Identifying Potential Antioxidant Properties from the Viscera of Sea Snails (Turbo cornutus)

Turbo cornutus, the horned turban sea snail, is found along the intertidal and basaltic shorelines of Jeju Island, Korea. T. cornutus feeds on seaweeds (e.g., Undaria sp., and Ecklonia sp.) composed of diverse antioxidants. This study identified potential antioxidant properties from T. cornutus viscera tissues. Diverse extracts were evaluated for their hydrogen peroxide (H2O2) scavenging activities. T. cornutus viscera protamex-assisted extracts (TVP) were purified by gel filtration chromatography (GFC), and potential antioxidant properties were analyzed for their amino acid sequences and its peroxidase inhibition effects by in silico molecular docking and in vitro analysis. According to the results, T. cornutus viscera tissues are composed of many protein contents with each over 50%. Among the extracts, TVP possessed the highest H2O2 scavenging activity. In addition, TVP-GFC-3 significantly decreased intracellular reactive oxygen species (ROS) levels and increased cell viability in H2O2-treated HepG2 cells without cytotoxicity. TVP-GFC-3 comprises nine low molecular bioactive peptides (ELR, VGPQ, TDY, ALPHA, PAH, VDY, WSDK, VFSP, and FAPQY). Notably, the peptides dock to the active site of the myeloperoxidase (MPO), especially TDY and FAPQY showed the MPO inhibition effects with IC50 values of 646.0 ± 45.0 µM and 57.1 ± 17.7 µM, respectively. Altogether, our findings demonstrated that T. cornutus viscera have potential antioxidant properties that can be used as high value-added ingredients.


Introduction
Turbo cornutus, an edible gastropod species with a horned turban, is found along the intertidal and basaltic shorelines of Jeju Island, Korea. T. cornutus is a major source of income for Jeju Haenyeo (women divers; Intangible Cultural Heritage, 2016). The muscle tissues of T. cornutus are used as local foods in Jeju, Korea, but most of its viscera tissues are discarded because of low consumer preference and awareness. Some studies published in the 1970s-2000s presented the physioecology of T. cornutus [1][2][3]. T. cornutus is an herbivorous marine animal that feeds on seaweeds composed of diverse antioxidants [4]. However, the nutritional and functional ingredients of T. cornutus remain unknown.
Under normal physiological conditions, intracellular reactive oxygen species (ROS) are maintained at a constant low level by the balance between the generation and elimination of ROS [5]. However, ROS generated without control can induce oxidative damage to intracellular biomacromolecules, such as protein, membrane lipid, RNA, and DNA [5,6]. Hydrogen peroxide (H 2 O 2 ) is a ROS that, when present in excess, can be harmful to cells [7]. In addition, H 2 O 2 can be converted the hypohalous acids, causing oxidative damage by the Myeloperoxidase (MPO)/H 2 O 2 system [8,9]. Thus, the removing H 2 O 2 is very important to combat oxidative stress and MPO-dependent ROS [10,11].
Several antioxidants prevent and relieve oxidative damage caused by ROS [12]. Exogenous antioxidants are widely distributed in food and medicinal plants and food processing by-products, such as seafood viscera [13][14][15][16]. From this, many studies are being conducted on search for natural antioxidant compounds.
Yearly, a considerable amount of world fishery resources are discarded as processing leftovers, such as viscera, gonads, bones, and skin [17]. These marine by-products cause problems, such as environmental pollution. Thus, efforts to explore the possibilities for the further use of marine by-products have become more important than the methods of their disposal [18][19][20][21]. Recently, much research is conducted to explore the possible uses of different by-products; many studies have presented that marine by-products contain valuable protein fractions, including surimi [22], gelatin/collagen [23], and bioactive peptides [24]. Producing functional food materials and other value-added products from marine by-products is a way to obtain attention because marine by-products contain valuable protein and lipid fractions, pigments, minerals, enzymes, and nutraceuticals or pharmacological [18,20].
The objective of this study is to explore valuable application process that can reuse the discarded viscera of T. conutus. The potential antioxidant properties were purified from T. cornutus viscera through enzymatic hydrolysis and gel filtration chromatography (GFC); also, its antioxidant activities were assessed in H 2 O 2 -treated HepG2 cells. Furthermore, the bioactive peptides that composed the potential antioxidant properties were analyzed for their peroxidase inhibition effect.

Amino Acid Composition of T. cornutus
The amino acid compositions of T. cornutus viscera and muscle are listed in Table 2. The most abundant amino acids in the T. cornutus viscera are aspartic acid (10.3 ± 0.0%), glutamic acid (13.1 ± 0.2%), and taurine (11.3 ± 0.1%), each of which comprises more than 10% of the T. cornutus viscera, followed by arginine (7.1±0.5%), leucine (6.5 ± 0.1%), and proline (6.2 ± 0.3%). Alternatively, the most abundant amino acids in the T. cornutus muscle are aspartic acid (9.5 ± 0.2%), glutamic acid (16.4±0.1%), and arginine (9.6 ± 0.0%), followed by glycine (8.8 ± 0.2%), taurine (8.1 ± 0.0%), and leucine (6.3 ± 0.1%). Aspartic acid, glutamic acid, arginine, and glycine are the most abundant amino acids in other marine animals, such as abalone [25]. Both the viscera and muscle contain the most abundant aspartic and glutamic acid. The viscera and muscle contain about 30% of the essential amino acid composition, such as histidine, threonine, valine methionine, phenylalanine, isoleucine, leucine, and lysine, for humans. Therefore, both of them are high-quality protein sources. To assess the H 2 O 2 scavenging activity of T. cornutus viscera and muscle, each viscera and muscle tissues was hydrolyzed with nine proteases: alcalase, flavourzyme, neutrase, protamex, pepsin, trypsin, α-chymotrypsin, bromelain, and papain. The extraction yields of diverse T. cornutus enzyme-assisted extracts are summarized in Figure 1A. The T. cornutus viscera enzyme-assisted extracts showed more than 40% extraction yields, with T. cornutus viscera protamex-assisted extracts (TVP) having the highest extraction yield (70%). In contrast, the T. cornutus muscle enzyme-assisted extracts showed higher extraction yields than those of the viscera. The H 2 O 2 scavenging activities of the enzymatic extracts of T. cornutus viscera and muscle were shown in Figure 1B,C. The viscera extracts indicated higher H 2 O 2 scavenging activities than those of the muscle extracts in a concentration ranging from 0.25 to 2 mg/mL. The viscera extracts showed more than 80% H 2 O 2 scavenging activities at 2 mg/mL. In addition, the viscera extracts showed approximately three times higher IC 50 values of H 2 O 2 scavenging activities against each muscle extract, with TVP having the lowest IC 50 value of 0.435 mg/mL.

Effect of Viscera and Muscle Extracts on H 2 O 2 -Induced Oxidative Stress in HepG2 Cells
The liver is a vital organ that plays a major role in metabolism, excretion, and detoxification in the human body. Liver impairment is caused by different factors, such as infection, drugs, and excessive ethanol intake, leading to the accumulation of ROS and various liver injuries by oxidative stress. Thus, oxidative stress prevention is needed for hepatoprotection [26]. ROS are broadly defined as chemically reactive molecules containing oxygen; these include hydroxyl radical (OH·

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The liver is a vital organ that plays a major role in metabolism, excretion, and detox-118 ification in the human body. Liver impairment is caused by different factors, such as in-119 fection, drugs, and excessive ethanol intake, leading to the accumulation of ROS and var-120 ious liver injuries by oxidative stress. Thus, oxidative stress prevention is needed for hepa-121 toprotection [26]. ROS are broadly defined as chemically reactive molecules containing 122 oxygen; these include hydroxyl radical (OH〮◌), superoxide anion (O2 − ), singlet oxygen 123 (O2), and H2O2 (H2O2) [5]. ROS react with molecules by reversible oxidative modifications 124 and factors in cellular signaling pathways, such as metabolism, growth, differentiation, 125 and death signaling [5]. However, ROS overproduction without control can result in oxi-126 dative damage to cell structures, including lipids and membranes, proteins, and DNA 127 [5,26]. Therefore, the MTT assay was performed in H2O2-exposed HepG2 cells to evaluate 128 the potential antioxidant effect of viscera and muscle extract s. As shown in Figure 2 [5]. ROS react with molecules by reversible oxidative modifications and factors in cellular signaling pathways, such as metabolism, growth, differentiation, and death signaling [5]. However, ROS overproduction without control can result in oxidative damage to cell structures, including lipids and membranes, proteins, and DNA [5,26]. Therefore, the MTT assay was performed in H 2 O 2 -exposed HepG2 cells to evaluate the potential antioxidant effect of viscera and muscle extract s. As shown in Figure 2, significant cell death was observed in the H 2 O 2 -treated cells. However, TVP and the muscle protamex extract markedly increased cell viability. Especially, TVP showed a higher protective effect than did muscle protamex-assisted extracts against H 2 O 2 in HepG2 cells. In addition, TVP inhibited intracellular ROS production, and aspartate aminotransferase (AST) levels increased by treating H 2 O 2 in HepG2 cells. These results indicated that T. cornutus viscera tissues possess a high value than did T. cornutus muscle tissues by protamex-assisted hydrolysis processing.

Characterization of Antioxidant Peptides from TVP
Depending on the H 2 O 2 scavenging activity and protective effect on H 2 O 2 in HepG2 cells, TVP was selected for the next separation step by GFC on Sephadex G-25 column. TVP was fractionated to four kinds of fractions according to their molecular sizes ( Figure 3A). Among them, GFC-Fr.3 (TVP-GFC-3) had the highest H 2 O 2 scavenging activity at a concentration of 0.25 mg/mL ( Figure 3B,C). TVP-GFC-3 significantly increased IC 50 values than did TVP. In addition, TVP-GFC-3 significantly decreased ROS generation and increased protective effects in H 2 O 2 -exposed HepG2 cells without cytotoxicity (Figure 4). To identify the amino acid sequences of the separated fraction, TVP-GFC-3 was analyzed using MicroQ-time-of-flight (TOF) tandem mass spectrometry. TVP-GFC-3 comprises nine small molecule peptides, and the amino acid sequences of the peptides were evaluated as ELR, VGPQ, TDY, ALPHA, PAH, VDY, WSDK, VFSP, and FAPQY (Table 3, Figures S1-S10).

In Silico Analysis of Antioxidant Peptides on MPO Inhibition
Several molecular docking studies targeting specific enzyme inhibition effects have been recently published [27,28]. Among the docking tools, CDOCKER, a CHARMm-based docking algorithm [29], found favorable docking poses between small molecules and target proteins using their structural characteristics, such as unshared electron pairs, double bonds, hydrophobicity, and charge.
To verify the antioxidant activity of bioactive peptides purified from TVP-GFC-3, the biological network dynamics of bioactive peptides and MPO were simulated in a computational space, and its binding energies were compared with thiocyanate ion, a pseudohalide anion, and 4-aminobenzoic acid hydrazide (4-ABH), an inhibitor of MPO. In silico analysis was performed using the crystalline structure of MPO (PDB ID 7LAL) and 4-ABH (PubChem CID 21450). Bioactive peptide structures were drawn using a CDOCKER tool. Each amino acid of bioactive peptides forms diverse hydrogen and pi bonds; also, all bioactive peptides dock to the active site of MPO with a more stable binding energy than that of thiocyanate ion ( Figure 5, Table 4). All of the bioactive peptides form the hydrogen bonds and/or pi bonds with a heme group. The activation of MPO is required a heme group as a cofactor [30].     4-ABH is one of hydrazide with the formula H 2 NC 6 H 4 C(O)NHNH 2 containing two amino groups and benzene ring. 4-ABH is docking to the active site of MPO with hydrogen bond and pi bond between the amino groups and benzene ring. Among the bioactive peptides, TDY and FAPQY having a benzene ring bound to MPO, with low binding energy of −368.111 and −387.049 kcal/mol, respectively. The top hit docking poses were presented as two-dimensional (2D) diagrams and three-dimensional (3D) to confirm the biological network dynamics of the complexes (Figures 5 and 6). MPO was expressed as a line model, and the active site including a heme group (green part), was expressed as a thin stick model ( Figure 6). TDY ( Figure 6A) and FAPQY ( Figure 6B) was shown as a gray and red stick model. The complexes displayed favorable hydrogen bond interactions, with the pink section as a donor and the green section as an acceptor ( Figure 6A,B). The docking of TDY was performed through interaction with the active site, including a heme group and PHE99, THR100, GLU102, ARG239, GLU242, PHE366, PHE407, and MET411 ( Figures 5C  and 6A). In addition, the docking of FAPQY was performed through interaction with the active site, including a heme group and HIS95, THR100, GLU116, ARG239, and GLU242 ( Figures 5I and 6B). Especially, FAPQY formed the similar binding pose with 4-ABH-MPO complex by combining as pi bonds between a benzene ring and a heme group and ARG239 (Figures 5K and 6C).

In Vitro Analysis of Antioxidant Peptides on Myeloperoxidase (MPO) Inhibition
To confirm the in silico prediction results on the docking of peptides to MPO, in vitro MPO inhibition effects of the peptides were assessed. Both TDY and FAPQY inhibited MPO in a concentration-dependent manner, and the IC50 values were calculated to be 646.0 ± 45.0 µM and 57.1 ± 17.7 µM, respectively ( Figure 6D,E). These results indicated that these bioactive peptides possessed the values of the natural MPO inhibitors. MPO promotes oxidative stress by involving the generation of radicals [31]. As with many radical species, H2O2 can cause the oxidative stress, directly reacting the cells and/or indirectly inducing the production other radical species. Especially, the hypohalous acids were produced by the MPO with H2O2; these radicals cause the stronger oxidative stress. In addition, the scavenging of H2O2 affects to inhibit the MPO activities. Thus, the components having both H2O2 scavenging activity and MPO inhibition effects can be considered as valuable antioxidant. Therefore, the protamex-assisted extracts and peptide from T. cornutus having both H2O2 scavenging activity and MPO inhibition effects can be used as functional food components for human health.

Materials
T. cornutus was purchased from a fishing village in Taeheung in May 2019 (Jeju, Korea) and was washed thrice with tap water to remove salt, epiphytes, and sand attached

In Vitro Analysis of Antioxidant Peptides on Myeloperoxidase (MPO) Inhibition
To confirm the in silico prediction results on the docking of peptides to MPO, in vitro MPO inhibition effects of the peptides were assessed. Both TDY and FAPQY inhibited MPO in a concentration-dependent manner, and the IC 50 values were calculated to be 646.0 ± 45.0 µM and 57.1 ± 17.7 µM, respectively ( Figure 6D,E). These results indicated that these bioactive peptides possessed the values of the natural MPO inhibitors. MPO promotes oxidative stress by involving the generation of radicals [31]. As with many radical species, H 2 O 2 can cause the oxidative stress, directly reacting the cells and/or indirectly inducing the production other radical species. Especially, the hypohalous acids were produced by the MPO with H 2 O 2 ; these radicals cause the stronger oxidative stress. In addition, the scavenging of H 2 O 2 affects to inhibit the MPO activities. Thus, the components having both H 2 O 2 scavenging activity and MPO inhibition effects can be considered as valuable antioxidant. Therefore, the protamex-assisted extracts and peptide from T. cornutus having both H 2 O 2 scavenging activity and MPO inhibition effects can be used as functional food components for human health.

Materials
T. cornutus was purchased from a fishing village in Taeheung in May 2019 (Jeju, Korea) and was washed thrice with tap water to remove salt, epiphytes, and sand attached to its surface. The viscera and muscle tissues were separated and carefully rinsed using fresh water and stored at −20 • C. Finally, the T. cornutus viscera and muscle tissues were freeze-dried and finely ground before hydrolysis. Commercial food-grade proteases, including alcalase 2.4 L FG, neutrase 0.8 L, flavourzyme 500 MG, and protamex, were purchased from Novo Co. (Novozyme Nordisk, Bagasvaerd, Denmark). Other proteases that contain pepsin, trypsin, α-chymotrypsin bromelain, and papain were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The characterized peptide was synthesized by Anygen Co., LTD. (Gwangju, Korea) based on its amino acid sequence. Other chemicals and reagents used were of analytical grade.

Proximate Composition of T. cornutus
The proximate composition of T. cornutus was determined following the AOAC method [32]. Crude protein was determined using the Kjeldahl method, and crude lipid was performed using the Soxhlet method. In addition, moisture was determined by keeping the sample in a dry oven, and crude ash was prepared at 550 • C in a dry-type furnace.

Preparation of T. cornutus Enzyme-Assisted Extracts
T. cornutus viscera and muscle enzyme-assisted hydrolysis was performed according to the method used by Ko et al. [34] and Heo et al. [35]. Hydrolytic enzymes were prepared using four food-grade proteases (alcalase, flavourzyme, neutrase, and protamex), three digestive enzymes (pepsin, trypsin, and α-chymotrypsin), and two plant-derived digestive enzymes (bromelain and papain). Two grams of the dried ground T. cornutus viscera and muscle powder was homogenized with buffer (100 mL) and hydrolyzed with enzymes in a substrate/enzyme ratio of 100:1 (w/w). The pH of the homogenates was adjusted to its optimal pH value before enzymatic hydrolysis. Enzymatic reactions were performed for 24 h to achieve an optimal degree of enzymatic hydrolysis. Then, the extracts were boiled for 10 min at 100 • C in a water bath to inactivate the enzyme. Each extract was clarified by centrifugation (3500 rpm, 20 min at 4 • C) to remove the residue. All extracts were freeze-dried and kept at −20 • C. The yields of each T. cornutus viscera and muscle enzyme-assisted extracts were calculated as the percentage of dry weight compared with the hydrolyzed sample weight. Briefly, extract yields were determined by subtracting the dried weight of the residue from 1 mL of dried extracts and were expressed as a percentage.

Separation of Fractions and Radical Scavenging Properties
Radical scavenging properties were separated as previously described by Kang et al. [6]. The extracts showing antioxidant activities were dissolved in distilled water, loaded onto a Sephadex G-25 gel filtration column (2.5 × 75 cm), and equilibrated with distilled water. The column was eluted with distilled water at a flow rate of 1.0 mL/min. Elution peaks were detected at 280 nm.

Characterization of the Separated Antioxidant Properties
The molecular mass and amino acid sequence of the separated antioxidant properties from T. cornutus was determined using a MicroQ-TOF tandem mass spectrometer (Bruker Daltonics, 255748 Bremen, Germany) coupled with a nanoelectrospray ionization (ESI) source. The fraction dissolved in water was infused into the ESI source, and the molecular weight was determined by doubly charged (M + 2H) 2+ state analysis in the mass spectrum. Following molecular weight determination, the peptides were automatically selected for fragmentation, and sequence information was obtained by tandem MS analysis.

H 2 O 2 Scavenging Activity
H 2 O 2 scavenging activity was determined according to the method of Müller [36]. One hundred microliters of 0.1-M phosphate buffer (pH 5.0) and twenty microliters of the sample solution were mixed in a 96-well plate. Twenty microliters of H 2 O 2 was added to the mixture and then incubated at 37 • C for 5 min. After incubation, 30 µL of 1.25 mM ABTS and 30 µL of peroxidase (1 unit/mL) was added to the mixture and then incubated at 37 • C for 10 min. The absorbance was read with a microplate reader at 405 nm.

Cell Line and Cell Culture
HepG2 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). HepG2 cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 1% streptomycin (100 µg/mL), and penicillin (100 unit/mL) and maintained at 37 • C in a 5% CO 2 incubator.

Determination of Cell Viability and ROS Generation in H 2 O 2 -Treated HepG2 Cells
Potential antioxidant activities were evaluated under H 2 O 2 -induced oxidative conditions. Briefly, HepG2 cells were plated in 96-well plates at a concentration of 1 × 10 5 cells/mL and incubated for 24 h. After 24 h of incubation, the samples were treated before activating them with H 2 O 2 (1 mM) for 1 h. After 24 h of incubation, cell viability was measured using the MTT assay [37]. The intracellular ROS scavenging activity was analyzed using the DCF-DA assay [38]. The HepG2 cells were seeded as shown before, treated with H 2 O 2 and different concentrations of samples, and incubated for 24 h. After 24 h of incubation, 500 µg/mL of DCF-DA was added to each well. Finally, DCF-DA fluorescence was measured using a Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Winooski, VT, USA) at excitation and emission wavelengths of 485 and 535 nm, respectively.

In Silico Analysis of MPO Inhibition
For molecular docking studies, the crystal structures of MPO (ID: 7LAL) were provided by the Protein Data Bank. The structures of nine bioactive peptides derived from TVP were drawn using a CDOCKER tool. The docking of bioactive peptides to MPO was performed using the CDOCKER tool in Discovery Studio 2020 (Dassault Systemes, Velizy-Villacoublay, France). The simulation was performed as follows: (1) a 2D structure was converted into a 3D structure; (2) receptors were prepared, and the binding site was defined; and (3) the docking of compounds was performed using a CDOCKER tool [28]. The binding energies of the produced complexes were calculated to compare the optimal agents among the bioactive peptides, inhibitors, and existing ligand (thiocyanate ion). The docking poses of bioactive peptides to MPO were expressed as 2D diagrams and 3D crystalline structures.

MPO Inhibition Effect
MPO inhibition effects of the peptides were measured by using an MPO inhibitor screening assay kit (Abcam PLC, Cambridge, UK) following the instruction in the enclosed user manuals. Briefly, 10 µL of each peptide, 10 µL of 1.25 µL/mL MPO and 40 µl of assay buffer were mixed in a 96-well black plate. 50 µL of the peroxidation initiator solution was quickly added to all of the wells and then incubated for 5 min at RT. After incubation, the fluorescence intensity of the each well was read using an excitation wavelength of 530 nm and an emission wavelength of 590 nm.

Statistical Analysis
All data were represented as the mean ± standard deviation of three determinations. The statistical comparison of the mean values was performed by one-way ANOVA, followed by Tukey's multiple comparisons test. Statistical significance was considered at p < 0.01.

Conclusions
By the sea food industrial activities, a considerable amount of fishery resources are discarded as processing leftovers including viscera. Thus, the possibility to recover such a material and convert it in a value-added product would be highly desirable. In Korea, T. cornutus muscle tissues are used in local foods, but most of the viscera tissues were discarded. T. cornutus viscera is a rich protein source, with more than 50% of protein contents composed of essential amino acids, such as histidine, threonine, valine, methionine, phenylalanine, isoleucine, leucine, and lysine. In addition, the potential antioxidant properties from T. cornutus viscera extracts possessed H 2 O 2 scavenging activity and protective effects on oxidative stress in H 2 O 2 -treated HepG2 cells. The potential antioxidant properties were composed of nine bioactive peptides. In addition, in silico analysis predicted that the nine bioactive peptides inhibit peroxidase by interacting with the surface of MPO close to the active site. Especially, TDY and FAPQY showed the MPO inhibition effects with IC 50 values of 646.0 ± 45.0 µM and 57.1 ± 17.7 µM, respectively. These results indicated that the potential antioxidant properties from T. cornutus viscera could be used for functional food components for human health.