Abstract

INTRODUCTION

In biological systems, free radicals are typically derived from the oxygen, nitrogen, and sulfur molecules. Due to their unpaired electrons, free radicals exhibit a great deal of combination desire for reaction with other molecules. The most important free radical including Reactive Oxygen and Nitrogen Species (ROS/RNS) are naturally produced by various metabolic pathways such as the aerobic metabolism in mitochondrial respiratory chain, and play numerous physiological roles such as intracellular signaling, regulation of cell proliferation and apoptosis, induction of gene expression, and ion transferring (Sarmadi, Ismail, 2010Sarmadi BH, Ismail A. Antioxidative peptides from food proteins: a review. Peptides. 2010;31(10):1949-1956.).

However, excessive production of these compounds under certain conditions can exert harmful effects by causing oxidative damage to important cellular structures. The ROS and RNS radicals react with nucleic acids, the side chains of amino acid in proteins, and double bonds of unsaturated fatty acids, triggering and developing oxidative stress, which plays an important role in the pathogenesis of many human diseases, including cancer (Nikoo, Benjakul, 2015Nikoo M, Benjakul S. Potential application of seafood-derived peptides as bifunctional ingredients, antioxidant-cryoprotectant: A review. J Funct Foods. 2015;19(Part A):753-764.). ROS contribute to tumor development and progression through two possible pathways inducing mutation of key gene and/or alterations of signaling and transcriptional pathways. When the cell with oxidized or otherwise modified DNA is divided, its metabolism and proliferation are impaired and a mutation occurs which is an important factor for carcinogenesis. In addition, products of lipid peroxidation can react with metal ions and produce active compounds, such as epoxide and aldehyde, which cause mutations in the DNA of cells (Noda, Wakasugi, 2001Noda N, Wakasugi H. Cancer and oxidative stress. Jp Med Assoc J. 2001;44(12):535-539.).

Today, researchers have proven that daily diets play an important role in preventing, developing, and treating various types of cancers. The consumption of foods rich in natural antioxidants, such as vitamins E and C, can prevent the development of certain cancers by inhibition of free radicals and ROS (Terry et al., 2000Terry P, Lagergren J, Ye W, Nyrén O, Wolk A. Antioxidants and cancers of the esophagus and gastric cardia. Int J Cancer. 2005;87(5):750-754.; Venugopal, 2008Venugopal V. Marine products for healthcare: functional and bioactive nutraceutical compounds from the ocean. Boca Raton: CRC Press; 2008.). Increased concerns about the association between health and diet have led to growing consumer demand for the health-promoting and functional foods. Functional foods are defined as food products that provide health benefits in addition to meeting basic nutritional needs of the body (Shahidi, Alasalvar, 2011Shahidi F, Alasalvar C. Marine oils and other marine nutraceuticals. Handbook of seafood quality, safety and health applications. Oxford: Wiley-Blackwell; 2011, p. 444-454.). The global functional foods market size was 299 billion dollars in 2017 and is expected to reach 441 billion dollars in 2022 (Menrad, 2003Menrad K. Market and marketing of functional food in Europe. J Food Eng. 2003;56(2-3):181-188.).

Marine animals, that comprise about half of the world’s biodiversity, provide a valuable source of bioactive and functional compounds. Some of these compounds have a proteinaceous nature and includes proteins, peptides, and amino acids. Marine animals, in addition to being an important source of high-quality protein, are also used as the raw material for production of physiologically important peptides (Raghavan, Kristinsson, Leeuwenburgh, 2008Raghavan S, Kristinsson HG, Leeuwenburgh C. Radical scavenging and reducing ability of tilapia (Oreochromis niloticus) protein hydrolysates. J Agric Food Chem. 2008;56(21):10359-10367.). Bioactive peptides are specific protein fragments which remain inactive within the sequence of their parent protein until released by enzymatic hydrolysis (Harnedy, FitzGerald, 2012Harnedy PA, FitzGerald RJ. Bioactive peptides from marine processing waste and shellfish: A review. J Funct Foods. 2012;4(1):6-24.). Bioactive peptides derived from marine animal using enzymatic hydrolysis exhibit numerous physiological functions such as immunomodulatory, antimicrobial, anxiolytic, and hypotensive activity (Giri, Ohshima, 2012Giri A, Ohshima T. Bioactive marine peptides: Nutraceutical value and novel approaches. Adv Food Nutr Res. 2012;65:73-105.; Kumar, Nazeer, Jaiganesh, 2011Kumar NS, Nazeer R, Jaiganesh R. Purification and biochemical characterization of antioxidant peptide from horse mackerel (Magalaspis cordyla) viscera protein. Peptides. 2011;32(7):1496-1501.).

According to the FAO, the total catch amount of Mugilidae species from southern and southwestern waters of Iran was 9300 tons in 2017 and Klunzinger’s mullet (Liza klunzingeri) capture comprise about 2950 tons of this amount (FAO, 2016FAO. Global Aquaculture Production Statistics. 2016.). Klunzinger’s mullet is an inexpensive and low-value fish due to its small size and the presence of a dark brown to black peritoneum (Kiabi, Abdoli, Naderi, 1999Kiabi BH, Abdoli A, Naderi M. Status of the fish fauna in the South Caspian Basin of Iran. Zool Midd East. 1999;18(1):57-65.). The use of Klunzinger’s mullet for the production of protein hydrolysates provide added value and allows the optimal use of marine resources that are decreasing. In this study, L. klunzingeri muscle protein was hydrolysed by papain and the antioxidant and cytotoxic effects of protein hydrolysates were studied in vitro. In addition, the molecular weight and amino acid sequence of the hydrolysate with the highest antioxidant activity was determined using HPLC.

MATERIAL AND METHODS

Material

2,2’-azinobios-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferrozine (3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4′′-disulfonic acid sodium salt), Dimethyl sulfoxide (DMSO), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), Butylated hydroxytoluene (BHT), and Trypsin-EDTA solution were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Other chemicals were purchased from Merck Co. (Germany) and were of analytical grade. Papain from papaya latex (1.5-10 units/mg Solid, pH 6.0, 40 °C) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The enzyme was stored at 4 °C until use.

Methods

L. klunzingeri proximate chemical composition: Fresh L. klunzingeri was provided from the fish market and transferred to the laboratory in ice. The fish were first washed and filleted, and the fillets were then minced and stored at -20 °C until the experiments. In order to determine the moisture content, 2 g of fish mince were dried in an oven at 105 °C to reach constant weight, the moisture content was calculated by measuring the weight loss following heating. Ash content was determined by complete oxidation of organic matter at 550-600 °C in a furnace. The nitrogen of the samples was determined via the Kjeldahl method. Crude protein was calculated by multiplying the determined nitrogen content by a nitrogen-to-protein conversion factor (× 6.25). Fat content of sample was determined by AOAC Soxhlet procedures (AOAC, 1995AOAC. Official Method of Analysis. 16th Ed. Association of Official Analytical Chemists. Washington, USA; 1995.).

Preparation of protein hydrolysates

Samples of L. klunzingeri mince (50 g) was placed in an Erlenmeyer flask and then 100 mL of phosphate buffer (pH 6) was added to keep the pH constant throughout the incubation time. In order to inactivate the endogenous enzymes, the samples were heated in a water bath at 85 °C for 20 min. After cooling, samples were hydrolyzed using papain (enzyme to substrate ratio of 1:50 and 1:25) for 45, 90 and 180 at 55°C. The hydrolysis was performed using 250 mL glass vessels inside a shaking water bath (SWB 15 Precision). After the incubation time, samples were heated at 95 °C during 15 min to stop the enzymatic reaction. After cooling at room temperature for 15 min, the samples were centrifuged (8000 ×g at 10 °C for 30 min). After removing the surface oil using a micropipette, the supernatant was collected and freeze-dried at -50 ºC under vacuum (Labconco Freeze Dryer, USA). Obtained protein hydrolysates were named FPH_2-45 (E/S ratio of 1:50 and time of 45 min), FPH_2-90 (E/S ratio of 1:50 and time of 90 min), FPH_2-180 (E/S ratio of 1:50 and time of 180 min), FPH_4-45 (E/S ratio of 1:25 and time of 45 min), FPH_4-90 (E/S ratio of 1:25 and time of 90 min) and FPH_4-180 (E/S ratio of 1:25 and time of 180 min) and stored at -20 ºC until further analysis.

DPPH^* * 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity

BHT and FPHs at different concentrations were prepared in distilled water. Then, 1 mL of sample solution was added to 1 mL of 0.1 mM DPPH solution (prepared in 95% ethanol) and the absorbance of the mixture was recorded at 517 nm after 20 min of incubation in dark. The control was prepared using 1 mL of distilled water instead of sample. DPPH radical scavenging activity was expressed as percentage of inhibition using the following equation: %DPPH radical scavenging activit y = [(Ac-As)/Ac] ×100. Where Ac is the absorbance of control and As is the absorbance of the sample. The effective concentration of sample required to inhibits 50% of the DPPH radical (IC₅₀ value) was obtained by plotting a graph of concentration (X axis) versus percentage of inhibition (Y-axis) (Nikoo et al., 2014Nikoo M, Benjakul S, Ehsani A, Li J, Wu F, Yang N, Xu X. Antioxidant and cryoprotective effects of a tetrapeptide isolated from Amur sturgeon skin gelatin. J Funct Foods. 2014;7:609-620.).

Fe²⁺ chelating activity

One mL of BHT or FPHs solution at different concentrations was mixed with 0.1 mL of 2 mM FeCl₂ and 0.2 mL of 5 mM ferrozine and the final volume of the mixture was increased to 5 mL with addition of distilled water. After 20 min of incubation, the absorbance was recorded at 562 nm. For control sample, distilled water was used instead of the sample. Fe²⁺ chelating activity was calculated using the following formula. Fe²⁺ chelating activity (%) = [(Ac- As)/Ac] ×100 where Ac is the absorbance of control and As is the absorbance of the sample. IC₅₀ value was calculated from the plot of the chelating activity against the sample concentration (Nikoo et al., 2014Nikoo M, Benjakul S, Ehsani A, Li J, Wu F, Yang N, Xu X. Antioxidant and cryoprotective effects of a tetrapeptide isolated from Amur sturgeon skin gelatin. J Funct Foods. 2014;7:609-620.).

Ferric reducing activity

A volume of 2 mL of protein hydrolysate (5 mg/mL) or BHT (0.5 mg/mL) was mixed with 2 mL of phosphate buffer (0.2 M, pH 6.6) and 2 mL of 1% potassium ferricyanide. After incubation at 50 °C for 20 min, 2 mL of 10% Trichloroacetic acid (TCA) was added to the mixture. Following centrifugation at 3000 rpm for 10 min, 2 mL of the supernatant was mixed with 2mL of distilled water and 0.5 mL of 0.1% FeCl₃. Then the optical absorbance was recorded at 700 nm (Nikoo et al., 2014Nikoo M, Benjakul S, Ehsani A, Li J, Wu F, Yang N, Xu X. Antioxidant and cryoprotective effects of a tetrapeptide isolated from Amur sturgeon skin gelatin. J Funct Foods. 2014;7:609-620.).

ABTS^** ** 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity

The stock solution was prepared by mixing 7.4 mM ABTS⁺ and 2.6 mM potassium persulfate solution (1:1) and left to incubate for 12 h at room temperature in the dark. Before the experiment, freshly prepared ABTS solution was diluted with methanol to reach an absorbance of 1.1 ± 0.02 at 734 nm. Then, 150 µL of FPHs or BHT at different concentrations was mixed with 2850 µL of ABTS solution, and after incubation at room temperature for 2 h, the optical absorbance was recorded. Control sample was prepared using 150 µL of distilled water instead of sample. ABTS scavenging activity was determined using the following formula. ABTS scavenging activity (%) = [(Ac-As) / Ac × 100]; where Ac is the absorbance of control and As is the absorbance of the sample. IC₅₀ value was determined from the plot of the scavenging activity against the sample concentration (Nikoo et al., 2014Nikoo M, Benjakul S, Ehsani A, Li J, Wu F, Yang N, Xu X. Antioxidant and cryoprotective effects of a tetrapeptide isolated from Amur sturgeon skin gelatin. J Funct Foods. 2014;7:609-620.).

Hydroxyl radical scavenging activity

Briefly, 1, 10-phenanthroline solution (1.865 mM, 1 mL) and FPHs or BHT at different concentrations were added into a tube and mixed. Then, 1mL of the FeSO₄ solution (1.865 mM) was added to the mixture and the reaction was initiated by adding 1 mL of H₂O₂ (3% v/v). After incubation at 37 °C for 60 min in a water bath, the absorbance was recorded at 536 nm. Solution containing protein hydrolysis without hydrogen peroxide was considered as Blank and solution without protein hydrolysis was considered negative control. Hydroxyl radical scavenging activity was determined using the following formula. Hydroxyl radical scavenging activity (%) = [(As-An)/( Ab-An)]×100; where As is the absorbance of sample, An is the absorbance of the negative control and Ab is the absorbance of blank. IC₅₀ value was determined from the plot of the scavenging activity against the sample concentration (Nikoo et al., 2014Nikoo M, Benjakul S, Ehsani A, Li J, Wu F, Yang N, Xu X. Antioxidant and cryoprotective effects of a tetrapeptide isolated from Amur sturgeon skin gelatin. J Funct Foods. 2014;7:609-620.).

Evaluation of cytotoxic effects

MTT is a yellow water-soluble tetrazolium salt. It is reduced in the mitochondria of viable cells to generate a water-insoluble formazan salt. The MTT assay is a colorimetric method that evaluates the activity of cellular enzymes, in which yellow tetrazolium is converted to purple formazan. The assay is used to evaluate the proliferation of cells and the cytotoxic effects of drugs. 4T₁ carcinoma cells line was purchased from National Cell Bank of Iran (Pasteur Institute., Tehran, Iran) and cultured in DMEM medium containing 10% FBS. After reaching around 80% confluence, they were detached by trypsin/EDTA and the number of cells was counted using a homocytometric lam. Then, 200 µL of suspension containing 15 × 10³ cells was added to each well of a 96-well plate. In the next step, the cells were treated with FPHs/carboplatin at different concentration for 48 hours. After removing the medium of the well and washing by PBS, 60 µl of MMT solution in PBS was added to each well. The cells were then incubated at 37 °C in 5% CO₂ for 4 hours. After incubation, the medium was removed from the wells and 150 µL of DMSO was added to each well. The plate was then incubated for 30 minutes at 37 °C in the dark. Finally, the plates’ absorbance was read at 570 nm using an ELISA reader. The percentage of cytotoxicity was calculated by the formula below:

IC₅₀ value determined from the plot of the scavenging activity against the sample concentration

Determination of molecular-weight distribution

The molecular weight distribution of the hydrolysate with the highest antioxidant activity was determined by gel permeation chromatography using HPLC system (Agilent 1100, USA). A TSK gel 2000 SWXL (300 × 7.8 mm) column (Tosoh, Tokyo, Japan) was equilibrated with acetonitrile: water (40:60, v/v) in the presence of 0.1% trifluoroacetic acid (TFA). The absorbance was monitored at 225 nm with flow rate of 0.5 mL/min. Cytochrome C (12384 Da), bacitracin (1422 Da), Gly-Gly-Try-Arg (451 Da), and Gly-Gly-Gly (189 Da) were used as protein molecular weight standards. The logarithm of molecular weight tested and the respective retention time was shown in a linear relationship. The equation ∑ (Mn × Ai)/100 was used to calculate the average molecular weight of sample (Guo et al., 2013Guo G, Hou H, Li B, Zhang Z, Wang S, Zhao X. Preparation, isolation and identification of iron-chelating peptides derived from Alaska pollock skin. Process Biochem. 2013;48(1):988-993.).

Determination of the amino acid composition

Amino acids were determined according to the AOAC method with some modifications. One hundred and twenty milligrams of the hydrolysate powder were digested with 8 mL of 6 M HCl at 110 °C for 22 hours under nitrogen atmosphere. After cooling, 4.8 mL of 10 M NaOH was added, the volume was made up to 25 mL with distilled water, then filtered through two layers of filter paper No. 40, and finally centrifuged at 10,000g for 10min. Amino acids were analyzed by using the reverse-phase high performance liquid chromatography (Agilent 1100 HPLC; Agilent Ltd., Palo Alto, CA, USA). Each sample (1 µL) was injected into a Zorbax, 80A C-18 column (column size: 4.0 × 250 mm, 5 µm particle size; Agilent, USA) at 40 °C with detection at 338nm. The mobile phase A was 7.35 mM/L of sodium acetate/triethylamine/tetrahydrofuran (500:0.12:2.5, v/v/v), adjusted to pH 7.2 using acetic acid, while the mobile phase B (pH 7.2) was 7.35 mM/L of sodium acetate/methanol/ acetonitrile (1:2:2, v/v/v). The amino acid composition was expressed as grams of amino acids per 100 g of protein (Guo et al., 2013Guo G, Hou H, Li B, Zhang Z, Wang S, Zhao X. Preparation, isolation and identification of iron-chelating peptides derived from Alaska pollock skin. Process Biochem. 2013;48(1):988-993.).

Statistical analysis

Data were analyzed using SPSS version 20. Analysis of Variance (ANOVA) followed by Duncan’s test used to identify statistical differences between means. All data were presented as mean ± SD and p value less than 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Figures 1-3 show the antioxidant activities of BHT and FPHs made by enzymatic hydrolysis of L. klunzingeri muscle protein. As shown BHT showed strong DPPH, ABTS and hydroxyl radicals scavenging activities (IC50 values of 0.047±0.01, 0.021±0.009 and 0.24±0.02 mg/mL respectively), Fe²⁺ chelating capacity (IC50 value of 0.057±0.01 mg/mL) and ferric reducing activity (optical absorbance of 1.16±0.09 at 700 nm wavelength). Protein hydrolysates obtained by enzymatic hydrolysis of L. kludingeri muscle using two concentration of papain at 45, 90, and 180 min, exhibited good scavenging activity on DPPH (IC₅₀=2.08-3.18 mg/mL), ABTS (IC₅₀=0.12-0.60 mg/mL), and hydroxyl (IC₅₀= 2.07-4.13 mg/mL) radicals, moderate chelating activities on Fe²⁺ (IC₅₀=2.12-12.60 mg/mL), and relatively poor ferric reducing activities (optical absorbance of 0.01-0.15 at 700 nm wavelength). With increasing the hydrolysis duration and the concentration of papain, the antioxidant activities of the FPHs in inhibiting the DPPH, ABTS, and hydroxyl radicals were increased, so that the highest inhibitory activity obtained for the FPH_4-180 with IC₅₀ values of 2.08 ± 0.13, 0.12 ± 0.01, and 2.07 ± 0.31 mg/mL, respectively. Fe²⁺- chelating activity of FPHs decreased with increasing the hydrolysis duration and FPH₄-₁₈₀ sample with the highest inhibitory activity on the ABTS, DPPH, and hydroxyl radicals showed the lowest Fe²⁺-chelating activity (IC₅₀= 12.60 ± 0.02). As shown in figure 3, the increase of hydrolysis time increased ferric reducing activity and therefore, FPHs obtained after 90 and 180 min of hydrolysis demonstrated better activities than sample obtained after 45 min of hydrolysis.

The analysis of the molecular weight distribution of the hydrlysate with the highest antioxidant activity (FPH_4-180) by using HPLC, showed that 95% of the peptides in this sample had a molecular weight of less than 1000 Da. 30.56% of peptides in this sample had molecular weight of less than 180 Da, 47.26% had molecular weight of 180-500 Da, and 17.46% had molecular weight of 500-1000 Da (Figure 4).

It seems that the higher activity of FPH_4-180 sample in inhibition of DPPH, ABTS, and hydroxyl radicals is due to the presence of low-molecular-weight peptides, which increased with increasing hydrolysis duration. Several studies have suggested that high degrees of hydrolysis and low molecular weight have a positive correlation with the DPPH and ABTS radical scavenging activity (Bougatef et al., 2010Bougatef A, Nedjar-Arroume N, Manni L, Ravallec R, Barkia A, Guillochon D, Nasri M. Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinella aurita) by-products proteins. Food Chem. 2010;118(3):559-565.; Liu et al., 2010Liu Q, Kong B, Xiong YL, Xia X. Antioxidant activity and functional properties of porcine plasma protein hydrolysate as influenced by the degree of hydrolysis. Food Chem. 2010;118(2):403-410.; Phanturat et al., 2010Phanturat P, Benjakul S, Visessanguan W, Roytrakul S. Use of pyloric caeca extract from bigeye snapper (Priacanthus macracanthus) for the production of gelatin hydrolysate with antioxidative activity. LWT-Food Sci Technol. 2010;43(1):86-97.; Raghavan, Kristinsson, 2008Raghavan S, Kristinsson HG. Antioxidative efficacy of alkali-treated tilapia protein hydrolysates: a comparative study of five enzymes. J Agric Food Chem. 2008:56(4):1434-1441.), although some studies have reported an inverse relationship (Alemán et al., 2011aAlemán A, Giménez B, Pérez-Santin E, Gómez-Guillén M, Montero P. Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate. Food Chem. 2011a;125(2):334-341.; Theodore, Raghavan, Kristinsson, 2008Theodore AE, Raghavan S, Kristinsson HG. Antioxidative activity of protein hydrolysates prepared from alkaline-aided channel catfish protein isolates. J Agric Food Chem. 2008;56(16):7459-7466.).

Also, in the present study, the Fe²⁺ chelating activities of the FPH_S showed the opposite trend and FPH_4-180 had the lowest Fe²⁺-chelating activity. In a study by Pownall, Udenigwe and Aluko (2010)Pownall TL, Udenigwe CC, Aluko RE. Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions. J Agric Food Chem. 2010;58(8):4712-4718., the pea seed protein hydrolysate, which showed the highest Fe²⁺-chelating activity, had a poor inhibitory effect on the ABTS, DPPH, hydroxyl, and hydrogen peroxide radicals, which is consistent with our results (Pownall, Udenigwe, Aluko, 2010Pownall TL, Udenigwe CC, Aluko RE. Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions. J Agric Food Chem. 2010;58(8):4712-4718.). Alemán et al. (2011b)Alemán A, Pérez-Santín E, Bordenave-Juchereau S, Arnaudin I, Gómez-Guillén M, Montero P. Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity. Food Res Int. 2011b;44(4):1044-1051. argued that squid gelatin hydrolysates with a higher degree of hydrolysis and a lower molecular weight, exhibited better Fe²⁺-chelating activity (Alemán et al., 2011bAlemán A, Pérez-Santín E, Bordenave-Juchereau S, Arnaudin I, Gómez-Guillén M, Montero P. Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity. Food Res Int. 2011b;44(4):1044-1051.), while Bamdad, Wu and Chen (2011)Bamdad F, Wu J, Chen L. Effects of enzymatic hydrolysis on molecular structure and antioxidant activity of barley hordein. J Cereal Sci. 2011;54(1):20-28. reported that peptides with a higher molecular weight exhibited higher Fe²⁺-chelating activity, which is due to the trapping of iron ions in the peptide chain (Bamdad, Wu, Chen, 2011Bamdad F, Wu J, Chen L. Effects of enzymatic hydrolysis on molecular structure and antioxidant activity of barley hordein. J Cereal Sci. 2011;54(1):20-28.).

Furthermore, we found that the ferric reducing activities of FPHs increased with the increasing the time of hydrolysis and therefore breakdown of large peptides into smaller peptide units. Contradictory results have been reported regarding molecular weight relationship with ferric reducing activity, some of which, in agreement with the present study, have shown an inverse correlation between ferric reducing activity and molecular weight (Alemán et al., 2011aAlemán A, Giménez B, Pérez-Santin E, Gómez-Guillén M, Montero P. Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate. Food Chem. 2011a;125(2):334-341.), and others have indicated a positive correlation (Theodore et al., 2008Theodore AE, Raghavan S, Kristinsson HG. Antioxidative activity of protein hydrolysates prepared from alkaline-aided channel catfish protein isolates. J Agric Food Chem. 2008;56(16):7459-7466.).

The inconsistencies in the findings of various studies suggest that molecular weight is not the main determinant of the antioxidant activity of the protein hydrolysate and peptide samples. It has been reported that the antioxidant properties of the bioactive peptides depends on their size of peptides and also amino acid sequences, which are influenced by the source of substrate protein, type of enzyme used, enzyme to substrate ratio and hydrolysis conditions (temperature, pH and time) (Harnedy, FitzGerald, 2012Harnedy PA, FitzGerald RJ. Bioactive peptides from marine processing waste and shellfish: A review. J Funct Foods. 2012;4(1):6-24.).

In this study, the most abundant amino acids in FPH₄-₁₈₀ sample were serine (9.593%), tyrosine (8.43%), cysteine (7.197%), valine (6.60%), histidine (5.81%), and glutamine (4.914%) (Table I). It is reported that aromatic amino acids (phenylalanine, tryptophan and tyrosine) donate the electron to free radicals and make them stable molecules (Sarmadi, Ismail, 2010Sarmadi BH, Ismail A. Antioxidative peptides from food proteins: a review. Peptides. 2010;31(10):1949-1956.). Amino acids, such as histidine, leucine, tyrosine, methionine, and cysteine, neutralize free radicals by donating proton (Mendis et al., 2005Mendis E, Rajapakse N, Byun H-G, Kim SK. Investigation of jumbo squid (Dosidicus gigas) skin gelatin peptides for their in vitro antioxidant effects. Life Sci. 2005;77(17):2166-2178.), fat-soluble free radicals (peroxyl radicals) that are produced throughout the oxidation of unsaturated fatty acids are neutralized by hydrophobic amino acids such as leucine, valine, alanine, and proline (Kim, Mendis, 2006Kim SK, Mendis E. Bioactive compounds from marine processing byproducts-a review. Food Res Int. 2006;39(4):383-393.). Thus, it can be argued that natural protein hydrolysates may have inhibitory effects on several types of free radicals due to the presence of various amino acids, while purified peptide from a protein hydrolysate that contains fewer types of amino acids may exert low inhibitory effect on some free radicals. In the study on fractions derived from Cod protein hydrolysates, it was observed that the isolation of different fractions with strong DPPH scavenging effect resulted in a decrease of ferric reducing activity, which was due to an increase in the ratio of positively charged amino acids to sulfur-containing amino acids (Girgih et al., 2015Girgih AT, He R, Hasan FM, Udenigwe CC, Gill TA, Aluko RE. Evaluation of the in vitro antioxidant properties of a cod (Gadus morhua) protein hydrolysate and peptide fractions. Food Chem. 2015;173:652-659.).

As shown in Figure 5, the FPHs obtained from enzymatic hydrolysis of L. klunzingeri showed significant cytotoxic activities (IC₅₀=1.62-2.61 mg/mL) on 4T₁ breast cancer cell line. The cytotoxic activities of samples decreased with increasing the hydrolysis duration and FPH₄-₄₅ sample that was hydrolysed for a shorter period showed the highest cytotoxic activity (IC₅₀= 1.62 ± 0.10 mg/mL, Figure 5). In the study of Picot et al. (2006)Picot L, Bordenave S, Didelot S, Fruitier-Arnaudin I, Sannier F, Thorkelsson G, Piot J. Antiproliferative activity of fish protein hydrolysates on human breast cancer cell lines. Process Biochem. 2006;41(5),1217-1222. the cytotoxic effects of protein hydrolysates of 18 fish species were determined on MCF-7/6 and MDA-MB-231 cancer cells, and the highest cytotoxic effect (up to 40%) was exhibited by the Cod protein hydrolysate (1 mg/kg, 72 h) on the MCF-7 cell line (Picot et al., 2006Picot L, Bordenave S, Didelot S, Fruitier-Arnaudin I, Sannier F, Thorkelsson G, Piot J. Antiproliferative activity of fish protein hydrolysates on human breast cancer cell lines. Process Biochem. 2006;41(5),1217-1222.). Tuna muscle protein hydrolysate also showed a significant inhibitory effect on the MCF-7 cell line, and the highest inhibitory activity was obtained for the fraction with 390-1000 Da molecular weight (Hsu, Li-Chan, Jao, 2011Hsu KC, Li-Chan EC, Jao CL. Antiproliferative activity of peptides prepared from enzymatic hydrolysates of tuna dark muscle on human breast cancer cell line MCF-7. Food Chem. 2011;126(2):617-622.). In a study, fraction with low MW peptides (<3 kDa) isolated from Loach protein hydrolysate showed better cytotoxicity than fractions with high MW peptides (3-5 kDa, 5-10 kDa and > kDa) (Zhao, Liu, Regenstein, 2011). However, some high MW peptides derived from buckwheat seeds (approximately 4 kDa) (Leung, Ng, 2007Leung EH, Ng T. A relatively stable antifungal peptide from buckwheat seeds with antiproliferative activity toward cancer cells. J Peptide Sci. 2007;13(11):762-7.) and soybean protein hydrolysate (> 10 kDa) were also reported to show significant cytotoxicity (Marcela et al., 2016Marcela GM, Eva RG, del Carmen RRM, Rosalva ME. Evaluation of the antioxidant and antiproliferative effects of three peptide fractions of germinated soybeans on breast and cervical cancer cell lines. Plant Foods Hum Nutr. 2016;71(4):368-74.). Therefore, some researchers have claimed that the cytotoxicity of the peptides depends not only on the chain length, but also amino acid sequences, which are influenced by the source of substrate protein, type and amount of enzyme used, and hydrolysis conditions (Alemán et al., 2011bAlemán A, Pérez-Santín E, Bordenave-Juchereau S, Arnaudin I, Gómez-Guillén M, Montero P. Squid gelatin hydrolysates with antihypertensive, anticancer and antioxidant activity. Food Res Int. 2011b;44(4):1044-1051.; Picot et al., 2006Picot L, Bordenave S, Didelot S, Fruitier-Arnaudin I, Sannier F, Thorkelsson G, Piot J. Antiproliferative activity of fish protein hydrolysates on human breast cancer cell lines. Process Biochem. 2006;41(5),1217-1222.). In the present study, 64.72% of peptide in FPH_4-180 sample had molecular weight distribution less than 500 Da. It seems that the molecular weight of peptides and therefore the cytotoxic activity were reduced compared to the samples hydrolysed at shorter time.

Table II shows the proximate composition of L. klunzingeri muscle and protein hydrolysate (with highest antioxidant activity, FPH₄-₁₈₀ sample). The protein contents of L. klunzingeri muscle and protein hydrolysate were 87.84 ± 2.85% and 22.45 ± 3.39%. This indicates that protein hydrolysate obtained from enzymatic hydrolysis of L. klunzingeri muscle has a high nutritive value due to the presence of high level of protein/amino acids.

In this study, however, FPHs exhibited much lower antioxidant and cytotoxic activities than do BHT and carboplatin, but they are safer to eat, causing fewer adverse effects and provide the body a rich source of high-quality protein. BHT is a synthetic antioxidant commonly used in products such as food, cosmetics and pharmaceutics, but it might exert some toxic side effects on body’s tissues, leading to the development of cancer (Witschi, 1986Witschi HP. Enhanced tumour development by butylated hydroxytoluene (BHT) in the liver, lung and gastro-intestinal tract. Food Chem Toxicol. 1986;24(10):1127-1130.). It was also reported that the oral administration of BHT could induce oxidative stress by interfering with oxidative-antioxidant balance (Faine et al., 2006Faine LA, Rodrigues HG, Galhardi CM, Ebaid GM, Diniz YS, Fernandes AA, Novelli EL. Butyl hydroxytoluene (BHT)-induced oxidative stress: effects on serum lipids and cardiac energy metabolism in rats. Exp Toxicol Pathol. 2006;57(3):221-6.). Carboplatin is a potent chemotherapy medication used to treat a number type of cancer but, it often causes specific side effects such as anemia, nausea, electrolyte problems, allergic reactions and increased risk of another cancer (Tothilla et al., 1992Tothilla P, Klys HS, Matheson LM, McKay K, Smyth JF. The long-term retention of platinum in human tissues following the administration of cisplatin or carboplatin for cancer chemotherapy. Eur J Cancer. 1992;28(8-9):1358-1361.). So there is a growing trend to replace these synthetic compounds with natural ones to prevent or alleviate oxidative stress and associated diseases (Hsu, Li-Chan, Jao, 2011Hsu KC, Li-Chan EC, Jao CL. Antiproliferative activity of peptides prepared from enzymatic hydrolysates of tuna dark muscle on human breast cancer cell line MCF-7. Food Chem. 2011;126(2):617-622.).

Based on the findings of the present study, the protein hydrolysates of L. klunzingeri muscle exhibit significant antioxidant and cytotoxic properties in vitro. Besides, it has a high nutritional value because of its valuable content of protein and essential amino acid. However, in the body, peptides present in the protein hydrolysates may be metabolized due to enzymatic and digestive processes, and their structure and activates may be altered; therefore, it is recommended that the efficiency of protein hydrolysates obtained from L. klunzingeri muscle be investigated and confirmed in animal models, before suggesting them as complementary and health-promoting compounds. It is also recommended to optimize the hydrolysis conditions to reach the sample with maximum efficiency in vitro, and to use the sample with the highest antioxidant or cytotoxic activity in subsequent investigations to observe the maximum efficiency in animal models. After obtaining promising results in animal and human studies and the necessary approvals, antioxidant and cytotoxic protein hydrolysate can be commercially produced and used as functional compounds.

CONCLUSION

Production of protein hydrolysates from Klunzinger’s mullet represented an alternative way of upgrading this species. The antioxidant and cytotoxic properties of klunzinger’s mullet protein hydrolysates showed that these hydrolysates present potential as a functional food ingredient or as natural food supplement.

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