Food-derived peptidic antioxidants: A review of their production, assessment, and potential applications

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Food-derived peptidic antioxidants: A review of their production, assessment, and potential applications Anusha G.P. Samaranayaka, Eunice C.Y. Li-Chan* Food, Nutrition, and Health Program, Faculty of Land and Food Systems, 2205 East Mall, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4

A R T I C L E I N F O

A B S T R A C T

Article history:

Antioxidant properties of food-derived peptides have been described in an increasing

Received 22 February 2011

number of studies in recent years. Consequently, these peptides are being considered as

Received in revised form

potential sources to control various oxidative processes in the human body as well as in

17 May 2011

food. It is however difficult to compare results from various studies due to the diversity

Accepted 20 May 2011

of in vitro assay systems and inconsistency in the conditions used to evaluate antioxidative capacity of peptides and protein hydrolysates. Further, specific assays and biomarkers are yet to be established to confirm their bioactive potential. This review summarizes the liter-

Keywords:

ature on food sources and methods of antioxidative peptide production, and the reported

Antioxidative peptides

efficacies and mechanisms of their action. Furthermore, it presents a critical evaluation

Food-derived peptides

of methods used for assessing antioxidative activity of peptides. Examples of promising

Protein hydrolysates Antioxidant activity assays Antioxidant applications

applications of these peptides in food, nutraceuticals and cosmeceuticals are also discussed with an insight to the future research needs.  2011 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3. 4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Occurrence and production of antioxidative peptides from food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Naturally occurring antioxidative peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Hydrolysis of food proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Enzymatic production of antioxidative protein hydrolysates and peptides . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Formation of antioxidative peptides during fermentation, food processing or GI-digestion . . . . . . . . . . Molecular characteristics and antioxidant mechanisms of peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assessment of the antioxidative capacity of peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. In vitro chemical assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. In vitro biological assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. In vivo assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential applications of food-derived peptidic antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Functional ingredients to control oxidative deterioration of food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Functional foods and nutraceuticals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Cosmeceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author: Tel.: +1 604 822 6182; fax: +1 604 822 5143. E-mail address: [email protected] (E.C.Y. Li-Chan). 1756-4646/$ - see front matter  2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2011.05.006

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1.

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Challenges of using peptides as antioxidants and future research needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Introduction

Antioxidants play a vital role in both food systems as well as in the human body to reduce oxidative processes. In food systems, antioxidants are useful in retarding lipid peroxidation and secondary lipid peroxidation product formation, and thus help to maintain flavor, texture, and, in some cases, the color of the food product during storage. Antioxidants further reduce protein oxidation as well as the interaction of lipidderived carbonyls with proteins that leads to an alteration of protein functionality (Elias, Kellerby, & Decker, 2008). Natural antioxidants such as vitamin C, tocopherols, herbal extracts like rosemary and sage, as well as tea extracts have already been commercialized as alternatives to synthetic antioxidants in food systems (Shahidi, 2000). Proteins and protein hydrolysates derived from sources like milk, soy, egg, and fish have also been shown to exhibit antioxidant activity in various muscle foods (Hagen & Sandnes, 2004; Penˇa-Ramos & Xiong, 2003; Sakanaka & Tachibana, 2006). In the human body, endogenous antioxidants help to protect tissues and organs from oxidative damage caused by reactive oxygen and reactive nitrogen species such as hydroxyl radicals (OH), peroxyl radicals (OOR), superoxide anion  ðO 2 Þ, and peroxynitrite (ONOO ). These endogenous antioxidative systems in the body include enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, and various nonenzymatic compounds such as selenium, atocopherol, and vitamin C (Wojcik, Burzynska-Pedziwiatr, & Wozniak, 2010). Apart from these, amino acids, peptides, and proteins also contribute to the overall antioxidative capacity of cells and towards maintaining the health of biological tissues. For example, blood proteins are estimated to scavenge 10–50% of the peroxyl radicals formed in the plasma (Frei, Stocker, & Ames, 1988; Wayner, Burton, Ingold, Barclay, & Locke, 1987). Peptides such as carnosine, anserine, and glutathione are well-known for their endogenous antioxidative activity (Babizhayev et al., 1994). However, the antioxidant– prooxidant balance in human body can change with the progression of age and due to other factors such as environmental pollutants, fatigue, excessive caloric intake, and high fat diets. With advancing age, the plasma and cellular antioxidant potential as well as the absorption of nutrients, including antioxidants, gradually diminishes (Elmadfa & Meyer, 2008; Rizvi, Jha, & Maurya, 2006). Researches have in fact indicated an accumulation of protein carbonyls with the aging process in humans as a result of the action of free radicals on the proteins (Chakravarti & Chakravarti, 2007; Stadtman, 2006). Use of dietary antioxidants has been recognized as potentially effective to promote human health by increasing the body’s antioxidant load.

Dietary antioxidant supplements and functional foods containing antioxidants like a-tocopherol, vitamin C, or plant-derived phytochemicals such as lycopene, lutein, isoflavones, green tea extract, and grape seed extracts find a huge demand in the current marketplace. A great deal of attention has also appeared in the recent literature to identify and assess antioxidative potential of peptides derived from various food sources and their possible applications as functional foods and nutraceuticals. Even though there are few in vivo or in situ studies conducted to date, in vitro studies using various chemical assays have indicated the potential of these food-derived peptides to act as antioxidative agents to control various oxidative processes in the human body as well as in food. Multifunctional nature of peptidic antioxidants, for example having the ability to impart other bioactivities such as antihypertensive, opioid, and cholesterol lowering capacity (Davalos, Miguel, Bartolome, & Lopez-Fandino, 2004; Herna´ndez-Ledesma, Miralles, Amigo, Ramos, & Recio, 2005; Herna´ndez-Ledesma, Quiro´s, Amigo, & Recio, 2007; Nagai, Nagashima, Abe, & Suzuki, 2006), make them more attractive candidates than non-peptidic antioxidants as dietary ingredients in promoting human health. The objectives of this paper are to review the reported food sources and processes of antioxidative peptide production, to examine the molecular characteristics and possible mechanisms of antioxidative peptides in exerting their activity in food as well as in biological systems, to conduct a critical evaluation of the methods used in assessing their antioxidative potential, and finally, to present the opportunities and challenges for food and nutraceutical applications of foodderived peptidic antioxidants.

2. Occurrence and production of antioxidative peptides from food 2.1.

Naturally occurring antioxidative peptides

Some antioxidative peptides are naturally present as such in food. For example, glutathione (c-Glu-Cys-Gly), carnosine (b-alanyl-L-histidine), anserine (b-alanyl-L-1-methylhistidine), and ophidine (b-alanyl-L-3-methylhistidine) are antioxidative peptides naturally present in muscle tissues (Babizhayev et al., 1994; Chan & Decker, 1994). Glutathione acts as an electron donor to protect cells from free radicals (Bray & Taylor, 1994). Its reducing power helps maintain the reduced state of cysteinyl sulfhydryl groups in proteins and thereby reduces disulfide bond formation within cytoplasmic proteins (Bray & Taylor, 1994). Carnosine on the other hand can act as a free radical scavenger as well as a metal ion chelator (Kang et al., 2002), and has demonstrated both in vivo and in vitro antioxidative

Table 1 – Antioxidative capacity of food-derived protein hydrolysates and peptides (listed in chronological order of publication since 1991).

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Table 1 – (Continued).

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Table 1 – (Continued).

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Table 1 – (Continued).

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See above-mentioned references for further information

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activity in rat skeletal muscle lipid and protein components under the conditions of oxidative stress (Nagasawa, Yonekura, Nishizawa, & Kitts, 2001). A recent study reported that the administration of carnosine (2 g/l of drinking water) for three months to the experimental rats reduced the thioacetamide (TAA)-induced lipid peroxidation in rat liver, even though it was not able to prevent the development of TAA-induced cirrhotic process (Aydin et al., 2010). Vasodilatory action of carnosine was also reported by Ririe, Roberts, Shouse, and Zaloga (2000). Moreover, carnosine, anserine, and ophidine have been studied for potential physiological functions related to neurotransmitter synthesis (Snyder, 1980).

2.2.

Hydrolysis of food proteins

Antioxidative peptides can also be released from different proteins of plant or animal origin during preparation of protein hydrolysates using exogenous or endogenous enzymes, food processing or during microbial fermentation, as well as during gastro-intestinal (GI) digestion of food proteins (Korhonen & Pihlanto-Leppa¨la¨, 2003).

2.2.1. Enzymatic production hydrolysates and peptides

of

antioxidative

protein

According to the literature, hydrolysis using enzymes has been the main process for producing antioxidative peptides from food proteins (Table 1). Various food protein sources including fish, milk, egg, soybean, wheat and zein, among others, have been exploited to produce antioxidative protein hydrolysates and peptides (Table 1). Fish and other seafood sources have particularly gained much interest as potential antioxidative peptide sources mainly due to the abundance of raw materials in the form of processing discards and underutilized species, in conjunction with research findings indicating an array of other biological activities for fish protein hydrolysates and specific peptide sequences derived from these sources such as antihypertensive, immunomodulatory, neuroactive, antimicrobial, mineral and hormonal regulating properties (Bernet, Montel, Noel, & Dupouy, 2000; Duarte, Vinderola, Ritz, Perdigo´n, & Matar, 2006; Je, Qian, Byun, & Kim, 2007; Jun, Park, Jung, & Kim, 2004; Jung et al., 2006; Liu et al., 2008; Murray & Fitzgerald, 2007). Table 1 also lists different enzymes and/or processes that have been used in making these protein hydrolysates and peptides. Use of exogenous enzymes is preferred in most cases over the autolytic process (i.e., use of endogenous enzymes present in the food source itself), due to the shorter time required to obtain similar degree of hydrolysis as well as better control of the hydrolysis to obtain more consistent molecular weight profiles and peptide composition. Industrial food-grade proteinases such as Alcalase, Flavourzyme, and Protamex derived from microorganisms, as well as enzymes from plant (e.g. papain) and animal sources (e.g., pepsin and trypsin), have been widely used in producing antioxidative peptides (Table 1). Nevertheless, the use of endogenous enzymes has also been reported in the literature, especially for the production of various antioxidative fish protein hydrolysates (Table 1). Depending on the raw material, endogenous enzymes such as trypsin, chymotrypsin, pepsin, other enzymes of the viscera and digestive tract, as well as lysosomal

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proteases or catheptic enzymes in fish or other invertebrate muscle cells may contribute to the breakdown of proteins during autolysis (Kristinsson & Rasco, 2000). Peptides with antioxidative properties could be prepared from Alaska pollack (Theragra chalcogramma) and yellowfin sole (Limanda aspera) frame proteins by using a crude enzyme mixture from mackerel intestine (Je, Park, & Kim, 2005; Jun et al., 2004), while the increased level of cathepsin L-like proteases present in parasitized Pacific hake (Merluccius productus) muscle was successfully used in our laboratory to produce fish protein hydrolysates with antioxidative properties (Samaranayaka & Li-Chan, 2008; Samaranayaka, Kitts, & Li-Chan, 2010). Careful choice of a suitable enzyme and digestion conditions such as temperature and pH for the optimal activity of enzyme, as well as the control of hydrolysis time, are crucial for obtaining protein hydrolysates with desirable functional and bioactive properties. The crude protein hydrolysate may be further processed, for example by passage through ultrafiltration membranes, in order to obtain a more uniform product with the desired range of molecular mass (PihlantoLeppa¨la¨ & Korhonen, 2003). In large-scale production of hydrolysates, membrane technology may also be coupled with enzymatic hydrolysis in a continuous process, thereby reducing the cost by eliminating the need for heat or pH adjustment to inactivate the enzymes at the end of hydrolysis (Gue´rard, 2007). Low molecular mass membrane cut-offs are useful for concentrating antioxidative peptides from the higher molecular mass components remaining, including undigested polypeptide chains and enzymes. Other techniques such as nanofiltration, ion-exchange membranes, or column chromatographic methods can be used in further concentrating and purifying antioxidative peptides (Pihlanto-Leppa¨la¨ & Korhonen, 2003).

2.2.2. Formation of antioxidative peptides fermentation, food processing or GI-digestion

during

In fermented food products, antioxidative peptides can be produced due to the action of microbes and endogenous proteolytic enzymes (Kristinsson & Rasco, 2000; Yamamoto, Masahiro, & Mizuno, 2003). Rajapakse, Mendis, Jung, Je, and Kim (2005) identified the radical scavenging peptide HFGBPFH from fermented mussel sauce. Fermented milk products made using different strains of lactic acid bacteria have been found to be antioxidative (Virtanen, Philanto, Akkanen, & Korhonen, 2007), and several antioxidative and angiotensinI-converting enzyme (ACE) inhibitory peptide sequences were identified from fermented milk (Herna´ndez-Ledesma, Miralles, et al., 2005). Fermented soybean products such as natto, tempeh, and douche also contain antioxidative peptides due to the action of fungal proteases (Iwa, Nakaya, Kawasaki, & Matsue, 2002; Sheih, Chen, & Chiu, 2000; Wang et al., 2008). The peptide cyclo(His-Pro) is an antioxidative peptide found in processed food products like dried shrimp, fish sauce, tuna, ham, potted meat, non-dairy creamer, white bread, and noodles (Hilton, Prasad, Vo, & Mouton, 1992; Minelli, Bellezza, Grottelli, & Galli, 2008). This peptide is produced during thermal processing of food (Prasad, Hilton, Lohr, & Robertson, 1991) and, because of its cyclic structure, it has the ability to be absorbed through the GI tract and reduce oxidative stress-based neurodegeneration (Minelli

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et al., 2008). Antioxidative and ACE-inhibitory activities were also observed in pressure-cooked chum salmon cartilage and skin extracts (Nagai et al., 2006). GI digestion of food proteins by digestive enzymes or microbial enzymes from gut microflora (Mo¨ller, ScholzAhrens, Roos, & Schrezenmeir, 2008) is another possible pathway of producing peptides that can exert antioxidative activities directly in the gut, via receptors or cell signaling in the gut, or at other target sites inside the body, if the peptides have the ability to permeate the epithelial cell membrane and enter the blood circulation. Oligopeptides, especially di- and tri-peptides, have the potential to permeate through the upper GI tract depending on their structural characteristics. Peptide LVGDEQAVPAVCVP released from in vitro GI digestion of mussel (Mytilus coruscus) protein exhibited potent antioxidant potential, inhibiting the formation of reactive oxygen species from the peroxidation of polyunsaturated fatty acids (Jung et al., 2007). Sannaveerappa, Carlsson, Sandberg, and Undeland (2007) also reported that an enzymatic breakdown of herring (Clupea harengus) proteins under GI-like conditions increased the peroxyl radical scavenging activity and the potential to inhibit low-density lipoprotein (LDL) oxidation. The activities of pre-formed antioxidative hydrolysates and peptides may or may not be altered during passage through the GI tract. For example, Sheih, Wu, and Fang (2009) reported that the antioxidative peptide VECYGPNRPQF isolated from microalgae, Chlorella vulgaris, was resistant to GI digestion. On the other hand, significant increase in antioxidative activity was observed upon in vitro simulated GI digestion of Pacific hake protein hydrolysate and the fraction of antioxidative peptides with molecular weight in the 1000–3000 Da range (Samaranayaka et al., 2010). Zhu, Chen, Tang, and Xiong (2008) reported that the antioxidative activity of a zein hydrolysate, which had previously shown antioxidant activity in aqueous solutions and in food systems (Kong & Xiong, 2006), was either decreased or improved during the course of in vitro digestion, depending on the enzymes encountered and the duration of hydrolysis. Therefore, the possibility of modification or breakdown of peptides during the GI-digestion is one of the most important factors to be considered when evaluating potential food-derived antioxidative peptides for promotion of human health.

3. Molecular characteristics and antioxidant mechanisms of peptides In general, all 20 amino acids found in proteins can interact with free radicals if the energy of the free radical is high (e.g., hydroxyl radicals) (Elias et al., 2008). The most reactive include the nucleophilic sulfur-containing amino acids Cys and Met, the aromatic amino acids Trp, Tyr, and Phe, and the imidazole-containing amino acid His. However, free amino acids are not generally found to be effective as antioxidants in food and biological systems, and extensive proteolysis of food proteins in fact has been reported to result in decreased antioxidative activity (Chan, Decker, Lee, & Butterfield, 1994; Ostdal, Andersen, & Davies, 1999; Rival, Boeriu, & Wichers, 2001; Rival, Fornaroli, Boeriu, & Wichers, 2001; Zhou & Decker, 1999). The higher antioxidative activity

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of peptides compared to free amino acids is attributed to the unique chemical and physical properties conferred by their amino acid sequences, especially the stability of the resultant peptide radicals that do not initiate or propagate further oxidative reactions (Elias et al., 2008). Nevertheless, a recent study conducted by Tsopmo et al. (2009) claims that Trp released from human milk upon GI digestion may have potential to act as a powerful radical scavenger. Several mechanisms have been postulated for the antioxidative properties of peptides, including metal ion chelation, free radical scavenging, and aldehyde adduction (Chen, Muramoto, Yamauchi, & Nokihara, 1996; Zhou & Decker, 1999). Table 1 lists food-derived antioxidative peptides reported in the literature from 1991 to 2010, whereas Table 2 is a summary of peptides that were prepared by chemical synthesis based on the sequences of antioxidative peptides identified from food, in order to understand their structure– activity relationships. The majority of the antioxidative peptides derived from food sources have molecular weights ranging from 500 to 1800 Da (Je, Kim, & Kim, 2005; Je, Park, et al., 2005; Jun et al., 2004; Ranathunga, Rajapakse, & Kim, 2006; Wu, Chen, & Shiau, 2003); moreover, they often include hydrophobic amino acid residues such as Val or Leu at the N-terminus of the peptides, and Pro, His, Tyr, Trp, Met, and Cys in their sequences (Chen, Muramoto, & Yamauchi, 1995; Elias et al., 2008; Uchida & Kawakishi, 1992). Hydrophobic amino acid residues like Val or Leu can increase the presence of the peptides at the water–lipid interface and therefore facilitate access to scavenge free radicals generated at the lipid phase (Ranathunga et al., 2006). Although the structure–activity relationship of antioxidative His-containing peptides has not been well defined yet, the activity could be attributed to hydrogen donating ability, lipid peroxy radical trapping, and/or the metal ion-chelating ability of the imidazole group (Chan & Decker, 1994). The differences in the activity of individual His-containing peptides may be due to the environment surrounding the imidazole group, as indicated by various observations. Murase, Nagao, and Terao (1993) found that N-(longchain-acyl) histidine-containing compounds suppressed the oxidation of phosphatidylcholine liposomes and methyl linoleate. The hydrophobicity of the compounds was important for the accessibility to the hydrophobic targets. In another study, Tsuge, Eikawa, Namura, Yamamoto, and Sugisawa (1991) described a potent antioxidative peptide Ala-His-Lys isolated from the egg white albumin hydrolysate, in which neither the dipeptide His-Lys nor the mixture of constituent amino acids had any activity, but Ala-His was as potent as the parent tripeptide. Uchida and Kawakishi (1992) investigated the oxidation of NRVYIHPF mediated by copper(II)/ascorbate. The N-terminal NRVY sequence contributed significantly to the reactivity of the His residue, which was converted to the 2-imidazolone derivative upon oxidation. Without the N-terminal segment, the His residue in IHPF showed no reactivity against the oxidation. Chen et al. (1996) measured antioxidative activities of 28 synthetic peptides designed based on an antioxidative peptide (LLPHH, Table 2) derived from a proteolytic digest of soybean protein. Results indicated that removal of the C-terminal His residue decreased antioxidative activity, whereas removal of the N-terminal Leu had no effect. In the peptide

Table 2 – Research conducted on synthetic peptides designed on the basis of peptide sequences identified from food sources.

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See above-mentioned references for further information

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Table 3 – Antioxidative potential of food-derived peptides and several commonly used non-peptidic antioxidants as reported using different in vitro assay systems.

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(continued on next page)

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Table 3 – (Continued).

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See above-mentioned references for further information

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sequence, His and Pro played important roles in the antioxidative activity, and among the peptides tested, Pro-His-His showed the greatest antioxidative activity. Further, peptides with the Pro-His-His sequence had the greatest synergism with lipid-soluble antioxidants such as tocopherols and butylated hydroxyanisole (BHA). Saito et al. (2003) also studied antioxidative activity of peptides created in two tripeptide libraries (Table 2). According to their results, for the 114 peptides containing either His or Tyr residues, tripeptides containing two Tyr residues showed higher activity in the linoleic acid peroxidation system than tripeptides containing two His residues. Further, Tyr-His-Tyr showed strong synergistic effects with phenolic antioxidants. Similar to results reported earlier by Chen et al. (1996) for 28 synthetic peptides related to Leu-Leu-Pro-His-His, Saito et al. (2003) reported that Pro-His-His exhibited the greatest antioxidative activity in the linoleic acid peroxidation system, compared to other tripeptides containing Pro or His within the library of 108 structurally related tripeptides. Moreover, substitution of other amino acid residues at either the N-terminus or C-terminus of the Pro-His-His tripeptide did not significantly alter its antioxidative activity. In general, tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but weak peroxynitrite scavenging activity. Cys-containing tripeptides on the other hand showed a strong peroxynitrite scavenging activity. In another study, Tang et al. reported that the free radical scavenging capacity of Alcalase-treated zein hydrolysate was dependent on the radical species and was strongly related to the molecular weight and hydrophobicity of the constituting peptides (Tang et al., 2010). When the ultrafiltrated (1, 3, 5, and 10 kDa membrane cut-offs) fractions of zein hydrolysate were analyzed for their radical scavenging capacities, the water soluble 2,2 0 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity was not dependent on their molecular size. However, ethanol soluble 1,1diphenyl-2-picrylhydrazyl (DPPH) radical and O 2 scavenging capacity of these peptide fractions were molecular weight dependent (Tang et al., 2010). Furthermore, when the peptides of <1 kDa range were subjected to RP-HPLC, the fractions with high hydrophobicity values indicated strong O 2 and DPPH radical scavenging capacity and those with intermediate hydrophobicity displayed the maximum ABTS radical scavenging capacity (Tang et al., 2010). The results described above demonstrate the importance of amino acid composition, sequence, and size in determining the antioxidative potential of peptides. Furthermore, it is evident that different amino acid residues and peptide sequences are responsible for the inhibition of oxidative reactions that are initiated by different types of free radicals or pro-oxidants such as metal ions, as well as in different molecular environments (for example, aqueous, lipid, or emulsion systems, or different pH conditions, or the presence of other compounds in the food matrices or biological systems, etc.). Although a general idea of the importance of specific amino acid residues and peptide sequences for antioxidative action can be obtained based on literature findings, it will really be necessary to first identify the target use of a particular peptide(s) of interest, and then to select appropriate assays and model systems to establish the antioxidative potential considering possible

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mechanisms of action in the envisioned application. The use of different antioxidative assays as well as variations of the same assay by different research groups makes it difficult to compare antioxidative potential and mechanisms of reported peptide sequences. Further, only a few research studies have reported IC50 values that could be used for comparing antioxidative efficacy of reported peptides towards scavenging different radicals or reducing oxidative processes in different model systems. Table 3 is a summary of reported IC50 values for different antioxidative peptides and several commonly used antioxidants towards scavenging different types of radicals. In addition to the peptides derived from food proteins, proteins themselves have the ability to act as antioxidative agents by inactivating reactive oxygen species, scavenging free radicals, chelating prooxidative transition metals, reducing hydroperoxides, and enzymatically eliminating specific oxidants (Elias et al., 2008). Proteins such as casein, b-lactoglobulin, and lactoferrin have been found to be antioxidative in various food systems (Diaz & Decker, 2005; Elias et al., 2006; Kong & Xiong, 2006). In food emulsion systems, proteins and peptides in protein hydrolysates can locate at the oil–water interface due to their surface active property and can form a physical barrier to minimize the contact of lipids with oxidizing agents, and contribute to reducing lipid peroxidation in food systems. Donnelly, Decker, and McClements (1998) reported that the oxidative stability of emulsified Menhaden oil was improved by the formation of a thick protein membrane when whey proteins were used instead of Tween 20 as the emulsifier. Acidic conditions are also preferred in emulsions made with proteins due to the fact that protonated amino groups can repel cationic prooxidants such as Fe2+ and Cu2+ and thereby inhibit the initiation of lipid peroxidation (Kellerby, McClements, & Decker, 2006). However, due to their large size, proteins cannot pass through cellular membranes and will be unable to act as biologically active compounds unless smaller antioxidative peptides have been generated from them upon degradation by GI enzymes or brush border peptidases.

4. Assessment of the antioxidative capacity of peptides Specific assays have not yet been developed or standardized to measure the antioxidative capacity of peptides or peptide mixtures. Therefore, assays that are commonly used for measuring antioxidative capacity of non-peptidic antioxidants have been used in the literature to measure the antioxidative capacity of peptides as well. In vitro assays based on chemical reactions are widely used in quantifying antioxidative effectiveness of whole food, partially purified peptides, and/or individual peptides isolated from food mixtures in preventing oxidative processes occurring in the human body as well as in food systems during storage. Even though these chemical assays give an insight to the potential biological activity of these food-derived antioxidants, further analysis such as investigating the fate of peptides during GI digestion as described previously, their permeability through cellular membranes, as well as their in vivo stability and reactivity has to be conducted in order to confirm their biological efficacy. On the

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other hand, when considering applications of antioxidative compounds to control oxidative rancidity in complex food systems, their physical location is also an important parameter to be considered (e.g., does the antioxidative compound concentrate where the oxidative reactions are most prevalent) (Alamed, Chaiyasit, Mcclements, & Decker, 2009).

4.1.

In vitro chemical assays

Due to the complexity of oxidative processes occurring in food or biological systems as well as the different antioxidative mechanisms by which various compounds may act, finding one method that can characterize the overall antioxidative potential of food is not an easy task. Nevertheless, methods such as the Trolox equivalent antioxidant capacity (TEAC) assay, oxygen radical absorbance capacity (ORAC) assay, and the total radical-trapping antioxidant parameter (TRAP) assay have been widely reported in the literature for measuring antioxidative capacity of food and biological samples (Cao & Prior, 1998; Ghiselli, Serafini, Natella, & Scaccini, 2000; Re et al., 1999). Detailed discussion of the methodology and principles behind these assays and other commonly used in vitro antioxidative capacity assays are beyond the focus of this review. There are several comprehensive reviews covering this topic (Decker, Warner, Richards, & Shahidi, 2005; Huang, Ou, & Prior, 2005; Kyung, Kim, & Lee, 2007; Magalha˜es, Segundo, Reis, & Lima, 2008; Moon & Shibamoto 2009; Prior, Wu, & Schaich, 2005). Generally, based on the chemical reactions involved, these in vitro assays quantify the hydrogen atom donating, electron transferring (i.e., reducing capacity), or metal ion chelating ability of an antioxidant (Huang et al., 2005). Since the mechanism of antioxidative action measured and/or reaction conditions used

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are different from one assay to another, a test sample may show different results for antioxidative capacity depending on the assay system used. For example, a good metal ion chelator may not show any activity in radical scavenging assays. Moreover, the conditions of the assay may affect the antioxidative capacity. The FRAP, TEAC, and Folin–Ciocalteu reagent (FCR) assays used to measure the reducing capacity of antioxidants are conducted under acidic, neutral, and basic conditions, respectively (Huang et al., 2005), and the reducing capacity of an antioxidant can be affected by pH of the assay media. Solubility of antioxidants in the reaction media also plays an important role in their antioxidative capacity. Due to these reasons, more than one assay should be and is often used in measuring antioxidative capacity of a food or a food constituent of interest. Further, in evaluating antioxidants for food use, Decker et al. (2005) explained the importance of using model systems that provide the chemical, physical, and environmental (e.g., pH and ionic strength) conditions expected in food products. Model systems were outlined for three types of food lipids: bulk oil, oil-in-water emulsions, and muscle foods (Decker et al., 2005). In vitro chemical assays that have been reported in the literature for measuring antioxidative capacity of protein hydrolysates and peptides are listed in Tables 1 and 2. Methods such as the TEAC assay and ORAC assay that are widely used for phenolic compounds and other phytochemicals have also been applied to analyze peptides, but to a lesser extent. More commonly used assays include measuring the inhibition of lipid peroxidation in a linoleic acid model system and the capacity to scavenge the DPPH radical (Table 1). Electron spin resonance (ESR) spectrometry is a method that has emerged as a useful tool in studying radical-antioxidant interactions and estimating free radical scavenging capacities of

Table 4 – Antioxidative activity of peptides A–E and selected standards. Symbol Synthetic peptide PLFQNKLAHAK AEAQKQLR AHK PHH PHHADS Standard for assays Trolox (15 lM) BHA BHT a

A B C D E

Origin

Pacific hake fish fillete Pacific hake fish fillete Egg white albumenf Soybean proteing Tuna cooking juiceh

DPPH radical scavenging capacity (%)a,b

ORAC valuea,c

% Inhibition of lipid peroxidationa,d

4.19y 1.98z 1.57z 2.27z 2.73yz

0.126 NDi 0.007 ND 0.030

49.79y 32.13x 87.67z 91.85z 91.35z

34.8x NA NA

NAj NA NA

NA 57y 97z

Values shown are mean values of triplicate analysis conducted in our laboratory using synthetic peptides A–E. Different superscript letters (x–z) within a column are significantly different at p < 0.05 (Tukey’s test). b 1,1-Diphenyl-2-picrylhydrazyl. Final concentration of peptides in the assay was 150 lM. Method described in Samaranayaka and Li-Chan (2008). c Oxygen radical absorbing capacity (lmol Trolox equiv/lmol peptide). Method described in Kitts and Hu (2005). d Samples, BHA, and BHT at 2 · 104 M assay concentration. Method described in Chen et al. (1996). e Samaranayaka and Li-Chan (2008). f Tsuge et al. (1991). g Chen et al. (1996). h Jao and Ko (2002). i Activity not detected up to 1.1 and 2.6 mM assay concentration for peptides B and D, respectively. j Sample not applicable for this assay.

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30 % ABTS radicals scavenged

antioxidants (Je et al., 2007). This assay is specific with a clear reaction mechanism, simple, and free of interferences from sample matrix components such as pigments (Yu & Cheng, 2008). Direct ESR measurement can be performed using a stable radical species such as DPPH radical, whereas ESR spintrapping method with an exogenous spin-trapping molecule is used to measure short-lived reactive oxygen species (ROS) such as singlet oxygen, hydroxyl, and superoxide anion radicals (Yu & Cheng, 2008). Kim and co-workers have used the ESR technique to measure the ability of different protein hydrolysates and peptides to scavenge DPPH radicals and ROS such as hydroxyl, peroxy, and superoxide as well as carbon-centered radicals (Je, Kim, et al., 2005; Je, Park, et al., 2005; Je et al., 2007; Kim, Je, & Kim, 2007; Mendis, Rajapakse, Byun, & Kim, 2005; Mendis, Rajapakse, & Kim, 2005; Rajapakse, Mendis, Byun, & Kim, 2005; Rajapakse, Mendis, Jung, et al., 2005; Ranathunga et al., 2006). As mentioned previously in Section 3, peptidic antioxidants interact differently with free radical species used in different in vitro chemical assay systems for the assessment of the radical scavenging activity. The solvent systems used in some assays may not always be suitable for the solubilization of antioxidative peptide molecules. For example, Zhu et al. (2008) and Tang et al. (2010) reported the effect of the solvent system in different assays on the observed antioxidant activity of a zein hydrolysate and its different molecular weight fractions. Further, peptides with several branched chain amino acid residues may not have the ability to reach and scavenge the bulky DPPH radicals due to steric hindrance. Some literature reports have shown a decrease in DPPH radical scavenging capacity of protein hydrolysates with increasing extent of protein hydrolysis (Klompong, Benjakul, Kantachote, & Shahidi, 2007; Theodore, Raghavan, & Kristinsson, 2008). At the same time, some other studies have shown a marked increase in the DPPH radical scavenging activity with the extended protein hydrolysis (Li, Chen, Wang, Ji, & Wu, 2007; Raghavan, Kristinsson, & Leeuwenburgh, 2008). This finding suggests that other than peptide size and solubility, the amino acid composition, sequence, and in the case of protein hydrolysates, abundance of free amino acids (Theodore et al., 2008), may also have a key role in determining the DPPH radical scavenging capacity. In our laboratory, we tested the antioxidative capacity of five chemically synthesized peptides that were designed based on the peptide sequences identified from several food sources as indicated in Table 4 (unpublished results). DPPH radical scavenging capacity of these peptides at 150 lM assay concentration was very low compared to the Trolox standard at 15 lM (Table 4). All five peptides were water-soluble at concentrations up to 1 mg/ml, but the 45% ethanol media used during this assay may have compromised solubility as well as their electron donating ability towards the lipophilic DPPH radical. Chen, Muramoto, Yamauchi, Fujimoto, and Nokihara (1998) also reported that PHH (i.e., peptide D in Table 4) and other His-containing synthetic peptides in their study had very low DPPH radical scavenging activity when tested at 33 lM concentration. Further, the peptide LPHH did not show activity even at 200 lM assay concentration. Rival, Fornaroli, et al. (2001) reported that four synthetic peptides derived from b-casein (VKEAMAPK, AVPYPQR, KVLPVPQK, and VLPVPQK)

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A B C D E Trolox

20

10

0 8

20

30

60

Incubation time (min)

Fig. 1 – ABTS radical scavenging capacity of peptides A–E compared to Trolox. Final concentrations of peptides and Trolox in the assay were 100 and 5 lM, respectively. Values shown are mean values of duplicate analysis. Detailed methodology is described in Samaranayaka and Li-Chan (2008). significantly inhibited the lipid peroxidation initiated by 2,2 0 -azobis-(2-amidinopropane) dihydrochloride (AAPH), but could not scavenge DPPH radicals at 100 lM assay concentration. However, as indicated in Table 3, other peptides such as glutathione, carnosine, and the peptide YFYPEL derived from milk casein have been reported to scavenge 50% of the DPPH radicals in the assay system at concentrations of 6.12, 23.3, and 98 lM, respectively (Suetsuna, Ukeda, & Ochi, 2000). This study by Suetsuna et al. (2000) used the ESR technique in measuring the DPPH radical capacity compared to the colorimetric assay used during our study and the studies conducted by Chen et al. (1998) and Rival, Fornaroli, et al. (2001) as explained above. Differences in assay conditions and/or the peptide sequences involved during these studies may have contributed to the differences in DPPH radical scavenging efficacies. Reaction times and the mechanisms of peptidic antioxidants reacting with radical species might also be different from those of phenolic and other non-peptidic antioxidants. Having several hydrogen atoms and electron-donating amino acid residues in the same peptide sequence, there is a high possibility for some peptides to exert activities based on multiple reaction mechanisms. Aliaga and Lissi (2000) studied the kinetics underlying the mechanism of the reaction between ABTS radical cations and the amino acids Cys, Trp, His, and Tyr. They found that the relative reactivity followed the order Cys  Trp > Tyr > His, and that a labile hydrogen atom (e.g., SH, NH, and OH) was required for the reaction to occur. Aliaga and Lissi (2000) further proposed a reaction mechanism between these amino acids and ABTS radical cations that involved an initial pH-dependent reversible step, followed by secondary reactions for the substrate-derived radical with itself or with another ABTS radical. In the case of His, a very small, but fast-reacting initial response was reported, followed by a slow reaction process with significant autoacceleration at longer times (Aliaga & Lissi, 2000). Henriquez, Aliaga, and Lissi (2004) also reported a complex reaction

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Table 5 – Results of statistical analysis of the effect of incubation time on % inhibition of the absorbance of ABTS radical cation by peptides A–E and Trolox.a Incubation time (min)

Sample A

8 20 30 60 a

x xy y z

B

C

D

E

Trolox

x x xy y

x x x x

w x y z

x xy y z

x x xy y

Different letters in a column indicate that the % inhibition values are significantly different at p < 0.05 (Tukey’s test).

mechanism between the ABTS radical cations and polyphenols. They found that the reaction between substrate and ABTS radical occurs in several steps, where the most reactive hydroxyl groups donate hydrogen atoms first, followed by less reactive ones that are present in the polyphenol complex. Therefore, the total reaction in the case of the polyphenolic compounds appears to take a longer time to complete when compared to the reactivity of compounds such as Trolox, which happens almost instantaneously (Henriquez et al., 2004). During our study with the five synthetic peptides listed in Table 4, after 8 min incubation, all five peptides possessed marginal ABTS radical cation scavenging activity at 100 lM assay concentration (Fig. 1). Peptide D had the highest activity followed by peptide A (p < 0.05). Addition of the ADS tripeptide sequence to the C-terminal of peptide D (i.e., to yield peptide E, Table 4) resulted in a decreased reducing capacity. Increasing the incubation time in the assay resulted in different results for the ABTS radical scavenging activity of the peptides and Trolox (Fig. 1). For Trolox, the % radical scavenging capacity did not significantly change with increasing incubation time up to 30 min (Table 5). Peptide D however showed a rapid increase in its ABTS radical scavenging activity with the increased incubation time (Table 5), reaching a radical scavenging capacity that was similar to that of Trolox (5 lM) at 60 min (Fig. 1). The ABTS scavenging activity, as indicated by inhibition of absorbance by peptides A and E, also increased with incubation time (Table 5) and peptide E was found to scavenge more radicals than peptide A by 60 min (Fig. 1). The TEAC assay for ABTS radical scavenging capacity used during our study was a modified method of Re et al. (1999) where the ABTS radicals were first generated by mixing an ABTS solution with potassium persulfate prior to the addition of antioxidant source (Samaranayaka & Li-Chan, 2008). Since this is an end point assay, the present results suggest that longer incubation times are needed for assessing and comparing antioxidative capacity of peptidic antioxidants, compared to other food components, such as polyphenols. Furthermore, these results show that the peptide sequence greatly affects the ABTS radical scavenging capacity of peptides. The decrease in radical scavenging capacity in the present study observed when ADS was added to the C-terminal of peptide D (i.e., to yield peptide E, Table 4) may be due to the loss of more labile hydrogen at the C-terminal of peptide D. Of the five synthetic peptides analyzed during our study, peptide A was shown to possess the highest oxygen radical absorbing capacity (p < 0.05) (Table 4). Peptide D (PHH) had no activity when tested at up to 2.6 mM assay concentration, but

the addition of ADS to the C-terminal (i.e., to yield peptide E) resulted in some oxygen radical scavenging capacity (Table 4). However, ORAC values of all five peptides were much lower than the values reported for single amino acids such as Trp, Tyr, and Met (4.649, 1.574, and 1.126 lmol Trolox/lmol amino acid, respectively), as well as for the commonly used synthetic antioxidant BHA (2.43 lmol Trolox/lmol BHA) (Herna´ndez-Ledesma, Davalos, Bartolome, & Amigo, 2005). None of these amino acids (Trp, Tyr, and Met) were present in the peptide sequences used for our study. In contrast to the weak DPPH radical scavenging and ORAC values, all five synthetic peptides showed considerable ability to inhibit lipid peroxidation in the linoleic acid model system (Table 4). Tsuge et al. (1991) and Chen et al. (1996) also reported high inhibitory activity for peptides C and D, respectively in the linoleic acid peroxidation system. Since most peptides are amphiphilic in nature, they have the ability to stay in the lipid–water interface and effectively scavenge radicals present in both aqueous as well as oil phase of these model systems. Antioxidative peptides are therefore promising candidates to reduce lipid peroxidation in emulsion-type food products, as well as potentially in the human membrane systems. Considering the results we obtained from our study with synthetic peptides as well as the literature reports as discussed above, extrapolation of the results from these in vitro chemical assays to the real food or biological systems should be done with caution. Distinction should also be made between assays and model systems that can be used to test the antioxidative capacity of peptides for either food or biological use, while considering factors such as possible mechanisms of their action, ability to reach target sites, and so on. This will help to gain a better understanding of their antioxidative mechanisms and possible applications.

4.2.

In vitro biological assays

In vitro cultured cell model systems allow for rapid, inexpensive screening of antioxidative compounds for their bioavailability, metabolism, as well as bioactivity, compared to expensive and time-consuming animal studies and human clinical trials. Use of cell culture models for antioxidant research is particularly important since the studies to date have demonstrated that the mechanism of the action of antioxidants in human health promotion go beyond the antioxidant activity of scavenging free radicals (Liu & Finley, 2005). For example, antioxidants found in fruits and vegetables are found to have other functions including the following: regula-

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tion of gene expression in cell proliferation, cell differentiation, oncogenes, and tumor suppressor genes; induction of cell cycle arrest and apoptosis; modulation of enzyme activities in detoxification, oxidation, and reduction; stimulation of the immune system; regulation of hormone-dependent carcinogenesis; inhibition of arachidonic acid metabolism; antibacterial and antiviral effects in prevention of cancer (Chu, Ho, & Chow, 2002; Sun, Chu, Wu, & Liu, 2002; Waladkhani & Clemens, 1998). Further, dietary antioxidants have been shown to have roles in the reduction of platelet aggregation, modulation of cholesterol synthesis and absorption, reduction of blood pressure, and retarding the progression of atherosclerotic lesions in preventing cardiovascular diseases (Libby, Ridker, & Maseri, 2002). Various cell culture models are therefore an invaluable tool to assess these potential health benefits of food antioxidants, in vitro. Human adenocarcinoma colon cancer (Caco-2) cell monolayers have been the most commonly reported in the literature for studying intestinal permeability of bioactive compounds due to their similarity to the intestinal endothelium cells (Liu & Finley, 2005; Vermeirssen, Augustijns, Van Camp, Opsomer, & Verstraete, 2005). Upon culturing as a monolayer, Caco-2 cells differentiate to form tight junctions between cells to serve as a model of paracellular movement of compounds across the monolayer. Further, Caco-2 cells express transporter proteins, efflux proteins, and Phase II conjugation enzymes to model a variety of transcellular pathways as well as metabolic transformation of test substances (van Breemen & Li, 2005). Small di- and tri-peptides may be absorbed intact across the brush border membrane using H+-coupled PepT1 transporter system (Vermeirssen et al., 2002). Larger water-soluble peptides can cross the intestinal barrier paracellularly via the tight junction between cells, while highly lipid-soluble peptides may diffuse via the transcellular route (Miguel et al., 2008). Peptides may also enter the enterocytes via endocytosis, which entails membrane binding and vesiculisation of the material (Ziv & Bendayan, 2000). The intestinal basolateral membrane also possesses a peptide transporter, which facilitates the exit of hydrolysis-resistant small peptides from the enterocyte into the portal circulation (Gardner, 1984). Further, the contribution of each route and the ability of individual peptides to transport across the membrane, depend upon the molecular size, and other structural characteristics such as hydrophobicity, as well as their resistance to brush-border peptidases (Satake et al., 2002; Shimizu, Tsunogai, & Arai, 1997). Cell culture models can also be used to evaluate cytotoxicity of antioxidative compounds at concentrations to be used to exert the desired bioactivity in the body, as well as to study the potential to inhibit intracellular oxidation and to reduce inflammatory responses. The 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay, which measures the metabolic activity of cells through oxidation– reduction activities of mitochondria, is often used to measure the viability of cells during cytotoxicity assays (Mosmann, 1983). During experiments, intracellular oxidation of cells can be induced by using a peroxy radical generator or by using hydrogen peroxide (Elisia & Kitts, 2008). The 2 0 ,7 0 -dichlorofluorescein diacetate (DCFH-DA) probe can be used to measure

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the extent of intracellular radical formation with and without added antioxidative compound in order to assess the cellular antioxidant activity (CAA) (Wolfe & Liu, 2007). Upon cellular uptake, the DCFH-DA probe is hydrolyzed to DCFH by intracellular esterases. DCFH is capable of emitting fluorescence when oxidized by peroxy radicals during the assay. The extent of oxidation can therefore be measured by the fluorescence intensity with and without the presence of antioxidative compounds (Wolfe & Liu, 2007). Several studies have used this technique to measure the inhibition of intracellular ROS generation by peptides like PR, which is derived from a protamine hydrolysate (Wang, Zhu, Chen, Han, & Wang, 2009), and SS-31, which is a synthetically made cell-penetrating peptide (Zhao, Luo, Giannelli, & Szeto, 2005).

4.3.

In vivo assays

Once the antioxidative potential of a food constituent of interest is established using in vitro assay methods as described above, animal studies and human clinical trials can be conducted to confirm bioavailability and the desired biological function. Various biomarkers are used in measuring the ability of dietary antioxidants to protect lipids, proteins, and DNA from oxidative damages (Collins, 2005; Griffiths et al., 2002). Results from these in vivo assays are an essential part in gaining approval from federal agencies for a dietary component to be used in functional food and nutraceutical formulations. Results from several animal and human clinical trials with foodderived antioxidative peptides are described in Section 5.2.

5. Potential applications peptidic antioxidants 5.1. Functional ingredients deterioration of food

to

of

food-derived

control

oxidative

Protein hydrolysates and peptide fractions can be added as functional ingredients in food systems to reduce oxidative changes during storage. Several studies reported that the antioxidative activity of protein hydrolysates and isolated peptides prepared from sources like egg yolk, hoki skin gelatin, marine blue mussel, tuna back bone, and Pacific hake fish fillet is superior to that of a-tocopherol and, in some cases, similar or higher in activity to that of commonly used synthetic antioxidants such as BHA and BHT (Je et al., 2007; Mendis, Rajapakse, & Kim, 2005; Park, Jung, Shahidi, & Kim, 2001; Rajapakse, Mendis, Jung, et al., 2005; Samaranayaka & Li-Chan, 2008). Synergistic effects of some antioxidative peptides with tocopherols in food and model systems have also been reported (Bishov & Henick, 1975; Hatate, Nagata, & Kochi, 1990; Jun et al., 2004; Kim et al., 2001). Incorporation of a fish protein hydrolysate preparation made by autolysis of arrowtooth flounder protein into a coating of salmon fillets slowed down the lipid oxidation process (Sathivel, 2005). Further, a brine solution containing salmon fish protein hydrolysate injected into smoked salmon fish fillets was shown to reduce lipid oxidation measured as 2-thiobarbituric acid reactive substances (TBARS) during 6 weeks of cold storage (4 C) and 8 months of frozen storage (18 C) (Hagen & Sandnes, 2004). Protein hydrolysates made from

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whey, casein, egg yolk, and potato have also been shown to inhibit lipid oxidation in muscle foods (Diaz & Decker, 2005; Penˇa-Ramos & Xiong, 2003; Sakanaka & Tachibana, 2006; Wang & Xiong, 2005). Antioxidative caseinophosphopeptides, derived from tryptic digestion of casein, have been used in breakfast cereals, breads, pastry, chocolate, juices, tea, and mayonnaise (Fitzgerald, 1998). In addition to inhibiting lipid peroxidation, peptides produced by enzymatic protein hydrolysis are capable of preventing oxidative modification of intact proteins. For example, the presence of potato protein hydrolysate minimized amino acid side chain damage and structural changes in myofibrillar proteins exposed to a OH-generating oxidizing system (Wang & Xiong, 2008). Since food protein hydrolysates are produced from naturally occurring proteins for which no adverse or safety concerns have been identified, their applications in food products should not be limited by regulations on allowed dose and use in food products, such as those applying to BHA, BHT and other synthetic antioxidants. Various vegetable protein hydrolysates are in fact allowed to be incorporated into specific foods in the United States as food additives (FDA, 2011) and are allowed as food ingredients in most countries (Schaafsma, 2009). However, when the process of making a protein hydrolysate or specific peptide fraction leads to a significant change in composition, structure, or the level of undesirable substances that affects the nutritional value, metabolism, or safety, that product should be evaluated by an independent expert panel before introducing to the market (Schaafsma, 2009). Furthermore, bitterness and other potential organoleptic problems, as well as the stability of antioxidative peptides during food processing and storage should be assessed before commercializing a particular protein hydrolysate of interest for incorporation into a food product.

5.2.

Functional foods and nutraceuticals

Research during the past decade has provided extensive scientific evidence for the health benefits of food-derived proteins and peptides. While bioactivities like antihypertensive activity of low molecular weight food-derived peptides have been well established and several functional foods and natural health products are already available in the market (Hartmann & Meisel, 2007; Murray & Fitzgerald, 2007; Vercruysse, Camp, & Smagghe, 2005), only a limited number of studies have been conducted to date for assessing the biological antioxidative potential of peptide hydrolysates or isolated peptides using cell cultures, animal models or human clinical trials. Nevertheless, results from the studies conducted so far as described below provide great promise that these peptidic antioxidants could have an impact on reducing oxidative stress as well as the risk of various degenerative diseases such as cancer, cardiovascular disease, inflammatory diseases, etc. associated with oxidative stress. Rajapakse, Mendis, Jung, et al. (2005) reported that the radical scavenging peptide HFGDPFH derived from fermented mussel sauce could enhance the viability of oxidation induced cultured human lung fibroblast cells by 76%. This effect was dose dependent up to 75 lg/ml, but showed no further protection on cell survival when the peptide concentration

3 ( 2 0 1 1 ) 2 2 9 –2 5 4

was increased beyond that dose. Seacure is a commercially available fermented fish product made by controlled yeast fermentation of Pacific hake, which is claimed to be beneficial for a variety of gut conditions. Fitzgerald et al. (2005) studied the efficacy of this product using various models of epithelial injury and repair. When cultured rat epithelia and human colon cells were given the Seacure, cell growth was significantly increased (at 1 mg/ml concentration, p < 0.01) and the cell injury was significantly reduced (at 25 mg/ml concentration, p < 0.05) due to the action of ethanol-soluble di- and tri-peptides containing glutamine (Fitzgerald et al., 2005). A pilot human clinical trial using Seacure pointed out that it could reduce the degree of small intestinal damage caused by the non-steroidal anti-inflammatory drug, indomethacin (Marchbank, Limdi, Mahmood, Elia, & Playford, 2008). This study also suggested that glutamine present in FPH might have contributed to antioxidative activity via stimulation of glutathione production. In another in vivo study, the overall antioxidative status of hypertensive rats was improved by 35% when they were fed with a fish protein hydrolysate preparation compared to casein (Boukortt et al., 2004). Sun, He, and Xie (2004) reported that peptides isolated from medicinal mushroom Ganoderma lucidum showed antioxidant activity in the rat liver tissue homogenates and mitochondrial membrane peroxidation systems. The auto-hemolysis of rat red blood cells was also blocked by these peptides in a dosedependent manner. In a human intervention study, ingestion of a G. lucidum supplement was found to cause an acute increase in plasma antioxidant capacity in healthy human subjects. However, no significant change in any biomarkers of antioxidant status was observed (Wachtel-Galor, Tomlinson, & Benzie, 2004). Another study reported that egg white hydrolysates fed to spontaneously hypertensive rats (0.5 g/ kg/day) helped to elevate the radical scavenging capacity of plasma while dropping the elevated concentration of malonaldehyde in the aorta (Manso et al., 2008).

5.3.

Cosmeceuticals

Cosmeceuticals are topical products that are designed to improve the appearance of skin by various mechanisms of action (Lupo & Cole, 2007). Antioxidant flavonoids and other polyphenolic compounds from food sources such as green tea, grape seed, soybeans, pomegranate, as well as other antioxidants like vitamin C from citrus fruits, lycopene from different red fruits and vegetables, and vitamin E from different plant and animal sources are currently included in many cosmeceutical products (Allemann & Baumann, 2008; Choi & Berson, 2006). Even though there is currently a lack of controlled clinical trials in humans examining the role of antioxidants in preventing or delaying skin aging, it is hypothesized that the topical application of antioxidants may neutralize some of the free radicals, and consequently lessen or prevent the signs of aging skin. Topical antioxidants from different food sources are presently being marketed to prevent aging and UV-induced skin damage, as well as to treat wrinkles and erythema due to inflammation (Allemann & Baumann, 2008). However, to the authors’ knowledge, the use of peptide antioxidants as cosmeceuticals is not yet common.

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The silk protein, Sericin, extracted from silkworm Bombyx mori, has been reported to possess antioxidant activity with high Cu2+ chelating ability (Gorouhi & Maibach, 2009). This protein could reduce UV-B-induced acute damage and tumor promotion in the skin of hairless mouse (Zhaorigetu, Yanaka, Sasaki, Watanabe, & Kato, 2003). L-carnosine (b-alanylhistidine) and related peptidomimetics like N-acetylcarnosine and carcinine (b-alanylhistamine) are considered as antioxidants that are useful to protect the skin from oxidative attacks (Babizhayev, 2006; Stvolinsky et al., 2010). Babizhayev (2006) reported that these peptides can protect enzymes such as superoxide dismutase (SOD). L-Carnosine was also reported to increase the lifespan of cultured human fibroblast cells as well as to rejuvenate senescent cells (McFarland & Holliday, 1994, 1999). As summarized by Zhang and Falla (2009), peptides are used in different skin care products to perform various other functions than that of antioxidative action, such as modulation of cell proliferation, cell migration, inflammation, angiogenesis, melanogenesis, and protein synthesis and regulation. In the United States alone, 25 peptides are currently used in cosmetic products to perform these functions (Zhang & Falla, 2009). For example, the tripeptide glycyl-L-histidyl-Llysine-copper (Cu-GHK) complex is used to deliver copper into cells, which is a cofactor for the antioxidant enzyme superoxide dismutase as well as for the collagen and elastin synthesis, that help in the wound healing process (Canapp et al., 2003; Choi & Berson, 2006). The source for Cu-GHK bioactive peptide complex is the human serum (Zhang & Falla, 2009). The ‘‘Dipeptide-2’’ marketed by Sederma is obtained from rapeseed and the activity is defined as lymph drainage via ACEinhibition (Zhang & Falla, 2009). With the increasing scientific evidence for the role of different low molecular weight foodderived peptides as antioxidants in food and biological systems, it will be worth exploring the possibility of incorporating some of these peptides in topical cosmetic products in order to protect the skin from endogenous and exogenous oxidative attack.

6. Challenges of using peptides antioxidants and future research needs

as

Albeit the antioxidative potential of food-derived peptides evidenced by research conducted during last decade as discussed above, only a few commercial products are available to date, which may be attributed to a variety of reasons including a lack of clinical trials (to confirm bioactivity, efficacy, and safety), high production cost, problems in making a reproducible product, bitterness, color, or other organoleptic problems. It is important to study techno-functional properties of active peptide fractions and how these peptides can retain their antioxidant activities in different targeted food matrices. Antioxidant peptides have the ability to interact with other components of the food matrix like carbohydrates and lipids, and may also lose their activity during food processing operations like thermal processing (Korhonen, Pihlanto-Leppa¨la¨, Rantama¨ki, & Tupasela, 1998). Further, particular antioxidant peptide sequence(s) of interest may also have synergistic or antagonistic effects with other anti-

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oxidants and/or trace metals present in food and biological systems, and even act as pro-oxidants under certain conditions. These factors should carefully be taken into consideration when looking for possible applications of antioxidant peptides. It will also be important to identify the form in which the antioxidative peptides can be incorporated into food matrices. Compared to the pure isolated peptides, crude or semipurified peptide extracts will be more economically feasible to use in food products. Furthermore, crude extracts may contain several different peptides that can act synergistically to exert antioxidative action. On the other hand, other components like pigments and trace lipids in crude extracts may cause color and flavor problems. The most challenging task in the antioxidant peptide research area at present is the establishment of the bioactive potential of these peptides and identification of possible mechanisms by which they can exert antioxidant activities in biological systems. Since most of the food-derived peptides are around 500–1500 Da in size (Table 1), the majority of them may not have the ability to penetrate the intestinal cells and enter the blood circulation. It is likely that most of the food-derived antioxidant peptides will exert their activity in the gut lumen or through receptors in the intestinal cell wall. Digestion may however yield smaller peptide products from the parent peptide sequence with the ability to reach other target organs via the blood circulation. Identification of the fate of food-derived antioxidant peptides during their passage through the GI tract, as well as establishing biomarkers for the assessment of their in vivo antioxidant activity will therefore be of utmost importance. Possible strategies for increasing the cellular permeability of food-derived antioxidant peptides should also be investigated.

Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada. The authors thank Imelda W.Y. Cheung for her careful proof-reading of the manuscript.

R E F E R E N C E S

Ahn, C. B., Lee, K. H., & Je, J. Y. (2010). Enzymatic production of bioactive protein hydrolysates from tuna liver: Effects of enzymes and molecular weight on bioactivity. International Journal of Food Science and Technology, 45, 562–568. Alamed, J., Chaiyasit, W., Mcclements, D. J., & Decker, E. A. (2009). Relationship between free radical scavenging and antioxidant activity in foods. Journal of Agricultural and Food Chemistry, 57, 2969–2976. Aliaga, C., & Lissi, E. (2000). Reactions of the radical cation derived from 2,2 0 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) with amino acids. Kinetics and mechanism. Canadian Journal of Chemistry, 78, 1052–1059. Allemann, I. B., & Baumann, L. (2008). Antioxidants used in skin care formulations. Skin Therapy Letter, 13, 5–9.

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