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Biological activity of peptides purified from fish skin hydrolysates

Biological activity of peptides purified from fish skin hydrolysates Fish skin waste accounts for part of the solid waste generated from seafood processing. Utilization of fish skin by bioconversion into high-grade products would potentially reduce pollution and economic cost associated with treating fish processing waste. Fish skin is an abundant supply of gelatin and collagen which can be hydrolyzed to produce bioactive peptides of 2–20 amino acid sequences. Bioactivity of peptides purified from fish skin includes a range of activities such as antihypertensive, anti-oxidative, antimicrobial, neuroprotection, antihyperglycemic, and anti-aging. Fish skin acts as a physical barrier and chemical barrier through antimicrobial peptide innate immune action and other functional peptides. Small peptides have been demonstrated to possess biological activities which are based on their amino acid composition and sequence. Fish skin-derived peptides contain a high content of hydrophobic amino acids which contribute to the antioxidant and angiotensin-converting enzyme inhibitory activity. The peptide-specific composition and sequence discussed in this review can be potentially utilized in the development of pharmaceutical and nutraceutical products. Keywords: Fish skin, Peptides, Bioactivity, Antioxidant, Antihypertensive, Antimicrobial, Anti-Alzheimer’s Background crease the economic value of the catch but also would The marine environment is a source of functional bio- reduce the amount of marine processing waste. Thus, materials such as polyunsaturated fatty acids (PUFAs), reducing pollution and economic cost associated with polysaccharides, minerals and vitamins, antioxidants, treating the generated waste (Fang et al. 2017). enzymes, and bioactive peptides (Kim and Wijesekara Biological activity of peptides is based on their amino 2010; Kim et al. 2008; Pomponi 1999). By-products of acid composition and sequence (Pihlanto-Leppälä 2000). marine processing industries, i.e., skin, trimmings, This biological activity ranges from antioxidant, antihyper- viscera, and blood, contain a good amount of proteins tensive, immunomodulatory, and antimicrobial activity as which can be used as a source for bioactive peptides. demonstrated by several studies using different fish spe- These by-products are utilized as additives in animal cies like Pollack, skate, Nile tilapia, sea beam, yellow fish, husbandry as animal feed or in agriculture as fertilizers and skipjack (Chalamaiah et al. 2012). Bioactive peptides (Chalamaiah et al. 2012). Fish by-products like skin and are specific protein fragments derived from plants or ani- frame need to be processed into fish hydrolysates either mal sources which possess nutritional benefits and posi- by fermentation or by hydrolysis techniques before they tively influence health (Hartmann and Meisel 2007; can be effectively utilized. By so doing this increases pro- Korhonen and Pihlanto 2003). Bioactive peptides are in- duction costs (Chalamaiah et al. 2012). Therefore, the active in their parent protein sequence but can be released utilization of fish processing waste through bioconver- by enzymatic hydrolysis; however, for effective use, bio- sion into high-grade products like bioactive peptides active peptides must reach the target organ or receptors in would be a better alternative. This not only would in- the intestinal lumen intact and must survive enzymatic degradation (Adessi and Soto 2002). This review will high- light and discuss the different preparation methods of bio- active peptides from fish skin, their biological activity, and associated mode of action in regard to specific peptide * Correspondence: hgbyun@gwnu.ac.kr Department of Marine Biotechnology, Gangneung–Wonju National composition, sequence, and cell signaling pathways. University, Gangneung 25457, Republic of Korea © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 2 of 14 Preparation of bioactive peptides from fish skin as an initial extraction step. The step is followed by con- Fish protein hydrolysates contain peptides of 2–20 amino tinuous stirring at a controlled temperature for a set time. acid sequences after hydrolysis, and these peptides usually The procedure is repeated about 3 times, and it is carried have biological activity. Several extraction methods are out with an aim to remove non-collagenous proteins and utilized to liberate bioactive peptides from the parent pro- pigments (Jongjareonrak et al. 2005;Wang et al. 2008; tein, and these include acid-alkaline hydrolysis: extracting Wang et al. 2015). The skin is alternatively treated with an collagen by using acidic or alkaline reagent; enzymatic hy- acid (HCl) (Wu et al. 2017). After acid-alkali treatment, the drolysis: using enzymes to hydrolyze fish skin; and fer- skin was washed to neutralize the pH and further extrac- mentation method: using microorganisms as a source of tion carried out with distilled water at 65 °C for 4 h. Some the enzymes (Huang et al. 2015). extraction procedures include a defatting step (Mahboob 2014). Jongjareonrak et al. 2005 removed fat using butyl al- Enzymatic hydrolysis cohol for 24–48hwithgentlestirringand achangeofsolu- Enzymatic hydrolysis is the best way to hydrolyze fish tion every 8 h. The resultant matter was then subjected to skin without losing nutritional value (Huang et al. 2015). acid treatment with acetic acid for 24 h with gentle stirring. The method is preferred especially in the food and Collagen was extracted from fish skin, scale, and bone pharmaceutical industries because the hydrolysis process using a procedure described as follows (Wang et al. 2008). does not leave residual organic solvents or toxic chemi- The collagen was extracted with 0.5 M acetic acid at a sam- cals in its products (Kim and Wijesekara 2010). Steps in ple/solution ratio of 1:100 (w/v)for 24hwithcontinuous enzymatic hydrolysis involve substrate preparation, stirring. The extracts were centrifuged at 20,000g for 1 h at choice of the right enzyme, measuring the extent of 4 °C, and the extraction step was repeated using the ob- enzymatic hydrolysis, homogenization, and heating to tained residue, followed by centrifugation under the same inactivate endogenous enzymes, hydrolysis, and termin- conditions. The supernatants of the two extracts were ation of the enzymatic reaction. Commercial enzymes combined and precipitated by the addition of NaCl to a such as alcalase, trypsin, pepsin, papain, pancreatin, and final concentration of 0.9 M and centrifuged at 2500g for thermolysin are employed in the enzymatic hydrolysis 0.5 h to obtain a precipitate that was dissolved in 0.5 M (Bernardini et al. 2011). Conditions like enzyme concen- acetic acid. The precipitate was dialyzed for 48 h against 10 tration, pH, time, and temperature have to be well moni- volumes of 0.1 M acetic acid and distilled water, respect- tored and maintained during hydrolysis. Enzyme ively, which were changed every 8 h, before being lyophi- concentrations, pH, and temperature vary with the type lized. Antimicrobial peptides were purified from winter of enzyme used. Enzyme concentrations of 0.01–5.00% flounder epidermis and mucus extracts (Cole et al. 1997). (w/w) and pH range of 1.5–11 have been documented The mucus was obtained from the skin by scraping and (Halim et al. 2016). Black-barred halfbeak gelatin was further subjected to homogenization in a solution of 50 ml dissolved in DW and subjected to enzymatic hydrolysis of 0.2 M sodium acetate, 0.2% Triton X-100, and 1 mM with an enzyme/substrate ratio of 30:1, pH 10.0 and 50 ° phenyl methyl sulfonyl fluoride. The homogenate was cen- C. The enzymatic activity was evaluated by a method de- trifuged for 20 min at 20,000g, and the resultant super- scribed by (Kembhavi et al. 1993) using casein as a sub- natant was further purified. strate. The gelatin solution was equilibrated for 30 min before the enzyme addition. The pH was maintained by Fermentation addition of 2 N NaOH, and after 3 h, the enzymes were Fermentation is considered a more natural method of inactivated by heating the solution at 95 °C for 20 min protein hydrolysis. The technique has been employed for (Abdelhedi et al. 2017). Extraction of pepsin soluble col- centuries especially in East Asian countries as a lagen (PSC) from fish skin was performed by (Mahboob traditional preservation method. Fermentation not only 2014). Undissolved residue obtained after acid soluble enhances the flavor and taste of food but also increases collagen (ASC) extraction was utilized for the PSC ex- its nutraceutical value. During the fermentation process, traction as described by (Singh et al. 2011). bioactive peptides are released by the action of both microorganisms and endogenous proteolytic enzymes. Acid-alkaline hydrolysis Several studies have demonstrated the bioactivity of vari- During fish skin hydrolysis by acid-alkaline hydrolysis, ous marine products like Thai fermented shrimp paste, certain amino acids, i.e., tryptophan, serine, and threo- shrimp by-products, squid miso, and a variety of nine, can be destroyed at high pH. Therefore, the pH traditional fermented fish products (Bueno-Solano et al. and temperature of the hydrolysates must be closely ob- 2009; Giri et al. 2011; Kleekayai et al. 2015). Majumdar served during the hydrolysis process. Collagen extraction et al. 2016 examined the chemical and microbial proper- from fish skin by acid-alkaline hydrolysis involves treat- ties of shidal, a traditional fermented fish product of ment of pre-cleaned skin samples with an alkali (NaOH) Northeast India. A combination of both fatty acids Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 3 of 14 (eicosapentaenoic, docosahexaenoic, arachidonic, lino- and water into the blood, while at the same time causing lenic, and linoleic acid) and proteins or peptides of MW the excretion of potassium. Inhibition of ACE diminishes (molecular weight) range between 45 and 29 kDa and 45 angiotensin II-mediated aldosterone secretion from the and 6 kDa respectively were reported to be present in adrenal cortex, leading to a decrease in water and the fermented fish product. Hydrolysates were prepared sodium reabsorption and a reduction in extracellular from turbot skin by utilization of the fermentation volume sequentially leading to vasodilation and eventu- method using 3 microorganisms, i.e., Saccharomyces cer- ally reduced arterial blood pressure. Hypertension is evisiae, Aspergillus oryzae, and Streptococcus thermo- manageable with various synthetic ACE inhibitors such philes (Fang et al. 2017). as captopril (Dezsi 2000). Hypertension can also be counteracted by endothelial-dependent vasodilation Purification of peptides through the upregulation of nitric oxide (NO) expres- The biological activity of peptides is determined by proper- sion, phosphorylation of eNOS, and downregulation of ties like molecular weight, charge, and hydrophobicity. endothelin (ET-1) expression. Endothelial smooth Therefore, peptides are purified through a multi-step puri- muscle cell relaxation is achieved via the nitric oxide/ fication process based on such properties. Purification cyclic guanosine monophosphate (cGMP)-mediated based on molecular weight employs methods like ultrafil- intracellular signaling pathway. This pathway involves tration (UF), nanofiltration (NF), and gel filtration (GF) the upregulation of cGMP-dependent protein kinase I (Halim et al. 2016). Ion exchange chromatography (IEC) is (cGK-I) and a decrease in intracellular calcium levels via used to fractionate peptides basing on their net charge. downregulation of the inositol-1,4,5-trisphosphate (IP 3) Fractioned peptides are then further purified using receptor (Daiber and Münzel 2015). technologies like reversed-phase HPLC which separates Conventional antihypertensive drugs cause several ad- compounds based on hydrophobicity and hydrophilicity verse effects; therefore, the search for safer natural alter- (Conlon 2007). Peptide sequences of the most active natives is in progress. Among the natural alternatives, fractions from HPLC analysis are then analyzed and identi- interest has grown in the utilization of bioactive peptides fied using mass spectrometry methods like matrix-assisted in the prevention of hypertension and in the initial treat- laser deionization time-of-flight (MALDI-TOF), electro- ment of mild hypertension (Guang and Phillips 2009). spray ionization mass (ESI), matrix-assisted laser desorp- The antihypertensive activity of fish skin peptides is dis- tion/ionization mass spectrometry (MALDI-MS), etc. cussed and summarized below in Table 1. (Bernardini et al. 2011). Ultrafiltration of steelhead/rainbow hydrolysates yielded fractions of less than 3 kDa with ACE inhibitory activity Biological activities higher than the activity of the whole hydrolysates (Cheung Antihypertensive activity and Li-Chan 2017). The low MW fractions were prepared Hypertension is a chronic condition affecting millions of from whole hydrolysates using commercial enzyme kits. people around the world. According to a report by Two fractions showed ACE inhibition activity of 54% and World Health Organization 2011, the global prevalence 63%. The ACE inhibitory activity was affected by different of hypertension among adults ≥ 25 years stands at 40% conditions suggesting that the additional hydrolysis and it is estimated that 1.56 billion people will have the achieved with higher enzyme concentration and longer condition by 2025 (Kearney et al. 2005). Blood pressure duration effectively generated shorter peptides with higher is regulated by the renin–angiotensin–aldosterone sys- activity. The highest ACE inhibition was demonstrated in tem (RAS). Low renal blood flow or low plasma sodium samples produced after 6 h hydrolysis with 4% protease. concentration initiates the conversion of pro-renin into Similarly, fish skin peptide bioactivity has been shown to renin in juxtaglomerular cells in the kidneys. Renin in be associated with low MW peptides (Iwaniak et al. 2014; circulation catalyzes the conversion of angiotensinogen Power et al. 2014). to angiotensin I which is then subsequently converted to Black-barred halfbeak (Hemiramphus far) skin showed angiotensin II by the enzyme angiotensin-converting en- high protein content in the gelatin extracts, estimated at zyme (ACE) (Paul 2006). ACE is mainly produced in the 91.36% (Abdelhedi et al. 2017). The protein quantity in lungs and other sites including the endothelial lining of the gelatin was closely similar to that obtained in other vascular tissues, heart, brain, kidney, placenta, bone mar- fish skin species such as splendid squid, cuttlefish, row, pancreas, and testis. Angiotensin II is a potent thornback ray, and cobia (Jridi et al. 2013; Lassoued et vasoconstrictor that causes blood vessels to narrow al. 2014; Nagarajan et al. 2012; Silva et al. 2014). The resulting in increased blood pressure (Rogerson et al. ACE inhibitory activity was 36.51% for 1 mg/mL of 1992). Simultaneously, it stimulates the secretion of the whole gelatin while 1 mg/mL gelatin hydrolysate showed hormone aldosterone from the adrenal cortex causing a significantly greater activity of 80.76%. However, these the renal tubules to increase the reabsorption of sodium values were significantly lower than the positive control Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 4 of 14 Table 1 ACE inhibitory activity peptides purified from fish skin hydrolysates Species IC (μM) Peptide sequence Reference Nile tilapia (Oreochromis niloticus) 760–1490 GIV, GAP*GF, GFA*GPA, SGNIGFP*GPK, GIPGPIGPP*GRP Thuanthong et al. (2017) Pacific cod skin 6.9 GASSGMPG Ngo et al. (2016) 14.5 LAYA Thornback ray skin 27.9 GIPGAP Lassoued et al. (2015) 170 APGAP Skate 3.09 MVGSAPGVL Ngo et al. (2014b) 4.22 LGPLGHQ Salmon (Oncorhynchus keta) 8.7 GLPLNLP Lee et al. (2014) Skate 95 PGPLGLTGP Lee et al. (2011) 148 QLGFLGPR Alaska pollock 2.6 GPL Byun and Kim (2002) 17.13 GPM Cod (Gadus microcephalus) – TCSP, TGGGNV Ngo et al. (2011) captopril. The hydrolysate had an ACE inhibitory IC be attributed to factors such as Van der Waals force and value of 332.66 ± 16.57 mg/mL. Higher values of ACE in- hydrogen bonds of molecules that stabilize the ligand– hibitory activity of hydrolysate in comparison to those of protein. the gelatin are an indicator that bioactive peptides are Peptides IGPAG, FGYGG, GIPGAP, IGAPGATGPAG, released from the protein molecules upon enzymatic hy- AKGDS, GASGPRGPA, GQDGRPGPAG, and GEAGNPG- drolysis (Abdelhedi et al. 2017). In another study, five PAGP were purified from Thornback ray skin gelatin neu- novel ACE inhibitory peptides GIV, GAP∗GF, GFP∗GPA, trase hydrolysate (Lassoued et al. 2015). Peptide GIPGAP, SGNIGFP∗GPK, GIPGPIGPP∗GPR were identified from IC 27.9 μM, was the most potent ACE inhibitor among the most active fractions of Nile tilapia skin gelatin. The the purified peptides. A hydrolysate TRGH-A26 was pre- IC value of the active peptides ranged between 760 pared using crude proteases from Bacillus subtilis A26, and and 1490 μM (Thuanthong et al. 2017). peptides AVGAT, GGVGR, APGAP, GEPGAPGPA, and Pacific cod skin gelatin was hydrolyzed using several GPRGAPGPA were purified. The peptide APGAP, IC enzymes and the pepsin hydrolysate showed the highest 170 μM, was the most potent ACE inhibitor from this hy- ACE inhibitory effect of about 91% (Ngo et al. 2016). drolysate. The two most potent peptides GIPGAP and ACE inhibitory active peptides were identified as APGAP from Thornback ray skin gelatin hydrolysates had GASSGMPG, IC 6.9 μM, and LAYA, IC 14.5 μM. a strikingly similar sequence of PGAP at the C-terminus. 50 50 The MW of peptides GASSGMPG and LAYA was less Another peptide FGYGG with a high ACE inhibitory activ- than 1 kDa which indicates they can cross the intestinal ity with an IC value of 231 μM contained the aromatic barrier and exert biological effects. The ACE inhibitory residue phenylalanine. activity of GASSGMPG was higher than that of ASL, The antihypertensive effect of skate skin hydrolysates IC 102.15 μM from silkworm pupa (Bombyx mori) pro- in an animal model experiment using spontaneously tein; PVNNPQIH, IC 206.7 μM from small red bean hypertensive rats (SHRs) was evaluated (Ngo et al. Phaseolus vulgaris; GDLGK-TTTVSNWSPPKYKDTP, 2014b). Purified peptides were orally administered to IC 11.28 μM from tuna frame protein; and AHEPVK, SHRs, and changes in heart rate and blood pressure IC 63 μM from edible mushroom Agaricus bisporus were monitored over a period of 20 days. Systolic blood (Lau et al. 2014; Lee et al. 2010; Rui et al. 2013;Wuet pressure readings were monitored, and the maximal dec- al. 2015). A docking simulation of the ACE–ligand com- rements in systolic blood pressure observed were 127.2 plexes between ACE/peptides and ACE/captopril dem- mmHg at 20 days and 118.8 mmHg at 10 days in the onstrated the potential of these peptides as ACE purified peptide (1000 mg/kg of BW) and captopril treat- inhibitors. The binding sites of GASSGMPG and capto- ment groups respectively. The peptides were identified pril on the ACE molecule were observed to be the same as MVGSAPGVL, IC 3.09 μM, and LGPLGHQ, IC 50 50 at the Asn72 residue while LAYA and captopril shared 4.22 μM. Docking simulation of the ACE molecule and two binding sites on the ACE molecule at Asn72 and the purified peptide on the Docking Server revealed al- Arg348 residues. Overall molecular docking simulation most similar binding on the ACE molecule as captopril. demonstrated good protein–drug interaction which can The binding site between the ACE molecule and the Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 5 of 14 purified peptide had many residues including Trp67, Antioxidant activity Asn68, Thr71, Asn72, and Arg348. This suggested that Reactive oxygen species (ROS) and reactive nitrogen spe- blockades by the purified peptides on the ACE molecule cies (RNS) are produced as a result of the metabolism of may contribute to the ACE inhibitory potency of the oxygen and nitrogen. ROS and RNS can cause damage to purified peptides for preventing hypertension. cellular components in the body. During metabolism and Furthermore, the protective effect of the same peptides respiration, ROS are constantly produced. These include − − MVGSAPGVL and LGPLGHQ from skate skin peptides superoxide anion radials O , hydroxyl radical OH ,and was investigated against angiotensin II-induced endothe- non-free radical species like hydrogen peroxide and singlet lial dysfunction using human endothelial cells (Ngo et al. oxygen O . Excess amounts of ROS as in oxidative stress 2014a). Increased production of endothelial nitric oxide exert oxidative damage to cellular macromolecules like pro- synthase (eNOS) and inhibition of endothelin-1 ET-1 teins, lipids, and DNA by subtracting electrons. This starts production through upregulation of the PPAR-γ pathway a series of reactions which eventually leads to new radicals was observed. The enzyme eNOS upregulates the gener- attacking and damaging other cellular macromolecule com- ation of nitric oxide (NO) in the vascular endothelium ponents (Kaur and Kapoor 2001). Peptides are considered while NO maintains endothelial integrity and proper to be more potent antioxidants than free amino acids be- function through regulation of vascular tone, local blood cause of the increased stability of the resultant peptide rad- flow, platelet aggregation and adhesion, and leukocyte– ical (Elias et al. 2008). The exact structure–antioxidant endothelial cell interactions (Dessy and Feron 2004). activity relationship of peptides has not been established. Thus, endothelial dysfunction, a precursor of hyperten- However, the type, position, and hydrophobicity of amino sion and other health conditions like diabetes, aging, acids in the peptides are considered to play an essential and atherosclerosis, results from abnormalities in NO role. The most reactive amino acids in proteins are usually production by the vascular endothelium. Purified pep- those with nucleophilic sulfur-containing side chains like tides from skate skin not only showed ACE inhibitory taurine, cysteine, and methionine or aromatic side chains activity but also provided protection against endothelial like tryptophan, tyrosine, and phenylalanine (Elias et al. dysfunction in endothelial cells. Other peptides with 2008). Fish skin is an abundant supply of gelatin and colla- ACE inhibitory activity purified from similar studies in- gen. Gelatin contains an abundance of hydrophobic amino clude PGPLGLTGP, IC 95 μM, and QLGFLGPR, IC acids such as glycine, valine, alanine, proline, and hydroxy- 50 50 148 μM, from skate skin; GLPLNLP, IC 18.7 μM, from proline and could potentially contain a range of peptides salmon skin; and GPL, IC 2.6 μM, and GPM, IC with potent lipid–peroxidation inhibitory activity (Kim and 50 50 17.13 μM, from Alaska Pollock skin (Byun and Kim Mendis 2006). Several methods are used for assessing anti- 2002; Lee et al. 2014; Lee et al. 2011). oxidant activity, and these include oxygen radical absorb- Typically, ACE inhibitory has been attributed to small- ance capacity (ORAC), Ferric-reducing antioxidant power sized peptide residues with 2–12 amino acids (Yamamoto (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). Low et al. 1994). Peptides with tryptophan, proline, or phenyl- MW peptides have higher ORAC values and metal-chelat- alanine at the C-terminus or branched-chain aliphatic ing activities while high MW peptides have higher FRAP amino acids at the N-terminus are suitable to act as com- and DPPH radical scavenging (Theodore et al. 2008). The petitive inhibitors of ACE (Cushman and Cheung 1971). antioxidant activity of fish skinpeptidesissummarizedin Many of the peptides identified in ACE inhibitory frac- Table 2. tions contain proline at one of the three C-terminal posi- Abdelhedi et al. 2017 investigated the antioxidant ac- tions. Hydrophobic amino acids in the N-terminus may tivity of gelatin extract from black-barred halfbeak also contribute to the ACE inhibitory activity (Rho et al. (Hemiramphus far) skin. The DPPH radical scavenging 2009). Therefore, in addition to the presence of proline in activities of the gelatin extract (5 mg/mL) and the posi- the C-terminal position, the presence of alanine could also tive control vitamin C were 43.39% and 70.0% respect- be a contributing factor to ACE inhibitory activity ively. The antioxidant activity was lower than that of the (Yamamoto et al. 1994). Natural ACE inhibitory peptides positive control. However, lower MW fish skin protein and ACE substrates such as bradykinin and angiotensin I hydrolysates of cobia skin and Raja clavata skin have have been shown to contain aromatic amino acid residues been demonstrated to show higher antioxidant activity such as phenylalanine (Camargo et al. 2012; Cheung et al. than their protein molecule precursors (Lassoued et al. 1980;Hara et al. 1984). Peptides inhibiting ACE activity 2015). The ferric-reducing antioxidant power for whole can potentially be used as nutraceuticals to lower elevated gelatin, gelatin hydrolysate, and vitamin C was deter- blood pressure. As described, ACE inhibitory peptides mined to be 0.47, 1.03, and 2.01 respectively. Whole gel- have been successfully purified from fish skin and their atin and gelatin hydrolysate exhibited similar antioxidant biological activity has been demonstrated by both in vitro activity using the β-carotene bleaching assay with activ- and in vivo studies. ities of 53.73% and 78.47% respectively. Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 6 of 14 Table 2 Anti-oxidative activity of peptides purified from fish skin Species Radical scavenging activity (μg/mL) Peptide sequence Reference DPPH Hydroxyl (HO•) ABTS Thornback ray 1980 –– AVGAT Lassoued et al. (2015) Nile tilapia (Oreochromis niloticus) 8.82 μM 7.56 μM – DPALATEPDMPF Ngo et al. (2010) Horse Mackerel (Magalaspis cordyla) 72.3% 51.2% – NHRYDR Sampath Kumar et al. (2012) Crocker (Otolithes ruber) 79.6% 56.8% – GNRGFACRHA Sampath Kumar et al. (2012) Hoki (Johnius belengerii) 156.2 μM –– HGPLGPL Mendis et al. (2005b) Blue leatherjacket (Navodon 405 179 – GSGGL Chi et al. (2015) septentrionalis) 194 89 – GPGGFI 118 73 – FIGP Seabass (Lates calcarifer) –– 81.41* GLFGPR Sae-Leaw et al. (2017) –– 10.4* GATGPQGPLGPR –– 2.59* VLGPF –– 0.50* QLGLGPV Jumbo squid (Dosidicus gigas) –– – FDSGPAGVL Mendis et al. (2005a) –– – DGPLQAGQPGER Amur sturgeon 5380 890 8 PAGT Nikoo et al. (2015) Cod (Gadus microcephalus) –– – TCSP, TGGGNV Ngo et al. (2011) Nile tilapia (Oreochromis niloticus) – 4.61 – EGL Zhang et al. (2012) – 6.45 – YGDEY Pacific cod Iron-chelating activity GPAGPHGPPGKDGR, AGPHGPPGKDGR, Wu et al. (2017) AGPAGPAGAR *Units in millimole TE per millimole peptide Similar results have been recorded for thornback ray histidine (16.83%) in TRGH-A26 may be responsible for skin by (Lassoued et al. 2015). Whole gelatin, gelatin hy- the high antioxidant activity (Carrasco-Castilla et al. drolysates, and vitamin C inhibited the peroxidation of 2012). The anti-oxidative efficacy of 180 mol/ml α- linoleic acid by 15.91%, 34.78%, and 70.22% respectively tocopherol equivalents at 5 mg/mL in the phosphomolyb- after 3 days and 39.25%, 74.88%, and 99.2% respectively denum assay was also evaluated and TRGH-A26 exhibited after 9 days. Similarly, the gelatin hydrolysate from Nile the highest anti-oxidative efficacy. TRGH-alcalase gelatin tilapia skin exhibited 59.74% of lipid peroxidation inhib- hydrolysate was the most potent inhibitor of DNA oxida- ition after a 5-day incubation period while whole gelatin tion by hydroxyl radicals. Likewise, it also demonstrated had activity of only 7.12% (Choonpicharn et al. 2015). DNA protective effect as no degradations were observed The authors noted that antioxidant activity observed for the two forms of plasmid DNA utilized in the test. could be due to the presence of hydrophobic amino DNA oxidation inhibition activity may be attributed to acids. Other studies using black-barred and Acipenser hydrophobic amino acids which were present in schrenckii skin gelatin hydrolysates observed that glycine TRGH-alcalase and TRGH-neutrase peptide hydrolysates. and proline had high antioxidant activity (Ngo et al. Amino acids histidine, tyrosine, methionine, and phenyl- 2011; Nikoo et al. 2015). alanine were high in TRGH-A26 and TRGH-Crude at The antioxidant activity of thornback ray gelatin hy- total percentages of 13.22 and 13.09%, respectively. drolysates was assayed using various in vitro tests Nile tilapia (Oreochromis. niloticus) scale gelatin protein (Lassoued et al. 2015). Hydrolysis was carried out using was hydrolyzed using alcalase, pronase E, trypsin, and alcalase, neutrase, and Bacillus subtilis A26 proteases. A pepsin (Ngo et al. 2010). A peptide purified from the alca- hydrolysate obtained by treatment with Bacillus subtilis lase hydrolysate provided significant protection against A26 proteases (TRGH-A26) had a high DPPH scaven- the DNA oxidative damage when exposed to *OH gener- 2+ ging activity with an IC value of 1.98 mg/mL and ated by Fe /H O . The DNA damage was inhibited by 50 2 2 β-carotene bleaching inhibition activity of 70%. The high about 70%. The cell viability tests using mouse macro- content of positively charged amino acids lysine and phages (RAW 264.7) and human lung fibroblasts Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 7 of 14 (MRC-5) confirmed that gelatin alcalase hydrolysate was Fe2 binding activity of fish scales is probably derived non-cytotoxic. The purified peptide was identified as from the exposure of glycine during hydrolysis since fish DPALATEPDMPF. The ROS scavenging activity was de- scale collage is rich in glycine (Chaud et al. 2002; Fahmi et termined using a fluorescence probe, 2´,7´-dichlorofluor- al. 2004). However, to fully understand the actual mechan- escin diacetate (DCFH-DA). DCFH reacts with ROS to ism, more study is needed. Peptides purified from Alaska form a highly fluorescent compound DCF. Pre-treatment Pollock skin gelatin and jumbo squid (Dosidicus gigas) with the purified peptide decreased the DCF fluoresce in a skin gelatin demonstrated a protective effect against oxi- time-dependent manner. The antioxidant activity of the dative stress in rat liver cells and human fibroblasts re- purified peptide could be attributed to the presence of spectively (Byun and Kim 2002;Mendis et al. 2005b). several amino acids, such as tyrosine, methionine, lysine, While peptides from hoki skin (Johnius belengerii) gelatin and tryptophan. The peptide sequence analysis showed showed protection against oxidative stress by upregulating high hydrophobic amino acid content (> 69%). The activity the expression of antioxidant enzymes, i.e., glutathione of the purified Nile tilapia (O. niloticus) scale gelatin pep- peroxidase, catalase, and superoxide dismutase, in human tide could be attributed to the presence of non-aromatic hepatoma cells in vitro (Mendis et al. 2005b). amino acids such as alanine, proline, valine, and leucine Peptides with antioxidant activities from marine sources (Mendis et al. 2005a;Mendis et al. 2005b). Other peptides with high radical scavenging antioxidant activity contained with anti-oxidative activity purified from fish skin have amino acids histidine, tyrosine and methionine (Saiga et been purified from blue leatherjacket (Navodon septentrio- al. 2003). While peptides containing amino acids histidine, nalis), seabass (Lates calcarifer), horse mackerel (Magalas- glutamic acid, aspartic acid, phosphorylated serine, and pis cordyla), crocker (Otolithes ruber), and Nile tilapia threonine have been demonstrated to be active metal che- (Oreochromis niloticus)(Chiet al. 2015;Sae-Leaw et al. lators. Amino acid methionine is considered as central in 2017; Sampath Kumar et al. 2012; Zhang et al. 2012). antioxidant activity, and its antioxidant mechanism is at- Chelation of pre-oxidative transition metals like Fe2 , tributed to the action of two-electron transfer from the 2+ 2+ Cu ,and Pb is another antioxidant mechanism. There- sulfide of methionine’s thioester group (Garner et al. fore, peptides exhibiting metal-chelating activity are consid- 1998). Last but not least, peptides with antioxidant activity ered as potential antioxidants. Three novel iron-chelating also have potential anti-inflammatory activity, neuropro- peptides were purified from Pacific cod skin gelatin (Wu et tective activity, and anti-allergy activity which in some al. 2017). The sequences of the purified peptides were iden- cases it has been tested as with the neuroprotective effect tified as GPAGPHGPPGKDGR, AGPHGPPGKDGR, and of grass carp skin hydrolysates observed in MES 23.5 cells AGPAGPAGAR. The iron-chelating ability was evaluated (Cai et al. 2015). using ESI-MS and FTIR spectroscopy. The analysis showed that the amino and carboxylate terminal groups, peptide Antimicrobial activity bonds from peptide backbone, amino, and imine from ar- Fish live in an environment where a myriad of saprophytic ginine side chain were involved in the formation of a com- and pathogenic microbes flourish putting them in con- plex with iron. Amino acid side chain groups of GPAG stant direct contact with potential pathogens. Therefore, PHGPPGKDGR and AGPHGPPGKDGR, including amino the fish skin acts a physical barrier by providing immedi- (lysine), imine (histidine), and carboxylate (aspartic acid), ate protection from the environment and as a chemical provided additional iron-binding sites. barrier through several innate immune factors such as Scales of Lates calcarifer, Mugil cephalus, Chanos cha- antimicrobial peptides (AMPs) (Bergsson et al. 2005). nos, and Oreochromis spp. were hydrolyzed by papain AMPS are low MW peptides that have a net positive and flavourzyme, and the Fe2 -binding activity for the charge and are amphiphilic. They are involved in the nat- different species was compared (Huang et al. 2015). Fer- ural defense mechanism against pathogens (innate im- rous ion together with collagen peptides from four fish munity); however, their main role is modulation of scales was placed in a dialysis bag (MW cut off 500 Da) mammalian cell functions. AMPS can be majorly divided for 3–4 days of dialysis. A fraction from Chanos chanos into different families which include defensin, parasin, had the highest iron-binding capacity at approximately cathelicidin and hepcidin, and piscidin. These AMP fam- 22.1 ppm/mg based on Fe2 binding activity/peptide ilies are species-specific, with piscidin being unique to concentration. MW distributions of the collagen pep- teleost fish (Campoverde et al. 2017). Table 3 outlines the tides from the scales of the four fish were all less than specific MEC and MIC values for fish skin peptides with 10 kDa, with an average MW of 1.3 kDa. It is suggested antimicrobial activity along with their sequences. that the iron-binding ability of peptides is related to the The antibacterial activity of black-barred halfbeak gelatin net charge and the exposure of glycine residues. Glycine and its hydrolysate was evaluated against three Gram is important for ferrous ions and peptides to form stable negative (Klebsiella pneumonia, Salmonella enterica,and complexes (Lee and Song 2009; Wu et al. 2012). The Salmonella typhi) and three Gram positive (Micrococcus Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 8 of 14 Table 3 Antimicrobial activity of peptides purified from fish skin Species MECs (μg/ MIC (μg/ Microorganism Peptide sequence Reference mL) mL) Skipjack tuna (Katsuwonus pelamis)3 – B. subtilis SJGAP Seo et al. (2014) 26 – M. luteus 4.8 – S. iniae 25 – A. hydrophila 2.7 – E. coli 9 – V. parahaemolytics 16 – C. albicans Yellowfin tuna (Thunnus albacares) 1.2 – B. subtilis YFGAP Seo et al. (2012) 6.5 – M. luteus 17 – S. iniae 8 – A. hydrophila 3 – E. coli 3.2 – V. parahaemolytics Yellow catfish (Pelteobagrus fulvidraco) – 2 B. subtilis GKLNLFLSRLEILKLFVGA Su (2011) – 4 S. aureus – 16 E. coli – 64 C. albicans Winter flounder (Pleuronectes – 1.1–2.2 * B. subtilis GWGSFFKKAAHVGKHVGKAALTHYL Cole et al. americanus) (1997) – 4.4–8.8 * P. haemolytica – 17.7–2.2 * S. aureus – 2.2–3.3 * E. coli – 8.8–17.7 * S. typhimurium (I and II) – 17.7–35.0 * A. salmonicida *Units in micromolar luteus, Staphylococcus aureus,and Bacillus cereus) bacteria including three fish pathogens, Aeromonas hydrophila, (Abdelhedi et al. 2017). Black-barred halfbeak gelatin at 10 Streptococcus iniae,and Vibrio parahaemolyticus.Anti- mg/mL exhibited a slight inhibitory activity against Gram microbial activity of SJGAP and YFGAP showed MECs positive M. luteus and B. cereus, with inhibitor diameter value of 1.2–17.0 μg/mL against Gram-positive bacteria zones of 6.5 mm and 7.0 mm, respectively. Increasing con- while the MEC value against Gram-negative bacteria was centration from 10 to 25 mg/mL slightly increased activity 3.1–12.0 μg/mL. The SJGAP peptide purified from skipjack with inhibition zone diameter reaching 8.5 mm and 9.0 mm tuna had a higher antimicrobial activity as it showed activity against M. luteus and B. cereus, respectively. The gelatin hy- against Candida. Albicans with MEC value of 16.0 μg/mL drolysate showed higher activity and was able to inhibit all unlike the peptide YFGAP purified from yellowfin tuna. the tested bacteria strains with different potentialities with These results suggest that these peptides might be related S. aureus and B. cereus being the most sensitive. The posi- to the innate defense in tuna. Based on the secondary struc- tive control gentamicin was more potent against S. aureus ture prediction and the homology modeling, the peptides and B. cereus, and it showed effective inhibition against the formed an amphipathic structure and consisted of a β-α-β growth of all tested bacteria strains than the black-barred motif with three secondary structural motifs including one gelatin hydrolysate. α-helix, two parallel β-strands, and two loop regions. Se- The antimicrobial activity of peptides purified from quence analysis results showed that both peptides, YFGAP yellowfin tuna (Thunnus albacares) and skipjack tuna and SJGAP, had high similarities with the N-terminus of (Katsuwonus pelamis) skin was investigated (Seo et al. GAPDH from other fish species by 81–91% and 91–97% 2014; Seo et al. 2012). Two glyceraldehyde-3-phosphate de- respectively. GAPDH is a multifunctional protein that regu- hydrogenase (GAPDH)-related AMPs, YFGAP and SJGAP, lates the sixth step of glycolysis and mediates cell death were identified. Both peptides showed broad-spectrum ac- under oxidative stress as well. Involvement of GAPDH in tivity against Gram-positive and Gram-negative bacteria nuclear translocation and its aggregation under oxidative Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 9 of 14 stress have been proposed as processes leading to bacteria. Their antimicrobial activity is broad spectrum GAPDH-mediated cell death. Furthermore, oxidative against both human and fish pathogens Gram-positive stressors initiate amyloid-like GAPDH aggregation via and Gram-negative bacteria, parasites, and fungi intermolecular disulfide bonds at Cys-152 (Nakajima et al. (Katzenback 2015). 2017). However, the antimicrobial activity observed could be as a result of the peptides, YFGAP and SJGAP, acting as Anti-Alzheimer’s and neuroprotective activity analogs of GAPDH in the sixth step of glycolysis due to Alzheimer’s disease is a kind of neurodegenerative disease high similarities with its N-terminus. characterized by progressive loss of neurons. The preva- Aside from hydrolysates, AMPs have also been success- lence of such degenerative neuro-diseases has increased fully isolated from fish skin mucus as well. The antimicro- with an increase in life expectancy especially as seen in de- bial activity of yellow catfish (Pelteobagrus fulvidraco)skin veloped countries (Choi and Choi 2015). Anti-Alzheimer’s mucus was investigated, and a novel peptide GKLNL disease activity is profiled using β-secretase inhibitory activ- FLSRLEILKLFVGAL was identified and named pelteoba- ity. The enzyme β-secretase along with another enzyme grin (Su 2011). Structural analysis using Schiffer–Edmund- ϒ-secretase generate a peptide amyloid-β (Aβ)through son helical wheel modeling revealed that pelteobagrin endo-proteolytic reactions of the amyloid precursor protein forms an amphipathic alpha-helix composed of 10 out of (APP) (Choi and Choi 2015). Apolipoprotein enhances the 12 hydrophobic residues on the surface and 4 out of 6 breakdown of beta-amyloid; however, an isoform of apoli- hydrophilic residues on the opposing side. The peptide had poprotein, APOE4, ineffectively breakdowns beta-amyloid a positive charge + 2 and was made up of 60% hydrophobic and leads to an excess amyloid buildup in the brain. The amino acids. It displayed a broad-spectrum antimicrobial peptide Aβ molecules can aggregate to form flexible soluble activity against Gram-positive bacteria, Gram-negative bac- oligomers, some of which turn out misfolded. These mis- teria, and fungi. However, Gram-positive bacteria B. subtilis folded oligomers can induce other Aβ molecules to also was the most sensitive to the peptide at a minimal inhib- take the misfolded oligomeric form (Haass and Selkoe ition concentration (MIC) of 2 μg/mL. The peptide demon- 2007;Nussbaumet al. 2013;Pulawskietal. 2012). strated no hemolytic activity against rabbit red blood cells, Anti-Alzheimer’s and neuroprotective activity of fish skin and it was relatively salt tolerant to concentrations of NaCl hydrolysates is summarized in Table 4.A β-secretase in- up to 137 mM. Similarly, a novel peptide from skin mucous hibitor peptide was purified from skate skin hydrolysate secretions of the winter flounder (Pleuronectes americanus) (Lee et al. 2015). The peptide was purified from a neutrase was purified and characterized (Cole et al. 1997). The hydrolysate of skate skin on a Sephadex G-25 column and peptide was named pleurocidin, and it had an amino acid with reversed-phase HPLC. The peptide sequence was de- sequence of GWGSFFKKAAHVGKHVGKAALTHYL. It termined to be QGYRPLRGPEFL and showed β-secretase exhibited a broad-spectrum activity against a wide range of inhibitory activity with an IC value of 24.26 μM. The neu- Gram-positive and Gram-negative bacteria. Gram-positive roprotective effect of protein hydrolysates with antioxidant bacteria B. subtilis was the most sensitive to the peptide activity from grass carp (Ctenopharyngodon idella)skin with a MIC value of 1.1–2.2 μM. was demonstrated (Cai et al. 2015). The hydrolysates at the A study by Bergsson investigated antimicrobial com- degree of hydrolysis DH5, DH10, and DH15 showed the ponents from the skin mucus of healthy Atlantic cod most significant neuroprotective effect on 6-OHDA- (Gadus morhua) (Bergsson et al. 2005). Results revealed induced neurotoxicity in MES 23.5. Salmon (Oncorhynchus acidic extracts were active against both Gram-positive keta) skin enzymatic hydrolysate showed learning and and Gram-negative bacteria in conditions that likely memory enhancement in mice (Pei et al. 2010). Oxidative mimicked the natural environment of cod. This suggests stress was alleviated, apoptotic neurons reduced, and that the skin mucus layer of the Atlantic cod is an im- brain-derived neurotrophic factor (BDNF) expression was portant tissue in surface defenses of cod and most likely upregulated in treatment groups compared with the con- protects the fish from infections caused by pathogenic trol group. Similarly, another study showed that salmon microbes. Antimicrobial test results revealed Bacillus skin collagen peptides reduced oxidative damage and megaterium as the most sensitive to the extract at all acetylcholinesterase (AChE) while it increased phosphory- concentrations of NaCl. Antimicrobial peptides were lated cAMP-response element binding protein (p-CREB) identified as histone H2B and ribosomal proteins L40, and BDNF expression in mice (Xu et al. 2015). L36A, and L35. Histone-derived peptides originate from both the N-terminus and C-terminus of H1, H2A, H2B, Other biological activities and H6 histones. These histone peptides are found in Other biological activities including antihyperglycemic, the skin, skin mucus, and other tissues, including gills, MMP inhibitory activity, and adipogenic regulatory have the spleen, and the gut. They are produced in response been demonstrated using fish skin as shown in Table 5. to epidermal damage, LPS, or certain Gram-negative Antihyperglycemic activity of fish skin was evaluated Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 10 of 14 Table 4 Anti-Alzheimer’s and neuroprotective activity of peptides purified from fish skin Activity or mechanism Species Peptide sequence Reference β-Secretase inhibitory Skate (Raja kenojei) QGYRPLRGPEFL Lee et al. (2015) Anti-acetylcholinesterase Salmon (Oncorhynchus keta) – Xu et al. (2015) Neuroprotection Grass carp (Ctenopharyngodon idella) – Cai et al. (2015) Learning and memory Salmon (Oncorhynchus keta) – Pei et al. (2010) using dipeptidyl peptidase IV (DPP-IV) inhibitory assay. lowered blood glucose levels of diabetic rats after 28-day Steelhead (Oncorhynchus mykiss) skin gelatin hydroly- administration. The normal rats and diabetic rats treated sates were prepared, and the hydrolysate of 4% papain with HSGH showed similar plasma DPP-IV activity range had the highest DPP-IV inhibitory activity 40–45% of 86.6–94.6% while the diabetic rats treated with TSGH (Cheung and Li-Chan 2017). The hydrolysates were had a significantly lower DPP-IV activity of 71.6%. The ef- purified with ultrafiltration to obtain fraction of less than fect of peptides on GLP-1 levels was also evaluated. TSGH 3 kDa. Two fractions showed 42% and 44% DPP-IV in- exhibited the highest increase in the total GLP-1 level hibitory activity showing that the activity was not influ- (27.81 pM) while HSGH and sitagliptin exhibited a com- enced by ultrafiltration as the values of the fractions and parable effect on GLP-1 secretion (23.46–23.81 pM) in dia- the whole hydrolysates were similar. betic rats after 30-day treatment. However, the underlying In a similar study, the DPP-IV inhibitory and glucagon- mechanism of peptide GLP-1 stimulatory activity is not like peptide-1 (GLP-1) stimulating activity of fish skin gel- well defined. However, it is has been proposed that the atin from various warm- and cold-water fish skins were presence of amino acids leucine and glutamic acid can in- evaluated and compared (Wang et al. 2015). Results duce GLP-1 secretion (Chen and Reimer 2009;Reimer revealed that the DPP-IV inhibitory activity of gelatin hy- 2006). Gelatin hydrolysates of warm-water fish skins drolysates from warm-water fish was greater than that from (TSGH) exhibited greater in vitro and in vivo DPP-IV cold-water fish. Halibut and tilapia skin gelatin hydrolysate inhibitory activity in comparison to cold-water fish (HSGH and TSGH) fractions at a cutoff of < 1.5 kDa UF skins (HSGH). TSGH contained higher imino acid were used for peptide sequence identification and to com- contents which resulted in increased antihyperglyce- pare the in vivo antihyperglycemic effect. MS/MS spectra mic activity in STZ-induced diabetic rats. analysis revealed amino acid sequences of 6 active peptides Fish skin hydrolysates have also been demonstrated to as SPGSSGPQGFTG, GPVGPAGNPGANGLN, PPGPTGP show MMP-1 inhibitory activity and thus have great po- RGQPGNIGF, IPGDPGPPGPPGP, LPGERGRPGAPGP, tential use as cosmeceuticals. Two active peptides from and GPKGDRGLPGPPGRDGM. All these peptides pos- cod skin gelatin hydrolysates (CGH) with anti-photoaging sessed the amino acid proline as the second N-terminal activity were identified (Lu et al. 2017). The peptides were residue. Moreover, it has been reported that peptides with purified from CGH by ion exchange chromatography and DPP-IV inhibitory activity have amino acids proline, trypto- RP-HPLC. The peptide sequences were determined using phan, alanine, valine, lysine, and aspartate as the second QTOF mass spectrometer as EIGPSGGRGKPGKDG- N-terminal residues in their sequences (Lacroix and DAGPK and GFSGLDGAKGD. The purified peptides had Li-Chan 2012). The IC values against DPP-IV of the puri- a MMP-1 inhibitory activity of 16% and 15% respectively. fied peptides ranged from 65.4 to 146.7 μM, and these were The activity of the peptide GFSGLDGAKGD was achieved comparable to the peptides from other proteins with the through the downregulation of MMP-1, p-ERK, and IC values between 41.9 and 174 μM(Huangetal. 2012; p-p38 while GEIGPSGGRGKPGKDGDAGPK activity was Lacroix and Li-Chan 2014; Silveira et al. 2013). Halibut and by the downregulation of p-JNK in MAPK signaling path- tilapia skin gelatin hydrolysates (HSGH and TSGH) ways. A study by Chen et al. 2016 purified 23 polypeptides Table 5 Antihyperglycemic and MMP inhibitory activity of peptides purified from fish skin Activity or mechanism Species Peptide sequence Reference Antihyperglycemic Halibut (Hippoglossus stenolepis) SPGSSGPQGFTG, GPVGPAGNPGANGLN, Wang et al. (2015) PPGPTGPRGQPGNIGF Tilapia (Oreochromis niloticus) IPGDPGPPGPPGP, LPGERGRPGAPGP, GPKGDRGLPGPPGRDGM MMP inhibitory activity Cod EIGPSGGRGKPGKDGDAGPK, GFSGLDGAKGD Lu et al. (2017) Tilapia (Oreochromis niloticus) LSGYGP Sun et al. (2013) Sutchi catfish (Pangasius hypophthalmus) LMWCP Pyun et al. (2012) Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 11 of 14 from cod skin hydrolysates. Amino acid sequences of palmitate-induced intracellular lipid vacuole accumula- Gly-Po and Gly-Leu and the amino acid arginine predom- tion visualized by Nile red staining. The palmitate- inated at the C-terminus of the polypeptides. The hydroly- induced intracellular lipid vacuole accumulation was sates showed a protective effect against UV-induced greatly reduced in the presence of 1 mg/ml SWFCP. photo-damage to collagen. Expression and activities of SWFCP significantly affected other obesity-related fac- matrix metalloproteinases (MMP-1, MMP-3, MMP-9) tors like low serum cholesterol, low serum triglyceride, were downregulated through the elevation of tissue inhibi- and low-density lipoprotein; high serum high-density tor of matrix metalloproteinases (TIMPs) and suppression lipoprotein levels; and reduced size of epididymal of the activation of mitogen-activated protein kinase adipocytes. (MAPK) signaling pathway in the skin of mice treated with the hydrolysate. In another study, the anti- Conclusion photoaging effect of a peptide LSGYGP purified from As elaborately discussed in this review, biologically ac- tilapia skin (Oreochromis niloticus) was evaluated by Sun tive peptides have been effectively produced through the et al. 2013. In vivo experiments showed that the peptide bioconversion of fish skin. These different peptides with improved the skin condition of UV irradiation-induced a range of bioactivities such as antihypertensive, antioxi- photoaging mice through its antioxidant activity. Further- dants, antimicrobial, neuroprotection, antihyperglyce- more, the mechanism of action of the same peptide mic, and anti-aging have been demonstrated in vitro LSGYGP was studied using ultraviolet B (UVB)-induced experiments and to an extent in vivo as well. However, mouse embryonic fibroblasts (MEFs) (Ma et al. 2018). The further study is required in the preparation of fish skin peptide reduced the intercellular ROS generation and hydrolysates using the fermentation method for the pro- decreased superoxide dismutase (SOD) activity as well as duction of bioactive peptides. In addition, further study reduced MMP-1 and MMP-9 activities. Molecular dock- is also required, to determine the potential immunomo- ing simulation analysis showed that the peptide inhibited dulation activity of antioxidant peptides, i.e., anti-inflam- MMP activities by docking the active sites of MMP-1 and matory, and anti-allergy and the corresponding cell MMP-9. The anti-photoaging effect of a peptide LMWCP signaling pathways. More investigations are also required purified from catfish skin (Pangasius hypophthalmus) was to determine the neuroprotective effect of fish skin hy- evaluated both in animal models and in a clinical trial. drolysates especially with protection against amyloid- The peptide downregulated the expression of MMP-3 and β-associated neurotoxicity as observed in Alzheimer’s MMP-13, while it upregulated the expression of MMP-2 disease. Nevertheless, bioactive peptides purified from and MMP-9 (Pyun et al. 2012). In the clinical trial results, fish skin can potentially be utilized in the development treatment groups receiving a daily oral dosage of 1000 mg of pharmaceutical and nutraceutical products. of LMWCP for 12 weeks showed significantly improved skin and less wrinkling in comparison with the placebo Abbreviations group (Kim et al. 2018). ABTS: 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); ACE: Angiotensin-converting enzyme; AMPs: Antimicrobial peptides; Last but not least, fish skin has also been demon- APOE4: Apolipoprotein; APP: Amyloid precursor protein; Aβ: Amyloid-β; strated to have adipogenic regulatory activity. The effect BDNF: Brain-derived neurotrophic factor; C/EBP: Ccaat (cytosine-cytosine- of subcritical water-hydrolyzed fish collagen peptide adenosine-adenosine-thymidine)-enhancer-binding proteins; cGK-I: cGMP- dependent protein kinase I; Cgmp: Cyclic guanosine 3′5′monophosphate; (SWFCP) from tuna skin on the protein levels of the DCFH-DA: Dichlorofluorescin diacetate; DMI: 1 μM: Dexamethasone and 1 μg/ master adipogenic transcription factors C/EBP and ml insulin; DMPO: 5,5-Dimethyl-1-pyrroline-N-oxide; DPPH: Diphenyl-2- PPAR was investigated (Lee et al. 2017). This was done picrylhydrazyl; DPP-IV: Dipeptidyl peptidase IV; EMR: Enzymatic membrane reactor; eNOS: Endothelial nitric oxide synthase; ESI-MS: Electrospray with the aim of evaluating the underlying inhibitory ionization mass spectrometry; ET-1: Endothelin-1; FRAP: Ferric-reducing mechanism of SWFCP in the adipogenic differentiation antioxidant power; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; of 3T3-L1 pre-adipocytes. Results revealed that SWFCP HPLC: High-performance liquid chromatography; IP 3: Inositol-1,4,5- trisphosphate; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein downregulated the expression of the key adipogenic kinase; MMP: Matrix metalloproteinase (MMP-1, MMP-3, MMP-9); NO: Nitric target gene and transcription factors in 3T3-L1 oxide; ORAC: Oxygen radical absorbance activity; PPAR-γ: Peroxisome pre-adipocytes exposed to MDI. After 8 days of incuba- proliferator-activated receptor gamma; PUFAs: Polyunsaturated fatty acids; RAS: Renin–angiotensin–aldosterone system; RNS: Reactive nitrogen species; tion of 3T3-L1 cells with 1 μM dexamethasone and 1 μg/ ROS: Reactive oxygen species; RP-HPLC: Reverse phase high-performance li- ml insulin (MDI) and SWFCP, the expression levels of quid chromatography; SDS/PAGE: Sodium dodecyl sulfate polyacrylamide gel C/EBP and PPAR protein were greatly reduced com- electrophoresis; SHR: Spontaneously hypertensive rats; TIMPs: Tissue inhibitor of matrix metalloproteinase pared with cells stimulated with MDI alone. SWFCP was also shown to downregulate the expression of aP2 an Acknowledgements adipogenic target gene, hence inhibiting adipogenic dif- This research was supported by the Marine Biotechnology Program of the ferentiation. Furthermore, SWFCP reduced lipogenesis Korea Institute of Marine Science and Technology Promotion (KIMST) funded in hepatocytes. 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Biological activity of peptides purified from fish skin hydrolysates

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Springer Journals
Copyright
Copyright © 2019 by The Author(s)
Subject
Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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2234-1757
DOI
10.1186/s41240-019-0125-4
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Abstract

Fish skin waste accounts for part of the solid waste generated from seafood processing. Utilization of fish skin by bioconversion into high-grade products would potentially reduce pollution and economic cost associated with treating fish processing waste. Fish skin is an abundant supply of gelatin and collagen which can be hydrolyzed to produce bioactive peptides of 2–20 amino acid sequences. Bioactivity of peptides purified from fish skin includes a range of activities such as antihypertensive, anti-oxidative, antimicrobial, neuroprotection, antihyperglycemic, and anti-aging. Fish skin acts as a physical barrier and chemical barrier through antimicrobial peptide innate immune action and other functional peptides. Small peptides have been demonstrated to possess biological activities which are based on their amino acid composition and sequence. Fish skin-derived peptides contain a high content of hydrophobic amino acids which contribute to the antioxidant and angiotensin-converting enzyme inhibitory activity. The peptide-specific composition and sequence discussed in this review can be potentially utilized in the development of pharmaceutical and nutraceutical products. Keywords: Fish skin, Peptides, Bioactivity, Antioxidant, Antihypertensive, Antimicrobial, Anti-Alzheimer’s Background crease the economic value of the catch but also would The marine environment is a source of functional bio- reduce the amount of marine processing waste. Thus, materials such as polyunsaturated fatty acids (PUFAs), reducing pollution and economic cost associated with polysaccharides, minerals and vitamins, antioxidants, treating the generated waste (Fang et al. 2017). enzymes, and bioactive peptides (Kim and Wijesekara Biological activity of peptides is based on their amino 2010; Kim et al. 2008; Pomponi 1999). By-products of acid composition and sequence (Pihlanto-Leppälä 2000). marine processing industries, i.e., skin, trimmings, This biological activity ranges from antioxidant, antihyper- viscera, and blood, contain a good amount of proteins tensive, immunomodulatory, and antimicrobial activity as which can be used as a source for bioactive peptides. demonstrated by several studies using different fish spe- These by-products are utilized as additives in animal cies like Pollack, skate, Nile tilapia, sea beam, yellow fish, husbandry as animal feed or in agriculture as fertilizers and skipjack (Chalamaiah et al. 2012). Bioactive peptides (Chalamaiah et al. 2012). Fish by-products like skin and are specific protein fragments derived from plants or ani- frame need to be processed into fish hydrolysates either mal sources which possess nutritional benefits and posi- by fermentation or by hydrolysis techniques before they tively influence health (Hartmann and Meisel 2007; can be effectively utilized. By so doing this increases pro- Korhonen and Pihlanto 2003). Bioactive peptides are in- duction costs (Chalamaiah et al. 2012). Therefore, the active in their parent protein sequence but can be released utilization of fish processing waste through bioconver- by enzymatic hydrolysis; however, for effective use, bio- sion into high-grade products like bioactive peptides active peptides must reach the target organ or receptors in would be a better alternative. This not only would in- the intestinal lumen intact and must survive enzymatic degradation (Adessi and Soto 2002). This review will high- light and discuss the different preparation methods of bio- active peptides from fish skin, their biological activity, and associated mode of action in regard to specific peptide * Correspondence: hgbyun@gwnu.ac.kr Department of Marine Biotechnology, Gangneung–Wonju National composition, sequence, and cell signaling pathways. University, Gangneung 25457, Republic of Korea © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 2 of 14 Preparation of bioactive peptides from fish skin as an initial extraction step. The step is followed by con- Fish protein hydrolysates contain peptides of 2–20 amino tinuous stirring at a controlled temperature for a set time. acid sequences after hydrolysis, and these peptides usually The procedure is repeated about 3 times, and it is carried have biological activity. Several extraction methods are out with an aim to remove non-collagenous proteins and utilized to liberate bioactive peptides from the parent pro- pigments (Jongjareonrak et al. 2005;Wang et al. 2008; tein, and these include acid-alkaline hydrolysis: extracting Wang et al. 2015). The skin is alternatively treated with an collagen by using acidic or alkaline reagent; enzymatic hy- acid (HCl) (Wu et al. 2017). After acid-alkali treatment, the drolysis: using enzymes to hydrolyze fish skin; and fer- skin was washed to neutralize the pH and further extrac- mentation method: using microorganisms as a source of tion carried out with distilled water at 65 °C for 4 h. Some the enzymes (Huang et al. 2015). extraction procedures include a defatting step (Mahboob 2014). Jongjareonrak et al. 2005 removed fat using butyl al- Enzymatic hydrolysis cohol for 24–48hwithgentlestirringand achangeofsolu- Enzymatic hydrolysis is the best way to hydrolyze fish tion every 8 h. The resultant matter was then subjected to skin without losing nutritional value (Huang et al. 2015). acid treatment with acetic acid for 24 h with gentle stirring. The method is preferred especially in the food and Collagen was extracted from fish skin, scale, and bone pharmaceutical industries because the hydrolysis process using a procedure described as follows (Wang et al. 2008). does not leave residual organic solvents or toxic chemi- The collagen was extracted with 0.5 M acetic acid at a sam- cals in its products (Kim and Wijesekara 2010). Steps in ple/solution ratio of 1:100 (w/v)for 24hwithcontinuous enzymatic hydrolysis involve substrate preparation, stirring. The extracts were centrifuged at 20,000g for 1 h at choice of the right enzyme, measuring the extent of 4 °C, and the extraction step was repeated using the ob- enzymatic hydrolysis, homogenization, and heating to tained residue, followed by centrifugation under the same inactivate endogenous enzymes, hydrolysis, and termin- conditions. The supernatants of the two extracts were ation of the enzymatic reaction. Commercial enzymes combined and precipitated by the addition of NaCl to a such as alcalase, trypsin, pepsin, papain, pancreatin, and final concentration of 0.9 M and centrifuged at 2500g for thermolysin are employed in the enzymatic hydrolysis 0.5 h to obtain a precipitate that was dissolved in 0.5 M (Bernardini et al. 2011). Conditions like enzyme concen- acetic acid. The precipitate was dialyzed for 48 h against 10 tration, pH, time, and temperature have to be well moni- volumes of 0.1 M acetic acid and distilled water, respect- tored and maintained during hydrolysis. Enzyme ively, which were changed every 8 h, before being lyophi- concentrations, pH, and temperature vary with the type lized. Antimicrobial peptides were purified from winter of enzyme used. Enzyme concentrations of 0.01–5.00% flounder epidermis and mucus extracts (Cole et al. 1997). (w/w) and pH range of 1.5–11 have been documented The mucus was obtained from the skin by scraping and (Halim et al. 2016). Black-barred halfbeak gelatin was further subjected to homogenization in a solution of 50 ml dissolved in DW and subjected to enzymatic hydrolysis of 0.2 M sodium acetate, 0.2% Triton X-100, and 1 mM with an enzyme/substrate ratio of 30:1, pH 10.0 and 50 ° phenyl methyl sulfonyl fluoride. The homogenate was cen- C. The enzymatic activity was evaluated by a method de- trifuged for 20 min at 20,000g, and the resultant super- scribed by (Kembhavi et al. 1993) using casein as a sub- natant was further purified. strate. The gelatin solution was equilibrated for 30 min before the enzyme addition. The pH was maintained by Fermentation addition of 2 N NaOH, and after 3 h, the enzymes were Fermentation is considered a more natural method of inactivated by heating the solution at 95 °C for 20 min protein hydrolysis. The technique has been employed for (Abdelhedi et al. 2017). Extraction of pepsin soluble col- centuries especially in East Asian countries as a lagen (PSC) from fish skin was performed by (Mahboob traditional preservation method. Fermentation not only 2014). Undissolved residue obtained after acid soluble enhances the flavor and taste of food but also increases collagen (ASC) extraction was utilized for the PSC ex- its nutraceutical value. During the fermentation process, traction as described by (Singh et al. 2011). bioactive peptides are released by the action of both microorganisms and endogenous proteolytic enzymes. Acid-alkaline hydrolysis Several studies have demonstrated the bioactivity of vari- During fish skin hydrolysis by acid-alkaline hydrolysis, ous marine products like Thai fermented shrimp paste, certain amino acids, i.e., tryptophan, serine, and threo- shrimp by-products, squid miso, and a variety of nine, can be destroyed at high pH. Therefore, the pH traditional fermented fish products (Bueno-Solano et al. and temperature of the hydrolysates must be closely ob- 2009; Giri et al. 2011; Kleekayai et al. 2015). Majumdar served during the hydrolysis process. Collagen extraction et al. 2016 examined the chemical and microbial proper- from fish skin by acid-alkaline hydrolysis involves treat- ties of shidal, a traditional fermented fish product of ment of pre-cleaned skin samples with an alkali (NaOH) Northeast India. A combination of both fatty acids Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 3 of 14 (eicosapentaenoic, docosahexaenoic, arachidonic, lino- and water into the blood, while at the same time causing lenic, and linoleic acid) and proteins or peptides of MW the excretion of potassium. Inhibition of ACE diminishes (molecular weight) range between 45 and 29 kDa and 45 angiotensin II-mediated aldosterone secretion from the and 6 kDa respectively were reported to be present in adrenal cortex, leading to a decrease in water and the fermented fish product. Hydrolysates were prepared sodium reabsorption and a reduction in extracellular from turbot skin by utilization of the fermentation volume sequentially leading to vasodilation and eventu- method using 3 microorganisms, i.e., Saccharomyces cer- ally reduced arterial blood pressure. Hypertension is evisiae, Aspergillus oryzae, and Streptococcus thermo- manageable with various synthetic ACE inhibitors such philes (Fang et al. 2017). as captopril (Dezsi 2000). Hypertension can also be counteracted by endothelial-dependent vasodilation Purification of peptides through the upregulation of nitric oxide (NO) expres- The biological activity of peptides is determined by proper- sion, phosphorylation of eNOS, and downregulation of ties like molecular weight, charge, and hydrophobicity. endothelin (ET-1) expression. Endothelial smooth Therefore, peptides are purified through a multi-step puri- muscle cell relaxation is achieved via the nitric oxide/ fication process based on such properties. Purification cyclic guanosine monophosphate (cGMP)-mediated based on molecular weight employs methods like ultrafil- intracellular signaling pathway. This pathway involves tration (UF), nanofiltration (NF), and gel filtration (GF) the upregulation of cGMP-dependent protein kinase I (Halim et al. 2016). Ion exchange chromatography (IEC) is (cGK-I) and a decrease in intracellular calcium levels via used to fractionate peptides basing on their net charge. downregulation of the inositol-1,4,5-trisphosphate (IP 3) Fractioned peptides are then further purified using receptor (Daiber and Münzel 2015). technologies like reversed-phase HPLC which separates Conventional antihypertensive drugs cause several ad- compounds based on hydrophobicity and hydrophilicity verse effects; therefore, the search for safer natural alter- (Conlon 2007). Peptide sequences of the most active natives is in progress. Among the natural alternatives, fractions from HPLC analysis are then analyzed and identi- interest has grown in the utilization of bioactive peptides fied using mass spectrometry methods like matrix-assisted in the prevention of hypertension and in the initial treat- laser deionization time-of-flight (MALDI-TOF), electro- ment of mild hypertension (Guang and Phillips 2009). spray ionization mass (ESI), matrix-assisted laser desorp- The antihypertensive activity of fish skin peptides is dis- tion/ionization mass spectrometry (MALDI-MS), etc. cussed and summarized below in Table 1. (Bernardini et al. 2011). Ultrafiltration of steelhead/rainbow hydrolysates yielded fractions of less than 3 kDa with ACE inhibitory activity Biological activities higher than the activity of the whole hydrolysates (Cheung Antihypertensive activity and Li-Chan 2017). The low MW fractions were prepared Hypertension is a chronic condition affecting millions of from whole hydrolysates using commercial enzyme kits. people around the world. According to a report by Two fractions showed ACE inhibition activity of 54% and World Health Organization 2011, the global prevalence 63%. The ACE inhibitory activity was affected by different of hypertension among adults ≥ 25 years stands at 40% conditions suggesting that the additional hydrolysis and it is estimated that 1.56 billion people will have the achieved with higher enzyme concentration and longer condition by 2025 (Kearney et al. 2005). Blood pressure duration effectively generated shorter peptides with higher is regulated by the renin–angiotensin–aldosterone sys- activity. The highest ACE inhibition was demonstrated in tem (RAS). Low renal blood flow or low plasma sodium samples produced after 6 h hydrolysis with 4% protease. concentration initiates the conversion of pro-renin into Similarly, fish skin peptide bioactivity has been shown to renin in juxtaglomerular cells in the kidneys. Renin in be associated with low MW peptides (Iwaniak et al. 2014; circulation catalyzes the conversion of angiotensinogen Power et al. 2014). to angiotensin I which is then subsequently converted to Black-barred halfbeak (Hemiramphus far) skin showed angiotensin II by the enzyme angiotensin-converting en- high protein content in the gelatin extracts, estimated at zyme (ACE) (Paul 2006). ACE is mainly produced in the 91.36% (Abdelhedi et al. 2017). The protein quantity in lungs and other sites including the endothelial lining of the gelatin was closely similar to that obtained in other vascular tissues, heart, brain, kidney, placenta, bone mar- fish skin species such as splendid squid, cuttlefish, row, pancreas, and testis. Angiotensin II is a potent thornback ray, and cobia (Jridi et al. 2013; Lassoued et vasoconstrictor that causes blood vessels to narrow al. 2014; Nagarajan et al. 2012; Silva et al. 2014). The resulting in increased blood pressure (Rogerson et al. ACE inhibitory activity was 36.51% for 1 mg/mL of 1992). Simultaneously, it stimulates the secretion of the whole gelatin while 1 mg/mL gelatin hydrolysate showed hormone aldosterone from the adrenal cortex causing a significantly greater activity of 80.76%. However, these the renal tubules to increase the reabsorption of sodium values were significantly lower than the positive control Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 4 of 14 Table 1 ACE inhibitory activity peptides purified from fish skin hydrolysates Species IC (μM) Peptide sequence Reference Nile tilapia (Oreochromis niloticus) 760–1490 GIV, GAP*GF, GFA*GPA, SGNIGFP*GPK, GIPGPIGPP*GRP Thuanthong et al. (2017) Pacific cod skin 6.9 GASSGMPG Ngo et al. (2016) 14.5 LAYA Thornback ray skin 27.9 GIPGAP Lassoued et al. (2015) 170 APGAP Skate 3.09 MVGSAPGVL Ngo et al. (2014b) 4.22 LGPLGHQ Salmon (Oncorhynchus keta) 8.7 GLPLNLP Lee et al. (2014) Skate 95 PGPLGLTGP Lee et al. (2011) 148 QLGFLGPR Alaska pollock 2.6 GPL Byun and Kim (2002) 17.13 GPM Cod (Gadus microcephalus) – TCSP, TGGGNV Ngo et al. (2011) captopril. The hydrolysate had an ACE inhibitory IC be attributed to factors such as Van der Waals force and value of 332.66 ± 16.57 mg/mL. Higher values of ACE in- hydrogen bonds of molecules that stabilize the ligand– hibitory activity of hydrolysate in comparison to those of protein. the gelatin are an indicator that bioactive peptides are Peptides IGPAG, FGYGG, GIPGAP, IGAPGATGPAG, released from the protein molecules upon enzymatic hy- AKGDS, GASGPRGPA, GQDGRPGPAG, and GEAGNPG- drolysis (Abdelhedi et al. 2017). In another study, five PAGP were purified from Thornback ray skin gelatin neu- novel ACE inhibitory peptides GIV, GAP∗GF, GFP∗GPA, trase hydrolysate (Lassoued et al. 2015). Peptide GIPGAP, SGNIGFP∗GPK, GIPGPIGPP∗GPR were identified from IC 27.9 μM, was the most potent ACE inhibitor among the most active fractions of Nile tilapia skin gelatin. The the purified peptides. A hydrolysate TRGH-A26 was pre- IC value of the active peptides ranged between 760 pared using crude proteases from Bacillus subtilis A26, and and 1490 μM (Thuanthong et al. 2017). peptides AVGAT, GGVGR, APGAP, GEPGAPGPA, and Pacific cod skin gelatin was hydrolyzed using several GPRGAPGPA were purified. The peptide APGAP, IC enzymes and the pepsin hydrolysate showed the highest 170 μM, was the most potent ACE inhibitor from this hy- ACE inhibitory effect of about 91% (Ngo et al. 2016). drolysate. The two most potent peptides GIPGAP and ACE inhibitory active peptides were identified as APGAP from Thornback ray skin gelatin hydrolysates had GASSGMPG, IC 6.9 μM, and LAYA, IC 14.5 μM. a strikingly similar sequence of PGAP at the C-terminus. 50 50 The MW of peptides GASSGMPG and LAYA was less Another peptide FGYGG with a high ACE inhibitory activ- than 1 kDa which indicates they can cross the intestinal ity with an IC value of 231 μM contained the aromatic barrier and exert biological effects. The ACE inhibitory residue phenylalanine. activity of GASSGMPG was higher than that of ASL, The antihypertensive effect of skate skin hydrolysates IC 102.15 μM from silkworm pupa (Bombyx mori) pro- in an animal model experiment using spontaneously tein; PVNNPQIH, IC 206.7 μM from small red bean hypertensive rats (SHRs) was evaluated (Ngo et al. Phaseolus vulgaris; GDLGK-TTTVSNWSPPKYKDTP, 2014b). Purified peptides were orally administered to IC 11.28 μM from tuna frame protein; and AHEPVK, SHRs, and changes in heart rate and blood pressure IC 63 μM from edible mushroom Agaricus bisporus were monitored over a period of 20 days. Systolic blood (Lau et al. 2014; Lee et al. 2010; Rui et al. 2013;Wuet pressure readings were monitored, and the maximal dec- al. 2015). A docking simulation of the ACE–ligand com- rements in systolic blood pressure observed were 127.2 plexes between ACE/peptides and ACE/captopril dem- mmHg at 20 days and 118.8 mmHg at 10 days in the onstrated the potential of these peptides as ACE purified peptide (1000 mg/kg of BW) and captopril treat- inhibitors. The binding sites of GASSGMPG and capto- ment groups respectively. The peptides were identified pril on the ACE molecule were observed to be the same as MVGSAPGVL, IC 3.09 μM, and LGPLGHQ, IC 50 50 at the Asn72 residue while LAYA and captopril shared 4.22 μM. Docking simulation of the ACE molecule and two binding sites on the ACE molecule at Asn72 and the purified peptide on the Docking Server revealed al- Arg348 residues. Overall molecular docking simulation most similar binding on the ACE molecule as captopril. demonstrated good protein–drug interaction which can The binding site between the ACE molecule and the Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 5 of 14 purified peptide had many residues including Trp67, Antioxidant activity Asn68, Thr71, Asn72, and Arg348. This suggested that Reactive oxygen species (ROS) and reactive nitrogen spe- blockades by the purified peptides on the ACE molecule cies (RNS) are produced as a result of the metabolism of may contribute to the ACE inhibitory potency of the oxygen and nitrogen. ROS and RNS can cause damage to purified peptides for preventing hypertension. cellular components in the body. During metabolism and Furthermore, the protective effect of the same peptides respiration, ROS are constantly produced. These include − − MVGSAPGVL and LGPLGHQ from skate skin peptides superoxide anion radials O , hydroxyl radical OH ,and was investigated against angiotensin II-induced endothe- non-free radical species like hydrogen peroxide and singlet lial dysfunction using human endothelial cells (Ngo et al. oxygen O . Excess amounts of ROS as in oxidative stress 2014a). Increased production of endothelial nitric oxide exert oxidative damage to cellular macromolecules like pro- synthase (eNOS) and inhibition of endothelin-1 ET-1 teins, lipids, and DNA by subtracting electrons. This starts production through upregulation of the PPAR-γ pathway a series of reactions which eventually leads to new radicals was observed. The enzyme eNOS upregulates the gener- attacking and damaging other cellular macromolecule com- ation of nitric oxide (NO) in the vascular endothelium ponents (Kaur and Kapoor 2001). Peptides are considered while NO maintains endothelial integrity and proper to be more potent antioxidants than free amino acids be- function through regulation of vascular tone, local blood cause of the increased stability of the resultant peptide rad- flow, platelet aggregation and adhesion, and leukocyte– ical (Elias et al. 2008). The exact structure–antioxidant endothelial cell interactions (Dessy and Feron 2004). activity relationship of peptides has not been established. Thus, endothelial dysfunction, a precursor of hyperten- However, the type, position, and hydrophobicity of amino sion and other health conditions like diabetes, aging, acids in the peptides are considered to play an essential and atherosclerosis, results from abnormalities in NO role. The most reactive amino acids in proteins are usually production by the vascular endothelium. Purified pep- those with nucleophilic sulfur-containing side chains like tides from skate skin not only showed ACE inhibitory taurine, cysteine, and methionine or aromatic side chains activity but also provided protection against endothelial like tryptophan, tyrosine, and phenylalanine (Elias et al. dysfunction in endothelial cells. Other peptides with 2008). Fish skin is an abundant supply of gelatin and colla- ACE inhibitory activity purified from similar studies in- gen. Gelatin contains an abundance of hydrophobic amino clude PGPLGLTGP, IC 95 μM, and QLGFLGPR, IC acids such as glycine, valine, alanine, proline, and hydroxy- 50 50 148 μM, from skate skin; GLPLNLP, IC 18.7 μM, from proline and could potentially contain a range of peptides salmon skin; and GPL, IC 2.6 μM, and GPM, IC with potent lipid–peroxidation inhibitory activity (Kim and 50 50 17.13 μM, from Alaska Pollock skin (Byun and Kim Mendis 2006). Several methods are used for assessing anti- 2002; Lee et al. 2014; Lee et al. 2011). oxidant activity, and these include oxygen radical absorb- Typically, ACE inhibitory has been attributed to small- ance capacity (ORAC), Ferric-reducing antioxidant power sized peptide residues with 2–12 amino acids (Yamamoto (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). Low et al. 1994). Peptides with tryptophan, proline, or phenyl- MW peptides have higher ORAC values and metal-chelat- alanine at the C-terminus or branched-chain aliphatic ing activities while high MW peptides have higher FRAP amino acids at the N-terminus are suitable to act as com- and DPPH radical scavenging (Theodore et al. 2008). The petitive inhibitors of ACE (Cushman and Cheung 1971). antioxidant activity of fish skinpeptidesissummarizedin Many of the peptides identified in ACE inhibitory frac- Table 2. tions contain proline at one of the three C-terminal posi- Abdelhedi et al. 2017 investigated the antioxidant ac- tions. Hydrophobic amino acids in the N-terminus may tivity of gelatin extract from black-barred halfbeak also contribute to the ACE inhibitory activity (Rho et al. (Hemiramphus far) skin. The DPPH radical scavenging 2009). Therefore, in addition to the presence of proline in activities of the gelatin extract (5 mg/mL) and the posi- the C-terminal position, the presence of alanine could also tive control vitamin C were 43.39% and 70.0% respect- be a contributing factor to ACE inhibitory activity ively. The antioxidant activity was lower than that of the (Yamamoto et al. 1994). Natural ACE inhibitory peptides positive control. However, lower MW fish skin protein and ACE substrates such as bradykinin and angiotensin I hydrolysates of cobia skin and Raja clavata skin have have been shown to contain aromatic amino acid residues been demonstrated to show higher antioxidant activity such as phenylalanine (Camargo et al. 2012; Cheung et al. than their protein molecule precursors (Lassoued et al. 1980;Hara et al. 1984). Peptides inhibiting ACE activity 2015). The ferric-reducing antioxidant power for whole can potentially be used as nutraceuticals to lower elevated gelatin, gelatin hydrolysate, and vitamin C was deter- blood pressure. As described, ACE inhibitory peptides mined to be 0.47, 1.03, and 2.01 respectively. Whole gel- have been successfully purified from fish skin and their atin and gelatin hydrolysate exhibited similar antioxidant biological activity has been demonstrated by both in vitro activity using the β-carotene bleaching assay with activ- and in vivo studies. ities of 53.73% and 78.47% respectively. Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 6 of 14 Table 2 Anti-oxidative activity of peptides purified from fish skin Species Radical scavenging activity (μg/mL) Peptide sequence Reference DPPH Hydroxyl (HO•) ABTS Thornback ray 1980 –– AVGAT Lassoued et al. (2015) Nile tilapia (Oreochromis niloticus) 8.82 μM 7.56 μM – DPALATEPDMPF Ngo et al. (2010) Horse Mackerel (Magalaspis cordyla) 72.3% 51.2% – NHRYDR Sampath Kumar et al. (2012) Crocker (Otolithes ruber) 79.6% 56.8% – GNRGFACRHA Sampath Kumar et al. (2012) Hoki (Johnius belengerii) 156.2 μM –– HGPLGPL Mendis et al. (2005b) Blue leatherjacket (Navodon 405 179 – GSGGL Chi et al. (2015) septentrionalis) 194 89 – GPGGFI 118 73 – FIGP Seabass (Lates calcarifer) –– 81.41* GLFGPR Sae-Leaw et al. (2017) –– 10.4* GATGPQGPLGPR –– 2.59* VLGPF –– 0.50* QLGLGPV Jumbo squid (Dosidicus gigas) –– – FDSGPAGVL Mendis et al. (2005a) –– – DGPLQAGQPGER Amur sturgeon 5380 890 8 PAGT Nikoo et al. (2015) Cod (Gadus microcephalus) –– – TCSP, TGGGNV Ngo et al. (2011) Nile tilapia (Oreochromis niloticus) – 4.61 – EGL Zhang et al. (2012) – 6.45 – YGDEY Pacific cod Iron-chelating activity GPAGPHGPPGKDGR, AGPHGPPGKDGR, Wu et al. (2017) AGPAGPAGAR *Units in millimole TE per millimole peptide Similar results have been recorded for thornback ray histidine (16.83%) in TRGH-A26 may be responsible for skin by (Lassoued et al. 2015). Whole gelatin, gelatin hy- the high antioxidant activity (Carrasco-Castilla et al. drolysates, and vitamin C inhibited the peroxidation of 2012). The anti-oxidative efficacy of 180 mol/ml α- linoleic acid by 15.91%, 34.78%, and 70.22% respectively tocopherol equivalents at 5 mg/mL in the phosphomolyb- after 3 days and 39.25%, 74.88%, and 99.2% respectively denum assay was also evaluated and TRGH-A26 exhibited after 9 days. Similarly, the gelatin hydrolysate from Nile the highest anti-oxidative efficacy. TRGH-alcalase gelatin tilapia skin exhibited 59.74% of lipid peroxidation inhib- hydrolysate was the most potent inhibitor of DNA oxida- ition after a 5-day incubation period while whole gelatin tion by hydroxyl radicals. Likewise, it also demonstrated had activity of only 7.12% (Choonpicharn et al. 2015). DNA protective effect as no degradations were observed The authors noted that antioxidant activity observed for the two forms of plasmid DNA utilized in the test. could be due to the presence of hydrophobic amino DNA oxidation inhibition activity may be attributed to acids. Other studies using black-barred and Acipenser hydrophobic amino acids which were present in schrenckii skin gelatin hydrolysates observed that glycine TRGH-alcalase and TRGH-neutrase peptide hydrolysates. and proline had high antioxidant activity (Ngo et al. Amino acids histidine, tyrosine, methionine, and phenyl- 2011; Nikoo et al. 2015). alanine were high in TRGH-A26 and TRGH-Crude at The antioxidant activity of thornback ray gelatin hy- total percentages of 13.22 and 13.09%, respectively. drolysates was assayed using various in vitro tests Nile tilapia (Oreochromis. niloticus) scale gelatin protein (Lassoued et al. 2015). Hydrolysis was carried out using was hydrolyzed using alcalase, pronase E, trypsin, and alcalase, neutrase, and Bacillus subtilis A26 proteases. A pepsin (Ngo et al. 2010). A peptide purified from the alca- hydrolysate obtained by treatment with Bacillus subtilis lase hydrolysate provided significant protection against A26 proteases (TRGH-A26) had a high DPPH scaven- the DNA oxidative damage when exposed to *OH gener- 2+ ging activity with an IC value of 1.98 mg/mL and ated by Fe /H O . The DNA damage was inhibited by 50 2 2 β-carotene bleaching inhibition activity of 70%. The high about 70%. The cell viability tests using mouse macro- content of positively charged amino acids lysine and phages (RAW 264.7) and human lung fibroblasts Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 7 of 14 (MRC-5) confirmed that gelatin alcalase hydrolysate was Fe2 binding activity of fish scales is probably derived non-cytotoxic. The purified peptide was identified as from the exposure of glycine during hydrolysis since fish DPALATEPDMPF. The ROS scavenging activity was de- scale collage is rich in glycine (Chaud et al. 2002; Fahmi et termined using a fluorescence probe, 2´,7´-dichlorofluor- al. 2004). However, to fully understand the actual mechan- escin diacetate (DCFH-DA). DCFH reacts with ROS to ism, more study is needed. Peptides purified from Alaska form a highly fluorescent compound DCF. Pre-treatment Pollock skin gelatin and jumbo squid (Dosidicus gigas) with the purified peptide decreased the DCF fluoresce in a skin gelatin demonstrated a protective effect against oxi- time-dependent manner. The antioxidant activity of the dative stress in rat liver cells and human fibroblasts re- purified peptide could be attributed to the presence of spectively (Byun and Kim 2002;Mendis et al. 2005b). several amino acids, such as tyrosine, methionine, lysine, While peptides from hoki skin (Johnius belengerii) gelatin and tryptophan. The peptide sequence analysis showed showed protection against oxidative stress by upregulating high hydrophobic amino acid content (> 69%). The activity the expression of antioxidant enzymes, i.e., glutathione of the purified Nile tilapia (O. niloticus) scale gelatin pep- peroxidase, catalase, and superoxide dismutase, in human tide could be attributed to the presence of non-aromatic hepatoma cells in vitro (Mendis et al. 2005b). amino acids such as alanine, proline, valine, and leucine Peptides with antioxidant activities from marine sources (Mendis et al. 2005a;Mendis et al. 2005b). Other peptides with high radical scavenging antioxidant activity contained with anti-oxidative activity purified from fish skin have amino acids histidine, tyrosine and methionine (Saiga et been purified from blue leatherjacket (Navodon septentrio- al. 2003). While peptides containing amino acids histidine, nalis), seabass (Lates calcarifer), horse mackerel (Magalas- glutamic acid, aspartic acid, phosphorylated serine, and pis cordyla), crocker (Otolithes ruber), and Nile tilapia threonine have been demonstrated to be active metal che- (Oreochromis niloticus)(Chiet al. 2015;Sae-Leaw et al. lators. Amino acid methionine is considered as central in 2017; Sampath Kumar et al. 2012; Zhang et al. 2012). antioxidant activity, and its antioxidant mechanism is at- Chelation of pre-oxidative transition metals like Fe2 , tributed to the action of two-electron transfer from the 2+ 2+ Cu ,and Pb is another antioxidant mechanism. There- sulfide of methionine’s thioester group (Garner et al. fore, peptides exhibiting metal-chelating activity are consid- 1998). Last but not least, peptides with antioxidant activity ered as potential antioxidants. Three novel iron-chelating also have potential anti-inflammatory activity, neuropro- peptides were purified from Pacific cod skin gelatin (Wu et tective activity, and anti-allergy activity which in some al. 2017). The sequences of the purified peptides were iden- cases it has been tested as with the neuroprotective effect tified as GPAGPHGPPGKDGR, AGPHGPPGKDGR, and of grass carp skin hydrolysates observed in MES 23.5 cells AGPAGPAGAR. The iron-chelating ability was evaluated (Cai et al. 2015). using ESI-MS and FTIR spectroscopy. The analysis showed that the amino and carboxylate terminal groups, peptide Antimicrobial activity bonds from peptide backbone, amino, and imine from ar- Fish live in an environment where a myriad of saprophytic ginine side chain were involved in the formation of a com- and pathogenic microbes flourish putting them in con- plex with iron. Amino acid side chain groups of GPAG stant direct contact with potential pathogens. Therefore, PHGPPGKDGR and AGPHGPPGKDGR, including amino the fish skin acts a physical barrier by providing immedi- (lysine), imine (histidine), and carboxylate (aspartic acid), ate protection from the environment and as a chemical provided additional iron-binding sites. barrier through several innate immune factors such as Scales of Lates calcarifer, Mugil cephalus, Chanos cha- antimicrobial peptides (AMPs) (Bergsson et al. 2005). nos, and Oreochromis spp. were hydrolyzed by papain AMPS are low MW peptides that have a net positive and flavourzyme, and the Fe2 -binding activity for the charge and are amphiphilic. They are involved in the nat- different species was compared (Huang et al. 2015). Fer- ural defense mechanism against pathogens (innate im- rous ion together with collagen peptides from four fish munity); however, their main role is modulation of scales was placed in a dialysis bag (MW cut off 500 Da) mammalian cell functions. AMPS can be majorly divided for 3–4 days of dialysis. A fraction from Chanos chanos into different families which include defensin, parasin, had the highest iron-binding capacity at approximately cathelicidin and hepcidin, and piscidin. These AMP fam- 22.1 ppm/mg based on Fe2 binding activity/peptide ilies are species-specific, with piscidin being unique to concentration. MW distributions of the collagen pep- teleost fish (Campoverde et al. 2017). Table 3 outlines the tides from the scales of the four fish were all less than specific MEC and MIC values for fish skin peptides with 10 kDa, with an average MW of 1.3 kDa. It is suggested antimicrobial activity along with their sequences. that the iron-binding ability of peptides is related to the The antibacterial activity of black-barred halfbeak gelatin net charge and the exposure of glycine residues. Glycine and its hydrolysate was evaluated against three Gram is important for ferrous ions and peptides to form stable negative (Klebsiella pneumonia, Salmonella enterica,and complexes (Lee and Song 2009; Wu et al. 2012). The Salmonella typhi) and three Gram positive (Micrococcus Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 8 of 14 Table 3 Antimicrobial activity of peptides purified from fish skin Species MECs (μg/ MIC (μg/ Microorganism Peptide sequence Reference mL) mL) Skipjack tuna (Katsuwonus pelamis)3 – B. subtilis SJGAP Seo et al. (2014) 26 – M. luteus 4.8 – S. iniae 25 – A. hydrophila 2.7 – E. coli 9 – V. parahaemolytics 16 – C. albicans Yellowfin tuna (Thunnus albacares) 1.2 – B. subtilis YFGAP Seo et al. (2012) 6.5 – M. luteus 17 – S. iniae 8 – A. hydrophila 3 – E. coli 3.2 – V. parahaemolytics Yellow catfish (Pelteobagrus fulvidraco) – 2 B. subtilis GKLNLFLSRLEILKLFVGA Su (2011) – 4 S. aureus – 16 E. coli – 64 C. albicans Winter flounder (Pleuronectes – 1.1–2.2 * B. subtilis GWGSFFKKAAHVGKHVGKAALTHYL Cole et al. americanus) (1997) – 4.4–8.8 * P. haemolytica – 17.7–2.2 * S. aureus – 2.2–3.3 * E. coli – 8.8–17.7 * S. typhimurium (I and II) – 17.7–35.0 * A. salmonicida *Units in micromolar luteus, Staphylococcus aureus,and Bacillus cereus) bacteria including three fish pathogens, Aeromonas hydrophila, (Abdelhedi et al. 2017). Black-barred halfbeak gelatin at 10 Streptococcus iniae,and Vibrio parahaemolyticus.Anti- mg/mL exhibited a slight inhibitory activity against Gram microbial activity of SJGAP and YFGAP showed MECs positive M. luteus and B. cereus, with inhibitor diameter value of 1.2–17.0 μg/mL against Gram-positive bacteria zones of 6.5 mm and 7.0 mm, respectively. Increasing con- while the MEC value against Gram-negative bacteria was centration from 10 to 25 mg/mL slightly increased activity 3.1–12.0 μg/mL. The SJGAP peptide purified from skipjack with inhibition zone diameter reaching 8.5 mm and 9.0 mm tuna had a higher antimicrobial activity as it showed activity against M. luteus and B. cereus, respectively. The gelatin hy- against Candida. Albicans with MEC value of 16.0 μg/mL drolysate showed higher activity and was able to inhibit all unlike the peptide YFGAP purified from yellowfin tuna. the tested bacteria strains with different potentialities with These results suggest that these peptides might be related S. aureus and B. cereus being the most sensitive. The posi- to the innate defense in tuna. Based on the secondary struc- tive control gentamicin was more potent against S. aureus ture prediction and the homology modeling, the peptides and B. cereus, and it showed effective inhibition against the formed an amphipathic structure and consisted of a β-α-β growth of all tested bacteria strains than the black-barred motif with three secondary structural motifs including one gelatin hydrolysate. α-helix, two parallel β-strands, and two loop regions. Se- The antimicrobial activity of peptides purified from quence analysis results showed that both peptides, YFGAP yellowfin tuna (Thunnus albacares) and skipjack tuna and SJGAP, had high similarities with the N-terminus of (Katsuwonus pelamis) skin was investigated (Seo et al. GAPDH from other fish species by 81–91% and 91–97% 2014; Seo et al. 2012). Two glyceraldehyde-3-phosphate de- respectively. GAPDH is a multifunctional protein that regu- hydrogenase (GAPDH)-related AMPs, YFGAP and SJGAP, lates the sixth step of glycolysis and mediates cell death were identified. Both peptides showed broad-spectrum ac- under oxidative stress as well. Involvement of GAPDH in tivity against Gram-positive and Gram-negative bacteria nuclear translocation and its aggregation under oxidative Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 9 of 14 stress have been proposed as processes leading to bacteria. Their antimicrobial activity is broad spectrum GAPDH-mediated cell death. Furthermore, oxidative against both human and fish pathogens Gram-positive stressors initiate amyloid-like GAPDH aggregation via and Gram-negative bacteria, parasites, and fungi intermolecular disulfide bonds at Cys-152 (Nakajima et al. (Katzenback 2015). 2017). However, the antimicrobial activity observed could be as a result of the peptides, YFGAP and SJGAP, acting as Anti-Alzheimer’s and neuroprotective activity analogs of GAPDH in the sixth step of glycolysis due to Alzheimer’s disease is a kind of neurodegenerative disease high similarities with its N-terminus. characterized by progressive loss of neurons. The preva- Aside from hydrolysates, AMPs have also been success- lence of such degenerative neuro-diseases has increased fully isolated from fish skin mucus as well. The antimicro- with an increase in life expectancy especially as seen in de- bial activity of yellow catfish (Pelteobagrus fulvidraco)skin veloped countries (Choi and Choi 2015). Anti-Alzheimer’s mucus was investigated, and a novel peptide GKLNL disease activity is profiled using β-secretase inhibitory activ- FLSRLEILKLFVGAL was identified and named pelteoba- ity. The enzyme β-secretase along with another enzyme grin (Su 2011). Structural analysis using Schiffer–Edmund- ϒ-secretase generate a peptide amyloid-β (Aβ)through son helical wheel modeling revealed that pelteobagrin endo-proteolytic reactions of the amyloid precursor protein forms an amphipathic alpha-helix composed of 10 out of (APP) (Choi and Choi 2015). Apolipoprotein enhances the 12 hydrophobic residues on the surface and 4 out of 6 breakdown of beta-amyloid; however, an isoform of apoli- hydrophilic residues on the opposing side. The peptide had poprotein, APOE4, ineffectively breakdowns beta-amyloid a positive charge + 2 and was made up of 60% hydrophobic and leads to an excess amyloid buildup in the brain. The amino acids. It displayed a broad-spectrum antimicrobial peptide Aβ molecules can aggregate to form flexible soluble activity against Gram-positive bacteria, Gram-negative bac- oligomers, some of which turn out misfolded. These mis- teria, and fungi. However, Gram-positive bacteria B. subtilis folded oligomers can induce other Aβ molecules to also was the most sensitive to the peptide at a minimal inhib- take the misfolded oligomeric form (Haass and Selkoe ition concentration (MIC) of 2 μg/mL. The peptide demon- 2007;Nussbaumet al. 2013;Pulawskietal. 2012). strated no hemolytic activity against rabbit red blood cells, Anti-Alzheimer’s and neuroprotective activity of fish skin and it was relatively salt tolerant to concentrations of NaCl hydrolysates is summarized in Table 4.A β-secretase in- up to 137 mM. Similarly, a novel peptide from skin mucous hibitor peptide was purified from skate skin hydrolysate secretions of the winter flounder (Pleuronectes americanus) (Lee et al. 2015). The peptide was purified from a neutrase was purified and characterized (Cole et al. 1997). The hydrolysate of skate skin on a Sephadex G-25 column and peptide was named pleurocidin, and it had an amino acid with reversed-phase HPLC. The peptide sequence was de- sequence of GWGSFFKKAAHVGKHVGKAALTHYL. It termined to be QGYRPLRGPEFL and showed β-secretase exhibited a broad-spectrum activity against a wide range of inhibitory activity with an IC value of 24.26 μM. The neu- Gram-positive and Gram-negative bacteria. Gram-positive roprotective effect of protein hydrolysates with antioxidant bacteria B. subtilis was the most sensitive to the peptide activity from grass carp (Ctenopharyngodon idella)skin with a MIC value of 1.1–2.2 μM. was demonstrated (Cai et al. 2015). The hydrolysates at the A study by Bergsson investigated antimicrobial com- degree of hydrolysis DH5, DH10, and DH15 showed the ponents from the skin mucus of healthy Atlantic cod most significant neuroprotective effect on 6-OHDA- (Gadus morhua) (Bergsson et al. 2005). Results revealed induced neurotoxicity in MES 23.5. Salmon (Oncorhynchus acidic extracts were active against both Gram-positive keta) skin enzymatic hydrolysate showed learning and and Gram-negative bacteria in conditions that likely memory enhancement in mice (Pei et al. 2010). Oxidative mimicked the natural environment of cod. This suggests stress was alleviated, apoptotic neurons reduced, and that the skin mucus layer of the Atlantic cod is an im- brain-derived neurotrophic factor (BDNF) expression was portant tissue in surface defenses of cod and most likely upregulated in treatment groups compared with the con- protects the fish from infections caused by pathogenic trol group. Similarly, another study showed that salmon microbes. Antimicrobial test results revealed Bacillus skin collagen peptides reduced oxidative damage and megaterium as the most sensitive to the extract at all acetylcholinesterase (AChE) while it increased phosphory- concentrations of NaCl. Antimicrobial peptides were lated cAMP-response element binding protein (p-CREB) identified as histone H2B and ribosomal proteins L40, and BDNF expression in mice (Xu et al. 2015). L36A, and L35. Histone-derived peptides originate from both the N-terminus and C-terminus of H1, H2A, H2B, Other biological activities and H6 histones. These histone peptides are found in Other biological activities including antihyperglycemic, the skin, skin mucus, and other tissues, including gills, MMP inhibitory activity, and adipogenic regulatory have the spleen, and the gut. They are produced in response been demonstrated using fish skin as shown in Table 5. to epidermal damage, LPS, or certain Gram-negative Antihyperglycemic activity of fish skin was evaluated Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 10 of 14 Table 4 Anti-Alzheimer’s and neuroprotective activity of peptides purified from fish skin Activity or mechanism Species Peptide sequence Reference β-Secretase inhibitory Skate (Raja kenojei) QGYRPLRGPEFL Lee et al. (2015) Anti-acetylcholinesterase Salmon (Oncorhynchus keta) – Xu et al. (2015) Neuroprotection Grass carp (Ctenopharyngodon idella) – Cai et al. (2015) Learning and memory Salmon (Oncorhynchus keta) – Pei et al. (2010) using dipeptidyl peptidase IV (DPP-IV) inhibitory assay. lowered blood glucose levels of diabetic rats after 28-day Steelhead (Oncorhynchus mykiss) skin gelatin hydroly- administration. The normal rats and diabetic rats treated sates were prepared, and the hydrolysate of 4% papain with HSGH showed similar plasma DPP-IV activity range had the highest DPP-IV inhibitory activity 40–45% of 86.6–94.6% while the diabetic rats treated with TSGH (Cheung and Li-Chan 2017). The hydrolysates were had a significantly lower DPP-IV activity of 71.6%. The ef- purified with ultrafiltration to obtain fraction of less than fect of peptides on GLP-1 levels was also evaluated. TSGH 3 kDa. Two fractions showed 42% and 44% DPP-IV in- exhibited the highest increase in the total GLP-1 level hibitory activity showing that the activity was not influ- (27.81 pM) while HSGH and sitagliptin exhibited a com- enced by ultrafiltration as the values of the fractions and parable effect on GLP-1 secretion (23.46–23.81 pM) in dia- the whole hydrolysates were similar. betic rats after 30-day treatment. However, the underlying In a similar study, the DPP-IV inhibitory and glucagon- mechanism of peptide GLP-1 stimulatory activity is not like peptide-1 (GLP-1) stimulating activity of fish skin gel- well defined. However, it is has been proposed that the atin from various warm- and cold-water fish skins were presence of amino acids leucine and glutamic acid can in- evaluated and compared (Wang et al. 2015). Results duce GLP-1 secretion (Chen and Reimer 2009;Reimer revealed that the DPP-IV inhibitory activity of gelatin hy- 2006). Gelatin hydrolysates of warm-water fish skins drolysates from warm-water fish was greater than that from (TSGH) exhibited greater in vitro and in vivo DPP-IV cold-water fish. Halibut and tilapia skin gelatin hydrolysate inhibitory activity in comparison to cold-water fish (HSGH and TSGH) fractions at a cutoff of < 1.5 kDa UF skins (HSGH). TSGH contained higher imino acid were used for peptide sequence identification and to com- contents which resulted in increased antihyperglyce- pare the in vivo antihyperglycemic effect. MS/MS spectra mic activity in STZ-induced diabetic rats. analysis revealed amino acid sequences of 6 active peptides Fish skin hydrolysates have also been demonstrated to as SPGSSGPQGFTG, GPVGPAGNPGANGLN, PPGPTGP show MMP-1 inhibitory activity and thus have great po- RGQPGNIGF, IPGDPGPPGPPGP, LPGERGRPGAPGP, tential use as cosmeceuticals. Two active peptides from and GPKGDRGLPGPPGRDGM. All these peptides pos- cod skin gelatin hydrolysates (CGH) with anti-photoaging sessed the amino acid proline as the second N-terminal activity were identified (Lu et al. 2017). The peptides were residue. Moreover, it has been reported that peptides with purified from CGH by ion exchange chromatography and DPP-IV inhibitory activity have amino acids proline, trypto- RP-HPLC. The peptide sequences were determined using phan, alanine, valine, lysine, and aspartate as the second QTOF mass spectrometer as EIGPSGGRGKPGKDG- N-terminal residues in their sequences (Lacroix and DAGPK and GFSGLDGAKGD. The purified peptides had Li-Chan 2012). The IC values against DPP-IV of the puri- a MMP-1 inhibitory activity of 16% and 15% respectively. fied peptides ranged from 65.4 to 146.7 μM, and these were The activity of the peptide GFSGLDGAKGD was achieved comparable to the peptides from other proteins with the through the downregulation of MMP-1, p-ERK, and IC values between 41.9 and 174 μM(Huangetal. 2012; p-p38 while GEIGPSGGRGKPGKDGDAGPK activity was Lacroix and Li-Chan 2014; Silveira et al. 2013). Halibut and by the downregulation of p-JNK in MAPK signaling path- tilapia skin gelatin hydrolysates (HSGH and TSGH) ways. A study by Chen et al. 2016 purified 23 polypeptides Table 5 Antihyperglycemic and MMP inhibitory activity of peptides purified from fish skin Activity or mechanism Species Peptide sequence Reference Antihyperglycemic Halibut (Hippoglossus stenolepis) SPGSSGPQGFTG, GPVGPAGNPGANGLN, Wang et al. (2015) PPGPTGPRGQPGNIGF Tilapia (Oreochromis niloticus) IPGDPGPPGPPGP, LPGERGRPGAPGP, GPKGDRGLPGPPGRDGM MMP inhibitory activity Cod EIGPSGGRGKPGKDGDAGPK, GFSGLDGAKGD Lu et al. (2017) Tilapia (Oreochromis niloticus) LSGYGP Sun et al. (2013) Sutchi catfish (Pangasius hypophthalmus) LMWCP Pyun et al. (2012) Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 11 of 14 from cod skin hydrolysates. Amino acid sequences of palmitate-induced intracellular lipid vacuole accumula- Gly-Po and Gly-Leu and the amino acid arginine predom- tion visualized by Nile red staining. The palmitate- inated at the C-terminus of the polypeptides. The hydroly- induced intracellular lipid vacuole accumulation was sates showed a protective effect against UV-induced greatly reduced in the presence of 1 mg/ml SWFCP. photo-damage to collagen. Expression and activities of SWFCP significantly affected other obesity-related fac- matrix metalloproteinases (MMP-1, MMP-3, MMP-9) tors like low serum cholesterol, low serum triglyceride, were downregulated through the elevation of tissue inhibi- and low-density lipoprotein; high serum high-density tor of matrix metalloproteinases (TIMPs) and suppression lipoprotein levels; and reduced size of epididymal of the activation of mitogen-activated protein kinase adipocytes. (MAPK) signaling pathway in the skin of mice treated with the hydrolysate. In another study, the anti- Conclusion photoaging effect of a peptide LSGYGP purified from As elaborately discussed in this review, biologically ac- tilapia skin (Oreochromis niloticus) was evaluated by Sun tive peptides have been effectively produced through the et al. 2013. In vivo experiments showed that the peptide bioconversion of fish skin. These different peptides with improved the skin condition of UV irradiation-induced a range of bioactivities such as antihypertensive, antioxi- photoaging mice through its antioxidant activity. Further- dants, antimicrobial, neuroprotection, antihyperglyce- more, the mechanism of action of the same peptide mic, and anti-aging have been demonstrated in vitro LSGYGP was studied using ultraviolet B (UVB)-induced experiments and to an extent in vivo as well. However, mouse embryonic fibroblasts (MEFs) (Ma et al. 2018). The further study is required in the preparation of fish skin peptide reduced the intercellular ROS generation and hydrolysates using the fermentation method for the pro- decreased superoxide dismutase (SOD) activity as well as duction of bioactive peptides. In addition, further study reduced MMP-1 and MMP-9 activities. Molecular dock- is also required, to determine the potential immunomo- ing simulation analysis showed that the peptide inhibited dulation activity of antioxidant peptides, i.e., anti-inflam- MMP activities by docking the active sites of MMP-1 and matory, and anti-allergy and the corresponding cell MMP-9. The anti-photoaging effect of a peptide LMWCP signaling pathways. More investigations are also required purified from catfish skin (Pangasius hypophthalmus) was to determine the neuroprotective effect of fish skin hy- evaluated both in animal models and in a clinical trial. drolysates especially with protection against amyloid- The peptide downregulated the expression of MMP-3 and β-associated neurotoxicity as observed in Alzheimer’s MMP-13, while it upregulated the expression of MMP-2 disease. Nevertheless, bioactive peptides purified from and MMP-9 (Pyun et al. 2012). In the clinical trial results, fish skin can potentially be utilized in the development treatment groups receiving a daily oral dosage of 1000 mg of pharmaceutical and nutraceutical products. of LMWCP for 12 weeks showed significantly improved skin and less wrinkling in comparison with the placebo Abbreviations group (Kim et al. 2018). ABTS: 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); ACE: Angiotensin-converting enzyme; AMPs: Antimicrobial peptides; Last but not least, fish skin has also been demon- APOE4: Apolipoprotein; APP: Amyloid precursor protein; Aβ: Amyloid-β; strated to have adipogenic regulatory activity. The effect BDNF: Brain-derived neurotrophic factor; C/EBP: Ccaat (cytosine-cytosine- of subcritical water-hydrolyzed fish collagen peptide adenosine-adenosine-thymidine)-enhancer-binding proteins; cGK-I: cGMP- dependent protein kinase I; Cgmp: Cyclic guanosine 3′5′monophosphate; (SWFCP) from tuna skin on the protein levels of the DCFH-DA: Dichlorofluorescin diacetate; DMI: 1 μM: Dexamethasone and 1 μg/ master adipogenic transcription factors C/EBP and ml insulin; DMPO: 5,5-Dimethyl-1-pyrroline-N-oxide; DPPH: Diphenyl-2- PPAR was investigated (Lee et al. 2017). This was done picrylhydrazyl; DPP-IV: Dipeptidyl peptidase IV; EMR: Enzymatic membrane reactor; eNOS: Endothelial nitric oxide synthase; ESI-MS: Electrospray with the aim of evaluating the underlying inhibitory ionization mass spectrometry; ET-1: Endothelin-1; FRAP: Ferric-reducing mechanism of SWFCP in the adipogenic differentiation antioxidant power; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; of 3T3-L1 pre-adipocytes. Results revealed that SWFCP HPLC: High-performance liquid chromatography; IP 3: Inositol-1,4,5- trisphosphate; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein downregulated the expression of the key adipogenic kinase; MMP: Matrix metalloproteinase (MMP-1, MMP-3, MMP-9); NO: Nitric target gene and transcription factors in 3T3-L1 oxide; ORAC: Oxygen radical absorbance activity; PPAR-γ: Peroxisome pre-adipocytes exposed to MDI. After 8 days of incuba- proliferator-activated receptor gamma; PUFAs: Polyunsaturated fatty acids; RAS: Renin–angiotensin–aldosterone system; RNS: Reactive nitrogen species; tion of 3T3-L1 cells with 1 μM dexamethasone and 1 μg/ ROS: Reactive oxygen species; RP-HPLC: Reverse phase high-performance li- ml insulin (MDI) and SWFCP, the expression levels of quid chromatography; SDS/PAGE: Sodium dodecyl sulfate polyacrylamide gel C/EBP and PPAR protein were greatly reduced com- electrophoresis; SHR: Spontaneously hypertensive rats; TIMPs: Tissue inhibitor of matrix metalloproteinase pared with cells stimulated with MDI alone. SWFCP was also shown to downregulate the expression of aP2 an Acknowledgements adipogenic target gene, hence inhibiting adipogenic dif- This research was supported by the Marine Biotechnology Program of the ferentiation. Furthermore, SWFCP reduced lipogenesis Korea Institute of Marine Science and Technology Promotion (KIMST) funded in hepatocytes. This was demonstrated by the use of by the Ministry of Oceans and Fisheries (MOF) (No. 20140441). Abuine et al. Fisheries and Aquatic Sciences (2019) 22:10 Page 12 of 14 Funding Chen T, Hou H, Fan Y, Wang S, Chen Q, Si L, et al. Protective effect of gelatin The design of the study; collection, analysis, and interpretation of the data; peptides from pacific cod skin against photoaging by inhibiting the and writing of the manuscript were funded by a grant from the Ministry of expression of MMPs via MAPK signaling pathway. J Photochem Photobiol B Oceans and Fisheries (MOF) (No. 20140441). Biol. 2016;165:34–41. Cheung HS, Wang FL, Ondetti MA, Sabo EF, Cushman DW. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of Availability of data and materials the COOH-terminal dipeptide sequence. J Biol Chem. 1980;255:401–5. Not applicable. Cheung IWY, Li-Chan ECY. 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Fisheries and Aquatic SciencesSpringer Journals

Published: May 23, 2019

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