (KPV, proline–lysine–valine) was successfully delivered to the colon using PLA NPs transported to the colon in a hydrogel made of alginate and chitosan. The authors successfully coupled KPV to BSA in the inner phase of the NP to prevent the burst effect that leads to an early and inefficient drug delivery from the NPs. Time and bacterial enzyme effects specifically degraded the hydrogel in the colon and delivered KPV-loaded NPs. Once delivered to the colon, NPs interact with cells like epithelial cells and macrophages, and deliver KPV intracellularly (Laroui et al., 2010a).

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The ancient anti-inflammatory peptide α-MSH is produced by cells, such as phagocytes and keratinocytes, which have primary roles in host defence. Its concentration increases in the blood of patients with infectious or inflammatory disorders. However, concentrations of the peptide greater than those found in human plasma were much more effective in reducing cytokine production in in vitro and animal models, and had more pronounced antimicrobial influences. This suggests that α-MSH, given in pharmacological concentrations, could help in the treatment of inflammatory and infectious disorders in humans. A priori, one might fear a detrimental effect of large doses of α-MSH. However, there is, so far, no evidence for the transformation of nevi into malignant melanomas in response to pharmacological amounts of this peptide. Some puzzling aspects of the effect of α-MSH on the malignant transformation of human melanocytes in vitro are discussed above and in Ref. 23.

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3.4 Peptides with anti-inflammatory activity

Inflammation is a complex process characterized by detrimental effects and contributes to the pathophysiology of several diseases such as cancer, atherosclerosis, rheumatoid arthritis, ulcerative colitis, asthma, and type-2 diabetes [128]. Commonly, inflammation is a response to local injury or infection and it involves immune-systems cells [129]. It has been reported that inflammation also contributes to aging and age-related diseases, because continuous (chronic) upregulation of pro-inflammatory mediators are induced during the aging process as a result of an age-related redox imbalance and dysregulation of the immune system that activates many pro-inflammatory signaling pathways. Furthermore, it has been reported that some skin diseases such as atopic dermatitis, xerosis, rosacea and psoriasis are classified as chronic inflammatory diseases [95,130]. During inflammatory processes, a variety of cytokine factors are produced by macrophages and T lymphocytes cells to recruit leukocytes to the site of infection or injury [131,132]. Inflammation-promoting cytokines, involved in the up-regulation of the inflammatory reaction, are a series of immunoregulatory molecules which include IL1α, IL1β, IL2, IL6, IL8, IL12, TNFα, and IFNγ [131]. Therefore, the measurement of cytokines can be used as an indicator or biomarker of inflammatory status. In the majority of cases, the evaluation of anti-inflammatory activities of bioactive peptides have been evaluated using cell line models with lipopolysaccharides (LPS). In studies using cell lines with LPS-induced inflammatory processes, it has been reported that the anti-inflammatory mechanisms of bioactive peptides are related to their capacity to bind to the lipid A moiety of LPS and the interference with LPS–CD14 interactions by means of competition with the LPS-binding peptide [53].

Yu et al. [129] identified 17 anti-inflammatory peptide sequences from spent hen muscle proteins. However, only the sequence FLWGKSY showed the highest anti-inflammatory activity, which induced 79% reduction of IL-6 production in endotoxin-activated macrophage-like U937 cells. Furthermore, the authors found that the nature of hydrophobicity and the presence of a tryptophan residues within peptide sequences were important for IL-6 inhibitory activity. Bamdad et al. [53] reported bioactive peptides with anti-inflammatory properties obtained from β-Lactoglobulin treated with high hydrostatic pressure-assisted enzymatic hydrolysis. In related work, Meram and Wu [128] reported egg yolk livetin peptide fractions with anti-inflammatory effects in LPS-induced RAW 264.7 macrophages. Results showed inhibition on the production of nitric oxide (NO) (22.7–39.2%), pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) (36.9–43.2%), interleukin-1β (IL-1β) (26.1–50.9%), interleukin-6 (IL-6) (60.4–69.0%), and the expression of inducible nitric oxide synthase (iNOS) (58.6–62%). In addition, Ma, Liu, Shi, and Yu [133] reported that anti-inflammatory peptides were obtained from whey protein hydrolysates. The peptide DQWL showed a significant (P < 0.05) inhibitory ability on the mRNA expression of IL-1β, COX-2, and TNF-α, and the secretion of IL-1β and TNF-α proteins in LPS-induced RAW 264.7 mouse macrophages. The inhibitory effects of DQWL on proinflammatory mediators and cytokines were possibly mediated by the inhibition of NF-κB and p38 MAPK signaling pathways. In addition, Manavalan et al. [134] used a random forest (RF)-based method for predicting anti-inflammatory peptides. The method proposed was based on the contribution of individual composition (amino acid-, dipeptide composition, amino acid index, chain-transition-distribution, and physicochemical properties) in anti-inflammatory peptide prediction.

To the best of our knowledge, few studies have described the protective anti-inflammatory role of peptide fractions on cytokine-stimulated dermal fibroblasts, suggesting their potential as skin health enhancers. Offengenden et al. [135] reported that chicken collagen peptides showed an anti-inflammatory effect on TNF-α-induced inflammation in dermal fibroblasts by the decreasing expression of inflammatory molecules, namely, intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). A similar result was reported by Sun et al. [136] who found that egg ovomucin peptides exhibited anti-inflammatory activity on TNF-α-induced inflammation in dermal fibroblasts by the decreasing expression of ICAM-1. Scientific evidence supports that bioactive peptides possess anti-inflammatory capacity mediated by the inhibition and induction of the immune systems in cell lines. However, further research is required by using more suitable cell lines (e.g. epidermal cell lines) or ex vivo models (e.g. animal or human skin).

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3.5 Anti-inflammatory activity

Inflammation is a self-limiting part of the defence response against injuries. However, chronic inflammation can occur in some disorders, in which the inflammatory process becomes continuous. Chronic inflammation is involved in several diseases such as cardiovascular diseases, cancer, rheumatoid arthritis, atherosclerosis, asthma, diabetes, Alzheimer, pulmonary diseases and autoimmune diseases (Y. S. Kim, Ahn, & Je, 2016). Macrophages have an important role in immune responses, once they can secret, by stimulus, several inflammatory mediators as nitric oxide (NO), prostaglandin E2 (PGE2) and cytokines [tumour necrosis factor (TNF-α), interleukin-6 (IL-6) and interleukin-1 (IL-1)] (Ahn, Cho, & Je, 2015). Macrophages are activated by interferon-gamma (IFNγ), pro-inflammatory cytokines (like TNF-α, IL-6 and IL-1b) and bacterial lipopolysaccharides (LPS) from Gram-negative bacteria (Ahn et al., 2015). Nitric oxide (NO) was found to be involved in inflammation and carcinogenesis, whereby its inhibition can be of great importance for controlling inflammation (E. K. Kim, Kim, Hwang, Kang, et al., 2013). Several peptides with a NO inhibitory activity had been described from mussels and fishes. Most of the anti-inflammatory peptides found were obtained from molluscs. There is scarce information on those peptides isolated from algae, fish and crustaceans. From bivalves Ruditapes philippinarum (Lee et al., 2012), Mytilus coruscus (E. K. Kim, Kim, Hwang, Kang, et al., 2013) and Crassostrea gigas (Hwang et al., 2012) anti-inflammatory peptides were isolated by alcalase, flavourzyme and protamex hydrolysis, respectively. These three peptides showed in vitro ability to inhibit NO production, LPS-induced, in mouse macrophages cell line (RAW264.7). From salmon pectoral fin, an anti-inflammatory peptide was isolated by pepsin hydrolysis, with the amino acid sequence Pro-Ala-Tyr. This peptide showed several beneficial anti-inflammatory responses in LPS-stimulated RAW264.7 macrophage cells, as it inhibited the production of NO (63.80%) and PGE2 (45.33%), suppressed the protein expression of inducible NO synthase and cyclooxygenase-2, which are responsible for the production of NO and PGE2, and attenuated the production of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1b) (Ahn et al., 2015).

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3.9 Anti-inflammatory peptides

Inflammation has acquired an annoying reputation for many years since the strong correlation between the chronic inflammation and the development of many diseases. Inflammation is part of our body's immune response, which protects us from damaged cells, viruses, and other harmful stimuli. The process of acute inflammation is short-lived and usually subsides in a short time. However, the chronic inflammation is a systemic inflammation that lasts for months or years, and it is considered as a common underlying cause of major metabolic diseases, including coronary artery disease, diabetes, Alzheimer's disease, and so on (Knight-Sepulveda, Kais, Santaolalla, & Abreu, 2015). Diet has an important influence on the development and progression of inflammation (Lewis & Abreu, 2017). Results of epidemiological studies have shown that diets with a higher pro-inflammatory capacity are associated with a higher risk of CVDs, and that consumption of diets with anti-inflammatory effects is an effective strategy to prevent CVDs (Li, Lee, et al., 2020). Applying functional foods in the prevention of chronic inflammatory disorders has aroused appealing interest (Giménez-Bastida, Laparra-Llopis, Baczek, & Zielinski, 2018). Shi et al. (2019) revealed that QPHs prepared by enzymatic hydrolysis (e.g., pepsin, pancreatic, and papain) presented a strong immunomodulatory activity in the LPS-stimulated immune response. They also found that the intact quinoa protein had similar anti-inflammatory effects to hydrolysates. On the contrary, Moronta, Smaldini, Docena, and Añón (2016) found that amaranth protein hydrolysates exhibited stronger inhibitory effects on the bacterial flagellin-mediated immune response of epithelial cells than non-hydrolysed isolates, indicating that enzymatic digestion could improve anti-inflammatory activity of amaranth protein. 16 active peptides from amaranth protein hydrolysates were further identified, among which SSEDIKE and IADEDPDEANDK exhibited excellent anti-inflammatory ability by down-regulating the mRNA of CCL20 in Caco-2 cells (Moronta et al., 2016). Recently, Sandoval-Sicairos et al. (2021) isolated three fragments with different molecular weights from the pancreatic enzyme hydrolysate of germinated amaranth, and found that fragments with molecular weights greater than 10 KDa and molecular weights less than 3 KDa exhibited stronger anti-inflammatory activity in lipopolysaccharide-induced RAW 264.7 macrophages. The peptides viz. PQQEHSGEHQ, RFQDQHQ, AITGQVPRR, and QDMK that possesses 1 to 3 glutamine residues could be contribute for enhancing anti-inflammatory activity. The peptide sequence, like SEPFG, is imbedded in other anti-inflammatory peptides (HGSEPFGPR), which was observed earlier in extruded amaranth (Montoya-Rodríguez, de Mejía, Dia, Reyes-Moreno, & Milán-Carrillo, 2014). Moreover, it was reported that peptides released after hydrolysis of amaranth protein have ability to inhibit the NF-ĸB signaling pathway via suppressing phosphorylation of IKK-α and IĸB-α and reducing the expression of NF-ĸB transcription factors p-50 and p-65 (Montoya-Rodríguez, de Mejía, Dia, Reyes-Moreno, & Milán-Carrillo, 2014). Buckwheat constitutes plentiful bioactive phytochemicals that exhibit anti-inflammatory activity in vivo and in vitro (Zhu, 2016a, 2016b, 2020). Giménez-Bastida et al. (2018) revealed that digested buckwheat-enriched products (i.e., buckwheat sprouts, buckwheat-enriched white bread, and roasted buckwheat groats – non-fermented and fermented) significantly decreased the TNF-α-induced migration and attenuated the TNF-α-altered cell cycle in colon myofibroblasts. Further comparative studies demonstrated that active compounds but not rutin or quercetin in buckwheat exert anti-inflammatory activity. However, immunomodulatory peptides in buckwheat have not been reported so far. Over the past few decades, researchers have identified various inflammatory mediators and cell signaling networks. Complex compensatory mechanisms involved in the crosstalk between pathways make the anti-inflammatory strategies targeting single signaling pathways or inflammatory factors inefficient (Jantan et al., 2021). It was noted that during germinated amaranth GID, phenolic compounds were released. Thus, the anti-inflammatory effects may be a synergy of peptides and phenolics. However, some reports revealed that the phenolics free amaranth peptides have good anti-inflammatory activity than that of synergy of peptides with phenolic compounds (Sandoval-Sicairos et al., 2021; Montoya-Rodríguez & de Mejía, 2015). The above-mentioned findings suggested that pseudocereals-derived peptides have anti-inflammatory potential in vitro, but in vivo experiments are still needed to gain a deeper understanding of their effects on inflammation-associated signaling pathways, providing a solid theoretical basis for the development of functional foods for the prevention and treatment of inflammatory diseases.

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3.6 Production of peptides with anti-inflammatory activity

Inflammation is an essential, complex and highly regulated physiological adaptive response of the body to cell damage and tissue vascularization, that enables patient survival during infection or injury and maintains tissue homeostasis under different noxious conditions [620]. This response is part of the host defense mechanism against inflammatory inducers like chemical and noxious mechanical agents, microbial infections, and conditions such as infection and tissue injury [621,622]. During the early phases of inflammatory response, tissue-resident cells (inflammatory sensors) detect the inflammatory stimulus and release soluble inflammatory mediators, including cytokines, vasoactive amines, free radicals, chemokines and eicosanoids [620,621]. It is important to mention that, although a typical inflammatory response consists of four components (inflammatory inducers, the sensors that detect them, the inflammatory mediators induced by the sensors, and the target tissues that are affected by the inflammatory mediators), each component comes in multiple forms and their combinations function in distinct inflammatory pathways which depend on the nature of the inflammatory trigger. Thus, for example, bacterial pathogens are detected by receptors of the innate immune system, such as Toll-like receptors, which are expressed on tissue-resident macrophages and induce the production of inflammatory cytokines (e.g., tumor necrosis factor-α, interleukins-1, interleukins −6, interleukin-1β) and chemokines (e.g., chemokine CC ligand 2 and C-X-C chemokine 8), nitric oxide as well as prostaglandin-E2 [620,623]. Excessive and uncontrolled inflammation is harmful to all tissues, since it may cause many acute and chronic human diseases including obesity, atherosclerosis, type 2 diabetes, cancer and neurodegenerative diseases [620,624]. For example, dysregulated activation of some inflammatory enzymes such as cyclooxygenase-2, generating prostaglandin-E2 from arachidonic acid, and inducible nitric oxide synthase, which catalyzes the reaction that oxidizes L-arginine to nitric oxide and citruline, play important roles in the progression of oncogenesis [625]. Therefore, suppressing the overproduction of inflammatory mediators and the control of the abnormal up-regulations of the inflammatory enzymes (that promote excessive inflammation) is important for the treatment and prevention of inflammation and to reduce the risk of inflammation-derived diseases [626]. For this reason, some synthetic drugs have been employed to regulate the response of the immune system. Unfortunately, the prolonged use of these chemical anti-inflammatory drugs may result in cardiovascular, renal or gastrointestinal damage. Therefore, there is a growing interest on the use of non-toxic natural compounds to reach this goal [627,628]. In this regard, the anti-inflammatory activity of many plant and animal derived food proteins and protein hydrolysates has been demonstrated [629,630].

In order to improve the bioactivity of food proteins, enzymatic hydrolysis has been applied to many food proteins to release bioactive peptides with desired functional properties [626]. Alcalase is one of the protesase used to produce protein hydrolysates with anti-inflammatory activity [631].

3.6.1 Use of stand-alone Alcalase

Focusing on the use of Alcalase, Oseguera-Toledo et al. demonstrated that Alcalase hydrolysates of pinto Durango and Negro beans inhibit cyclooxygenase-2 expression, prostaglandin E2 production, inducible nitric oxide synthase expression and nitric oxide production [632]. For this reason, these hydrolysates from common beans can be used to treat inflammatory associated diseases [632]. In another paper, an anti-inflammatory peptide was identified in lupine protein hydrolysates obtained by Alcalase hydrolysis [633]. This peptide, with a sequence of Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg, was synthesized and its anti-inflammatory activity was tested. It was found that the peptide may help prevent chronic inflammation due to a significant reduction of the expression of tumor necrosis factors, interleukin-1β, and CC motif chemokine ligand 2, and the induction of the anti-inflammatory cytokine interleukin-10 expression, together with a decrease of nitric oxide production [633]. Lee et al. obtained velvet antler Alcalase hydrolysate and assessed their anti-inflammatory effects in zebrafish as well as in vitro, using different cell lines [634]. They found that the Alcalase hydrolysate inhibited the production of nitric oxide by lipopolysaccharide-induced cells in a dose-dependent manner and also reduced the expression of inflammatory mediators such as nitric oxide synthase and cyclooxygenase-2. In addition, the analysis of anti-inflammatory effects of velvet antler Alcalase hydrolysate using lipopolysaccharide-stimulated zebrafish showed that this hydrolysate significantly inhibited the extent of lipopolysaccharide-stimulated cell death and generation of nitric oxide and reactive oxygen species in zebrafish [634]. These authors emphasize that velvet antler Alcalase hydrolysate could be used as a natural and strong anti-inflammatory, and that enzymatic hydrolysis of velvet antler may be an effective process to produce antler derivatives that can be used in the preparation of health foods and nutraceutical products [634]. In another study, it was demonstrated that the low-molecular weight fractions prepared from ovomucin Alcalase hydrolysate may have potential applications for the maintenance of dermal health and treatment of skin diseases [635], due to the their anti-inflammatory activity regulated through the inhibition of tumor necrosis factor-mediated nuclear factor κ-light-chain-enhancer of activated B cells activity [635].

Alcalase has also been used in the hydrolysis of whey protein to produce, isolate and characterize anti-inflammatory peptides. In one study, eight peptides, including 2 new peptides (DYKKY and DQWL) were identified [636]. DQWL showed the strongest inhibitory ability on cyclooxygenase-2, interleukin-1β, and tumor necrosis factor-α mRNA expression and production of interleukin-1β and tumor necrosis factor-α proteins [636].

3.6.2 Comparison of Alcalase with other proteases

Ruditapes philippinarum protein extract was hydrolyzed using eight proteases, being Alcalase among them [637]. It was found that the Alcalase-produced hydrolysate exhibited the highest nitric oxide production inhibitory activity, and one of the produced peptides displayed potent anti-inflammatory activity through inhibition of the lipopolysaccharides-induced nitric oxide production in RAW264.7 cells [637]. In another research, tuna cooking juice was hydrolyzed by three commercial enzymes (Flavourzyme, Orientase and Alcalase) [629]. Among the evaluated enzymes, Alcalase hydrolysate exhibited the most potent anti-inflammatory capability, and its peptide fraction with molecular weight ranging from 204 to 1672.9 Da possessed the greatest activity [629]. O'Sullivan et al. reported the production of hydrolysates from bovine lung tissue using pepsin, papain or Alcalase, and they assessed the anti-inflammatory activity of these hydrolysates in RAW264.7 macrophages and Jurkat T cells [638]. They found that the cell treatment with the Alcalase hydrolysate significantly decreased the production of the pro-inflammatory cytokines interleukin-6 and interleukin-1β in a dose dependent manner in RAW264.7 cells, and the nitric oxide production; therefore, the authors concluded that the Alcalase hydrolysis of bovine lung may have potential as an anti-inflammatory agent [638]. Finally, Meram and Wu, evaluated the anti-inflammatory effects of egg yolk livetins (α, β, and γ-livetin) fraction and its hydrolysate, prepared by hydrolysis with Alcalase or pepsin, on lipopolysaccharide-induced RAW 264.7 macrophages as an in vitro model [626]. They found that the treatment with livetins and peptides from its hydrolysate significantly reduced the inflammatory response by the inhibition of production of nitric oxide, pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-6 and interleukin-1β, and the expression of inducible nitric oxide synthase. In addition, Alcalase hydrolysate showed more effects in inhibiting prostaglandin-E2 production as well as expression of cyclooxygenase-2 [626].

3.6.3 Combined use of Alcalase with other proteases

Regarding the use of Alcalase in combination with other proteases, there is just one example in the analyzed time frame. Alcalase and Izyme AL were used to hydrolyze lupine protein isolate to obtain protein hydrolysates with potential anti-inflammatory capacities through their in vitro inhibition capabilities of phospholipase A2, cyclooxygenase 2, thrombin, and transglutaminase, which are all enzymes that are involved in the inflammatory process [639]. The protein hydrolysates prepared after 15 min of hydrolysis with Alcalase and lupine protein hydrolysates obtained after 60 min of hydrolysis with Izyme followed by 15 min of hydrolysis with Alcalase, exhibited the best inhibitory activities [639].

Evidently, Alcalase hydrolysis of different proteins is an excellent tool for producing anti-inflammatory peptides which have potential to be used in the preparation of health foods and nutraceutical and pharmaceutical products that promote and protect global health, against acute or chronic diseases derived from the inflammatory response.

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4.2 Anti-inflammatory bioactive peptides

Bioactive peptides with known anti-inflammatory effects are potential candidates for improving endothelial dysfunction. In contrast to antioxidant peptides, nearly all anti-inflammatory ones have been validated in physiologically relevant systems like cultured cells and animal models, which improve their chances of in vivo biological actions. In spite of this, few of the anti-inflammatory peptides identified to date have been specifically tested for improvements in vascular/endothelial functions.

Research in our laboratory had identified an egg protein ovotransferrin derived tripeptide, Ile-Arg-Trp, as a potential ACE (angiotensin converting enzyme) inhibitory molecule [40]. Further testing of this peptide determined its additional anti-inflammatory and antioxidant effects on cultured endothelial cells [56]. These functions were validated in an in vivo study involving SHRs where this peptide reduced blood pressure, improved endothelium-dependent vasorelaxation (as shown in ex vivo study) and attenuated markers of inflammation, showing correlation between cell-based and whole animal studies [57]. Another ovotransferrin derived peptide, Ile-Gln-Trp also showed similar anti-inflammatory effects on endothelial cells and comparable vasculo-protective roles in SHRs [39,58]. It is likely that a combination of factors such as anti-inflammatory properties, ACE inhibition and improved NO bioavailability all contributed to the final outcomes in both cases.

Peptide-rich milk protein hydrolysates have shown anti-inflammatory effects in cultured endothelial cells and appear to inhibit leukocyte-endothelial interactions [59], which may explain some of their beneficial vascular/endothelial roles in addition to better known ACE inhibitory effects. A gastric enzyme hydrolysate of Spirulina (a type of sea weed) yielded 2 peptides (Leu-Asp-Ala-Val-Asn-Arg and Met-Met-Leu-Asp-Phe) with similar anti-inflammatory effects on endothelial cells [60], while other bioactive peptides such as Ser-Ser-Ser, Glu-Glu-Glu and Val-Pro-Leu have all been shown to attenuate leukocyte-endothelial interactions [61]. Given the crucial role for inflammatory leukocyte recruitment in atherosclerosis, such peptides may help maintain normal endothelial functions in an inflamed environment.

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2.4 Anti-inflammatory peptides nanomedicines

Anti-inflammatory peptides, which are small bioactive molecules containing up to 50 amino acids, are raising increasing interest [78]. For example, the anti-inflammatory peptide KAFAK, an inhibitor of mitogen-activated protein kinase-2, was assessed in a cartilage explant inflammation model. To increase the peptide’s therapeutic lifetime, KAFAK was first loaded into co-polymerized 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and N-isopropylacrylamide (NIPAM) monomers, to form pH-dependant poly(NIPAM-co-AMPS) nanoparticles [79,80]. These NPs demonstrated the ability to selectively diffuse through the degraded cartilage explants before to release the peptide, allowing a significant reduction of pro-inflammatory cytokine production. However, the authors noted that the high affinity of KAFAK towards the nanoparticles core and the lack of core degradation resulted in the release of less than half of the loaded peptides, thus limiting the therapeutic applications of this approach [80]. Then, the same authors developed a more complex delivery system, based on poly(NIPAM) nanoparticles with degradable disulfide crosslinks (abbreviated as NGPEGSS), loaded with KAFAK peptides. The system relies on the co-polymerization of NIPAM and AMPS monomers, coated with polyethylene glycol (PEG) and using the degradable crosslinker bis(acryloyl)cystamine (BAC) for more effective release into the intracellular compartment [80,81]. Ex vivo, a preferential accumulation of the NPs was observed throughout the inflamed aggrecan-depleted cartilage explants compared to their healthy counterparts and, simultaneously, a significant reduction of IL-6 pro-inflammatory cytokine was measured. Moreover, these NPs were stable and released less than 10% of the loaded KAFAK over 96h at pH=7.4 in a non-reducing environment. Thus, their ability to diffuse into the damaged cartilage, maintaining the majority of their payload prior to cell uptake by chondrocytes and macrophages, combined with a release of the peptide into these cells, represented an improvement comparatively to the treatment with the free peptide or with the previously described poly(NIPAM-co-AMPS) NPs [81].

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3.5 Anti-inflammatory

Inflammation is an immune system response to damage to the body. When homeostasis is unfavourable and this situation is maintained, macrophages can cause tissue damage by activating the formation of free radicals and other oxidizing elements that are toxic to the cell. If cells die, tissues are damaged and stop making their function giving rise to chronic diseases. Two non-specific anti-inflammatory in silico tools are PreAIP (Khatun et al., 2019), which can be found in http://kurata14.bio.kyutech.ac.jp/PreAIP/(last time accessed on June 2nd, 2023) and AIPpred (Manavalan et al., 2018), found in http://www.thegleelab.org/AIPpred/, last time accessed on June 2nd, 2023. The first tool incorporates manifold features like primary sequence, physicochemical properties, evolutionary features, and structural information by combining k-spaced amino acid pairs, amino acids index and k-spaced amino acid pairs acquired from position-specific scoring matrix through a random forest (machine learning algorithm) classifier, whereas the second one uses 4 different machine learning methods (ERT, RF, k-NN, and SVM) and sequence encoding features like amino acid composition, dipeptide composition, amino acid index, and physiochemical properties. The PreAIP and AIPpred tools have been recently validated by an analysis of several anti-inflammatory peptides proved to exert bioactivity in vitro (Rivera-Jiménez et al., 2022). The first conclusions found by the authors was that both tools were to some extent, comparable, based on the highest scores obtained, but overall, the correlation coefficient of the scores obtained for each peptide by both predictors was found to be 0.2337. This indicates the absence of correlation between both tools, demonstrating that in vitroassays are still necessary.

In addition to these tools, a specific one assessing IL-6 inducing peptides can be also found, called StackIL6 (Charoenkwan et al., 2021). This tool is available from http://camt.pythonanywhere.com/StackIL6 and it was constructed from twelve different feature descriptors (e.g., composition or physicochemical properties) and five popular machine learning algorithms (extremely randomized trees, logistic regression, multi-layer perceptron, support vector machine, and random forest). Similar to this one, an in silico tool to identify IL-2 inducing peptides has also been recently developed, which can be found in https://webs.iiitd.edu.in/raghava/il2pred, last time accessed on June 2nd, 2023. In this IL2Pred tool (Lathwal et al., n.d.), +4600 have been used for training, testing and validation of the models, achieving an accuracy of 73.25%. The complexity of the inflammatory response is evident, and therefore, there are several targets in which peptides can have an effect and lead to pro or anti-inflammatory responses. In this sense, more specific tools are still required in order to fully understand the underlying mechanisms by which a pool of peptides would lead to a modulation of the response to a certain extent. In Table 1, several examples on how these tools to predict the antiinflammatory potential of food-derived peptides have been used is described. As it can be observed, most of the authors are using these tools as supporting information to increase the quality of their reports. As a consequence, more validation analysis comparing in vitro and in silico assays are required, in order to enhance the functionality of these tools, specially considering the numerous mechanisms underlying the anti-inflammatory response.

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