Antioxidant Effects of Turbo cornutus By-Products Visceral Extract against Hydrogen Peroxide-Induced Oxidative Stress by Regulating MAPK and Akt Signaling Pathways in Vero Cells

Turbo cornutus, a marine gastropod mollusk commonly called sea snail, is found along the southern coast of Korea and holds considerable importance as a marine food resource, particularly on Jeju Island, Korea. Data are scarce on the antioxidant activity of hot water extracts from T. cornutus visceral tissue. Therefore, this study was performed to evaluate the antioxidant activities of T. cornutus visceral tissue hot water extract (TVE) and the underlying mechanisms against hydrogen peroxide-induced oxidative stress in Vero cells. The amino acid composition and antioxidant effects of TVE were evaluated. Furthermore, the impact of TVE on the expression of proteins within the mitogen-activated protein kinase (MAPK) pathway is investigated. TVE showed a concentration-dependent enhancement in its scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals (IC50 = 1.07 ± 0.06 mg/mL) and hydrogen peroxide (IC50 = 0.33 ± 0.03 mg/mL). TVE reduced intracellular reactive oxygen species (ROS) production and maintained cell viability under H2O2-induced oxidative stress by suppressing apoptosis in Vero cells. Additionally, TVE demonstrated regulatory effects on the MAPK and protein kinase B (Akt) signaling pathways activated by H2O2. In conclusion, the findings from our study propose that TVE holds potential as a bioactive component in the formulation of functional foods.


Introduction
Turbo cornutus, a marine gastropod mollusk, also known as sea snail, is distributed along the southern coast of Korea. T. cornutus found on Jeju Island is important in the food industry as a strategic local specialty. The total production of T. cornutus in Republic of Korea was estimated to be 2376 tons in 2020 [1]. As large quantities of T. cornutus are produced each year, the T. cornutus visceral tissue and shells are discarded after processing due to low consumer preference and awareness [2]. Gastropod-derived foods exhibit many beneficial properties such as anti-inflammatory and antioxidant properties [2][3][4].
Oxidative stress is caused by a disturbance in the balance between reactive oxygen species (ROS) production and antioxidant defenses [5]. Excessive ROS production can damage lipids, proteins, and DNA and is associated with the development of various diseases, including vascular disorders, autoimmune diseases, neurodegenerative diseases, and respiratory diseases [6]. To prevent ROS accumulation, cellular systems involve antioxidant mechanisms such as mitogen-activated protein kinase (MAPK) and protein kinase B (Akt)-mediated signaling pathways [7,8].
Previous studies have reported that Kim et al. [3] demonstrated the anti-inflammatory effect of an ethanolic extract of T. cornutus viscera on lipopolysaccharide-stimulated RAW Foods 2023, 12, 3660 2 of 10 264.7 cells and a zebrafish model. And the hydrolysates of T. cornutus viscera have potential antioxidant and bioactive peptide properties in the human hepatoblastoma (HepG2) cell line [2]. In addition, abalone (Haliotis discus hannai) viscera hydrolysates, a species of gastropod, have antioxidant activity and antioxidant amino acids [4]. As therapeutic candidates, peptides have been researched and applied in pharmaceutical fields such as drug discovery and antibacterial peptides [9]. Although hot water extraction is an efficient technique for extracting bioactive compounds in the food industry [10], studies on the antioxidant activity of the hot water extract of T. cornutus visceral tissue are lacking.
This study aims to investigate whether T. cornutus visceral tissue hot water extract (TVE) exhibited antioxidant activity that could ameliorate hydrogen peroxide (H 2 O 2 )-induced oxidative stress in Vero cells.

Sample Collection and Extraction
T. cornutus was obtained from a fishing village in Udo, Republic of Korea. The shells of T. cornutus were removed, and the muscle and visceral tissues were separated and washed with tap water. Muscle and visceral tissues were lyophilized and ground into a powder. Hot water or enzyme-assisted extraction is being used for the application of various natural resources for food and nutraceutical purposes [11]. Extraction was performed using distilled water via three methods: (1) For traditional extraction, a freeze-dried T. cornutus sample (2 g) was extracted with distilled water (100 mL) using a shaking incubator for 24 h at room temperature. (2) Artificial digestion for extraction of T. cornutus was performed according to the method described by Oh et al. [12]. (3) For hot water extraction, 2 g of freeze-dried sample was pressure-extracted with 100 mL of distilled water at 121 • C for 20 min. After extraction using each method, the extract was centrifuged at 3200× g rpm for 10 min and filtered through a filter paper (Whatman, Maidstone, Kent, United Kingdom). The filtrate was lyophilized and stored at refrigerator temperatures (−20 • C) until used in experiments.

Analysis of Chemical Composition
Bicinchoninic acid (BCA) method, a copper-based colorimetric assay, was used to determine the total protein content in TVE while using bovine serum albumin as the reference standard. Briefly, BCA protein reagents were added to samples and then reacted for 30 min at 37 • C. Absorbance was measured at 562 nm using a microplate reader (Multiskan; Thermo Fisher Scientific, Waltham, MA, USA). Carbohydrate content was analyzed by modifying the methods described by Han et al. [13] using D-glucose as the reference standard.

Measurement of Amino Acid
High-performance liquid chromatography (HPLC)-fluorescence detection system has been widely used for quantitative amino acid analysis [14]. The samples for amino acid analysis were hydrolyzed with 6 N hydrogen chloride following the method described by Kang et al. [2]. The amino acid composition of TVE was determined using an HPLC system (Ultimate 3000; Thermo-Dionex, Sunnyvale, CA, USA) consisting of a fluorescence detector (1260FLD; Agilent Technologies, Santa Clara, CA, USA). Amino acids were determined using an inno C18 column (5 µm, 4.6 × 150 mm; YoungJin Biochrom, Seongnam-si, Gyeoggido, Republic of Korea). The experimental conditions were as follows: mobile phase (water:acetonitrile:methanol = 10:45:45); flow rate (1.5 mL/min); and column temperature (40 • C).

Availability of Antioxidant Activity
Freeze-dried samples were dissolved in distilled water at various concentrations for antioxidant assays. To measure the antioxidant activity of TVE, DPPH radical scavenging and H 2 O 2 -scavenging assays were performed using modified methods of Chatatikun and Chiabchalard [15] and Kim et al. [16], respectively. The half-maximal inhibitory concentration (IC 50 ) values were obtained.

Measurement of Cell Viability
TVE cytotoxicity was evaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described by Kim et al. [3]. The cells were seeded in a 96-well plate at a density of 1 × 10 5 cells/mL and incubated for 24 h. To determine the cytoprotective effect of TVE, the cells were pre-incubated with 25, 50, 100, and 200 µg/mL TVE for 1 h before stimulation with 200 µM H 2 O 2 . After 24 h, the MTT reagent was added to each well for 3 h, and the formazan produced was dissolved using dimethyl sulfoxide (DMSO). The absorbance was measured at 540 nm using a microplate reader (BioTek Synergy HT; Agilent Technologies, Santa Clara, CA, USA).

Evaluation of Intracellular ROS Level
The cells were seeded in a 96-well plate at a density of 1 × 10 5 cells/mL and incubated for 24 h. The cells were then pre-treated with 25, 50, 100, and 200 µg/mL TVE for 1 h before stimulation with 200 µM H 2 O 2 . After 30 min, the DCFH-DA reagent was added. 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence was measured using a microplate reader (BioTek Synergy HT; Agilent Technologies, Santa Clara, CA, USA) and used to calculate the intracellular ROS levels.

Apoptosis Analysis
Nuclear morphology was confirmed using Hoechst 33342 (NucBlue™ Live ReadyProbes™ Reagent, Invitrogen, Waltham, MA, USA), a cell-permeable DNA-staining sample. Vero cells were seeded in a four-well chamber slide (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 24 h. The cells were pretreated with TVE for 1 h and stimulated with H 2 O 2 (200 µM) for 6 h. Nuclei were stained with Hoechst 33342 solution according to the manufacturer's specifications. The stained cells were observed under a fluorescence microscope.

Western Blotting
The cells were seeded in 60 mm dishes for 24 h and then pre-treated with different concentrations of TVE for 1 h; all dishes, except the control, were subsequently stimulated with H 2 O 2 for 10 min. Cells were washed twice with phosphate-buffered saline, harvested, washed, and lysed in radioimmunoprecipitation assay buffer. The protein content of the cell lysates was measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal volumes of lysates were electrophoresed on 4-12% Bis-Tris protein gels and transferred to nitrocellulose membranes. Primary antibodies used were as follows: anti-phosphorylated (phospho)-p38, anti-p38, anti-phospho-extracellular signal-regulated kinase (ERK), anti-ERK, anti-phospho-c-Jun N-terminal kinase (JNK), anti-JNK, anti-phospho-Akt, anti-Akt, and anti-β-actin. The secondary antibodies used were anti-rabbit and anti-mouse IgG. Antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), Santa Cruz Biotechnology (Dallas, TX, USA), and Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were detected using a chemiluminescence detection kit (EZ-Western Lumi Pico; DoGen Bio, Seoul, Republic of Korea) on a Davinch-Chemi Imaging System (CAS400SM; Davinch-K, Seoul, Republic of Korea). Ex-pression levels were quantified using ImageJ version 1.53t (National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis
All experiments were performed in triplicate. Statistical significance was assessed using one-way ANOVA followed by Dunnett's multiple comparison test using GraphPad Prism software version 9 (GraphPad Software, San Diego, CA, USA), and p values < 0.05 were considered significant.

H 2 O 2 -scavenging Activity of Various T. cornutus Extracts
The yield and activity of the extracts depend significantly on the solvent used, temperature, pressure, and extraction time [17]. To investigate the impact of various water-based extraction methods on the H 2 O 2 -scavenging effect, extraction techniques using distilled water, artificial digestion, and hot water were selected. The overall H 2 O 2 -scavenging activity of T. cornutus hot water extracts was higher than that of the distilled water and artificial digestion extracts ( Figure 1). In addition, in the hot water extract, the H 2 O 2 -scavenging activity of the visceral tissue-derived extract (IC 50 = 0.34 ± 0.00 mg/mL) was higher than that of the muscle tissue-derived extract (IC 50 = 0.57 ± 0.02 mg/mL). These results showed that TVE had the highest antioxidant activity. Therefore, subsequent experiments were performed using TVE.

Statistical Analysis
All experiments were performed in triplicate. Statistical significance was assessed using one-way ANOVA followed by Dunnett's multiple comparison test using GraphPad Prism software version 9 (GraphPad Software, San Diego, CA, USA), and p values < 0.05 were considered significant.

H2O2-scavenging Activity of Various T. cornutus Extracts
The yield and activity of the extracts depend significantly on the solvent used, temperature, pressure, and extraction time [17]. To investigate the impact of various waterbased extraction methods on the H2O2-scavenging effect, extraction techniques using distilled water, artificial digestion, and hot water were selected. The overall H2O2-scavenging activity of T. cornutus hot water extracts was higher than that of the distilled water and artificial digestion extracts ( Figure 1). In addition, in the hot water extract, the H2O2-scavenging activity of the visceral tissue-derived extract (IC50 = 0.34 ± 0.00 mg/mL) was higher than that of the muscle tissue-derived extract (IC50 = 0.57 ± 0.02 mg/mL). These results showed that TVE had the highest antioxidant activity. Therefore, subsequent experiments were performed using TVE.  Figure 1. Hydrogen peroxide-scavenging activity of various T. cornutus extracts. The hydrogen peroxide-scavenging activity was evaluated in the muscle and visceral tissues of T. cornutus extracts using three extraction methods. Data represent the mean ± standard deviation from triplication. IC 50 , half-maximal inhibitory concentration.

Extraction Yield and Proximate Composition of TVE
The composition of TVE is presented in Table 1. The extraction yield of TVE was 37.31 ± 0.41%, which was plentiful protein (41.08 ± 1.01%) and carbohydrate Foods 2023, 12, 3660 5 of 10 (19.69 ± 1.70%). Han et al. [13] have found that the hydrolysate from the gastropod Batillus cornutus meat tissue has a protein content of 37.94 ± 0.17%. A comparison of these results showed that TVE had a similar protein content. Therefore, it is considered that TVE will be an excellent protein source.

Amino Acid Composition in TVE
It was confirmed that the main component of TVE is protein. Therefore, TVE analyzed amino acids, which are constituents of proteins. The amino acid composition of TVE is presented in Table 2. The most abundant amino acid was taurine (2-aminoethanesulfonic acid), accounting for 40.75 ± 1.65% of the total amino acid pool. Additionally, TVE was abundant in glutamic acid (10.98 ± 0.75%), aspartic acid (7.01 ± 0.00%), and glycine (6.65 ± 1.00%). The results confirmed that TVE is a good source of taurine. Previous studies reported that the alkaline protease hydrolysate derived from octopuses is rich in taurine (23.16%) and exhibits antioxidant activity [18]. Taurine is a non-proteinogenic sulfur-containing amino acid that exerts cytoprotective effects [19]. Taurine has several biological functions including anti-inflammatory, antioxidant, vasodilatory, and anti-apoptotic effects [20,21]. These results demonstrated that TVE contained higher amounts of taurine with various biological activities than octopus hydrolysate. Seafood is an excellent source of taurine, which improves human health by exerting cytoprotective actions via various mechanisms, including antioxidants, energy production, neuromodulation, Ca 2+ homeostasis, and osmotic regulation [22].

Antioxidant Activities of TVE
The antioxidant activity of TVE was measured using DPPH radical and H 2 O 2 -scavenging assays and compared to that of the positive control, NAC (Figure 2). TVE showed a concentration-dependent increase in DPPH radical and H 2 O 2 -scavenging activities with IC 50 values of 1.07 ± 0.06 and 0.33 ± 0.03 mg/mL, respectively. The DPPH radical and H 2 O 2 -scavenging effects of 4 mg/mL TVE (99.35 ± 8.96 and 98.93 ± 0.52%, respectively) were similar to those of NAC (89.8 ± 0.5 and 98.78 ± 0.45%, respectively).

Antioxidant Activities of TVE
The antioxidant activity of TVE was measured using DPPH radical and H2O enging assays and compared to that of the positive control, NAC (Figure 2). TVE s a concentration-dependent increase in DPPH radical and H2O2-scavenging activitie IC50 values of 1.07 ± 0.06 and 0.33 ± 0.03 mg/mL, respectively. The DPPH radical and scavenging effects of 4 mg/mL TVE (99.35 ± 8.96 and 98.93 ± 0.52%, respectively similar to those of NAC (89.8 ± 0.5 and 98.78 ± 0.45%, respectively).
Kang et al. [2] reported that T. cornutus viscera protamex-assisted extracts had scavenging activity with an IC50 value of 0.435 mg/mL. It has been demonstrated th possessed higher H2O2-scavenging than T. cornutus viscera hydrolysate.
These results indicated that TVE has excellent inhibitory activity against DPP icals and H2O2.

Effect of TVE on Cell Viability and ROS Generation in H2O2-Stimulated Vero Cells
Based on the antioxidant activity of TVE, further experiments were perform evaluate the viability and intracellular ROS levels in H2O2-stimulated Vero cells. H2 concentration of 200 µM reduced cell viability to approximately 58.35 ± 8.83%, whi considered optimal for inducing cytotoxicity in the subsequent experiments (Figu TVE at concentrations ranging from 25 to 200 µg/mL was not cytotoxic to Vero cel ther experiments were conducted using these concentrations ( Figure 3B). H2O2 st tion reduced cell viability and increased intracellular ROS levels compared to the c ( Figure 3C,D). TVE increased cell viability and reduced intracellular ROS levels in stimulated cells in a concentration-dependent manner. The intracellular ROS levels pre-treated with 25, 50, 100, and 200 µg/mL TVE were 85.8 ± 4.0, 83.3 ± 2.2, 74.5 ± 0 65.6 ± 3.2%, respectively. These results indicated that TVE exerts a cytoprotective against H2O2-induced oxidative stress. Kang et al. [2] reported that T. cornutus viscera protamex-assisted extracts had H 2 O 2 -scavenging activity with an IC 50 value of 0.435 mg/mL. It has been demonstrated that TVE possessed higher H 2 O 2 -scavenging than T. cornutus viscera hydrolysate.
These results indicated that TVE has excellent inhibitory activity against DPPH radicals and H 2 O 2 .

Effect of TVE on Cell Viability and ROS Generation in H 2 O 2 -Stimulated Vero Cells
Based on the antioxidant activity of TVE, further experiments were performed to evaluate the viability and intracellular ROS levels in H 2 O 2 -stimulated Vero cells. H 2 O 2 at a concentration of 200 µM reduced cell viability to approximately 58.35 ± 8.83%, which was considered optimal for inducing cytotoxicity in the subsequent experiments ( Figure 3A). TVE at concentrations ranging from 25 to 200 µg/mL was not cytotoxic to Vero cells; further experiments were conducted using these concentrations ( Figure 3B). H 2 O 2 stimulation reduced cell viability and increased intracellular ROS levels compared to the control ( Figure 3C,D). TVE increased cell viability and reduced intracellular ROS levels in H 2 O 2 -stimulated cells in a concentration-dependent manner. The intracellular ROS levels in cells pre-treated with 25, 50, 100, and 200 µg/mL TVE were 85.8 ± 4.0, 83.3 ± 2.2, 74.5 ± 0.9, and 65.6 ± 3.2%, respectively. These results indicated that TVE exerts a cytoprotective effect against H 2 O 2 -induced oxidative stress.

Effect of TVE on H2O2-Induced Apoptosis in Vero Cells
ROS overproduction can activate cell death processes such as apoptosis [23]. Hoechst 33342 is taken up by both dead and live cells, and it is used to detect chromatin condensation and DNA fragmentation in apoptotic cells [24]. H2O2 treatment increased apoptotic body formation, as observed by nuclear condensation and fragmentation, according to Hoechst 33342 staining ( Figure 4). TVE treatment suppressed apoptotic body formation in a concentration-dependent manner. The results suggested that TVE prevents H2O2-induced cell death by suppressing apoptosis via ROS scavenging.

Effect of TVE on H 2 O 2 -Induced Apoptosis in Vero Cells
ROS overproduction can activate cell death processes such as apoptosis [23]. Hoechst 33342 is taken up by both dead and live cells, and it is used to detect chromatin condensation and DNA fragmentation in apoptotic cells [24]. H 2 O 2 treatment increased apoptotic body formation, as observed by nuclear condensation and fragmentation, according to Hoechst 33342 staining ( Figure 4). TVE treatment suppressed apoptotic body formation in a concentration-dependent manner. The results suggested that TVE prevents H 2 O 2 -induced cell death by suppressing apoptosis via ROS scavenging.

Effect of TVE on Regulation of MAPK and Akt Signaling Pathways
Antioxidants can scavenge ROS and reduce the oxidation of cellular molecules [25]; ROS play vital roles in mediating MAPK and Akt activation [8]. MAPK and Akt play essential roles in transmitting cell signals, consequently influencing the expression of genes that regulate various cellular processes such as cell growth, proliferation, and apoptosis [26]. To investigate the molecular mechanisms underlying the antioxidant effects of TVE, the levels of phosphorylated MAPKs, including p38, ERK, JNK, and Akt, were analyzed via Western blotting. H 2 O 2 treatment markedly increased the phosphorylation of p38, ERK, JNK, and Akt ( Figure 5). In addition, TVE pretreatment attenuated the phosphorylation of MAPK and Akt signaling. These results indicated that TVE protects cells against H 2 O 2 -induced oxidative stress by inhibiting the MAPK and Akt signaling pathways.
ROS overproduction can activate cell death processes such as apoptosis [23]. Hoechst 33342 is taken up by both dead and live cells, and it is used to detect chromatin condensation and DNA fragmentation in apoptotic cells [24]. H2O2 treatment increased apoptotic body formation, as observed by nuclear condensation and fragmentation, according to Hoechst 33342 staining (Figure 4). TVE treatment suppressed apoptotic body formation in a concentration-dependent manner. The results suggested that TVE prevents H2O2-induced cell death by suppressing apoptosis via ROS scavenging.

Effect of TVE on Regulation of MAPK and Akt Signaling Pathways
Antioxidants can scavenge ROS and reduce the oxidation of cellular molecules [25]; ROS play vital roles in mediating MAPK and Akt activation [8]. MAPK and Akt play essential roles in transmitting cell signals, consequently influencing the expression of genes that regulate various cellular processes such as cell growth, proliferation, and apoptosis [26]. To investigate the molecular mechanisms underlying the antioxidant effects of TVE, the levels of phosphorylated MAPKs, including p38, ERK, JNK, and Akt, were analyzed via Western blotting. H2O2 treatment markedly increased the phosphorylation of p38, ERK, JNK, and Akt ( Figure 5). In addition, TVE pretreatment attenuated the phosphorylation of MAPK and Akt signaling. These results indicated that TVE protects cells against H2O2-induced oxidative stress by inhibiting the MAPK and Akt signaling pathways. Western blotting and β-actin was used as a loading control. (b) Relative protein expression of phosphorylated proteins in H2O2−stimulated Vero cells. ImageJ software was used to determine the intensity of the bands. Data represent the mean ± standard deviation from triplication. ### p < 0.001 as compared to the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with the H2O2−treated group. TVE, T. cornutus visceral tissue hot water extract; H2O2, hydrogen peroxide; MAPK, mitogenactivated protein kinase.

Conclusions
The visceral tissue of T. cornutus is considered a post-harvest by-product. Several studies have reported that visceral tissues of T. cornutus have antioxidant and anti-inflammatory properties. The present study demonstrated that TVE, a taurine-rich protein source, protected cells against H2O2-induced oxidative stress by scavenging intracellular ROS and inhibiting apoptosis via deactivation of the MAPK and Akt pathways in Vero cells. In the future, additional experiments are needed, such as how taurine, an amino acid abundant in TVE, acts on cytoprotection, ROS generation, and antioxidant mechanisms. In conclusion, this study suggests that T. cornutus visceral tissue, a marine by-product, can stimulated Vero cells. ImageJ software was used to determine the intensity of the bands. Data represent the mean ± standard deviation from triplication. ### p < 0.001 as compared to the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with the H 2 O 2 −treated group. TVE, T. cornutus visceral tissue hot water extract; H 2 O 2 , hydrogen peroxide; MAPK, mitogen-activated protein kinase.

Conclusions
The visceral tissue of T. cornutus is considered a post-harvest by-product. Several studies have reported that visceral tissues of T. cornutus have antioxidant and anti-inflammatory properties. The present study demonstrated that TVE, a taurine-rich protein source, protected cells against H 2 O 2 -induced oxidative stress by scavenging intracellular ROS and inhibiting apoptosis via deactivation of the MAPK and Akt pathways in Vero cells. In the