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Seahorse, a syngnathidae fish, is one of the important organisms used in Chinese traditional medicine. Hippocampus abdominalis, a seahorse species successfully cultured in Korea, was validated for use in food by the Ministry of Food and Drug Safety in February 2016; however. the validation was restricted to 50% of the entire composition. Therefore, to use H. abdominalis as a food ingredient, H. abdominalis has to be prepared as a mixture by adding other materials. In this study, the effect of H. abdominalis on muscles was investigated to scientifically verify its potential bioactivity. In addition, the anti-fatigue activity of a mixture comprising H. abdominalis and red ginseng (RG) was evaluated to commercially utilize H. abdominalis in food industry. H. abdominalis was hydrolyzed using Alcalase, a protease, and the effect of H. abdominalis hydrolysate (HH) on the muscles was assessed in C2C12 myoblasts by measuring cell proliferation and glycogen content. In addition, the mixtures comprising HH and RG were prepared at different percentages of RG to HH (20, 30, 40, 50, 60, 70, and 80% RG), and the anti-fatigue activity of these mixtures against oxidative stress was assessed in C2C12 myoblasts. In C2C12 myoblasts, H O -induced oxidative stress caused a 2 2 decrease in viability and physical fatigue-related biomarkers such as glycogen and ATP contents. However, treatment with RG and HH mixtures increased cell viability and the content of fatigue-related biomarkers. In particular, the 80% RG mixture showed an optimum effect on cell viability and ATP synthesis activity. In this study, all results indicated that HH had anti-fatigue activity at concentrations approved for use in food by the law in Korea. Especially, an 80% RG to HH mixture can be used in food for ameliorating fatigue. Keywords: Hippocampus abdominalis, Anti-fatigue activity, C2C12 myoblast, Mixture of Hippocampus abdominalis hydrolysate and red ginseng Background seahorses have a putative free radical scavenging effect in Seahorse is a well-known ingredient in traditional Chinese controlling aging process (Kumaravel et al. 2012). However, medicine and is used as an invigorant for the treatment of the natural source of seahorse has dramatically reduced erectile dysfunction, impotence, wheezing, and nocturnal owing to overfishing, unsustainable trade, and habitat de- enuresis. Modern scientific research has proven the struction (Qian et al. 2012). Therefore, seahorses became pharmaceutical effects of seahorse. Hippocampus kuda has the first commercially valuable marine genus to be pro- various bioactivities such as anti-tumor, anti-aging, and tected and included in Appendix II of the Convention on 2+ anti-fatigue as well as Ca channel blocking properties International Trade in Endangered Species (CITES) in 2004 (Kumaravel et al. 2010). A peptide derived from H. kuda (Segade et al. 2015). hasbeenshown to be effectiveinchondrocytesand Hippocampus abdominalis is one of the largest sea- inflammatory arthritis (Kumaravel et al. 2012). In addition, horse species growing up to 35 cm in length (Perera et al. 2016). It was validated for use as a food ingre- dient by the Ministry of Food and Drug Safety in * Correspondence: firstname.lastname@example.org; email@example.com February 2016. However, the validation was restricted Equal contributors to 50% of the entire composition. For use in food, we Department of Marine Life Sciences, Jeju National University, Jeju 63243, Korea should try to prepare a mixture of H. abdominalis by Full list of author information is available at the end of the article © The Author(s). 2017 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. Kang et al. Fisheries and Aquatic Sciences (2017) 20:3 Page 2 of 8 adding other materials. In addition, biological lyophilized at −70 °C using a freeze dryer. The lyophilized activities of H. abdominalis have rarely been reported red ginseng powder was stored at −80 °C until use. Alca- so far. lase, a commercial food-grade protease, was purchased Fatigue is a common distressing condition accompanied from Novozyme Co. (Novozyme Nordisk, Bagsvaerd, by a feeling of extreme physical or mental tiredness that Denmark). The other chemicals and reagents used were of often results in diverse disorders such as anemia, thyroid analytical grade. dysfunction, premature aging, and depression. It could also have adverse effects on work efficiency, physical activ- Preparation of H. abdominalis hydrolysate (HH) ities, life quality, and social relationships (Huang et al. The enzymatic hydrolysis of H. abdominalis was per- 2011). Fatigue is caused by sleep deprivation, inadequate formed using Alcalase under optimal conditions (50 °C rest, low mood, stress, nutritional imbalance, insufficient andpH8). Thedried H. abdominalis powder was homog- exercise, as well as side effects of medications. Chronic fa- enized in distilled water and hydrolyzed using the enzyme tigue is a persistent unexplainable fatigue lasting for more at an enzyme/substrate (E/S) ratio of 1:100 for enzymatic than 6 months, and it is considered a complex symptom reactions. The optimal pH of the homogenates was ad- of various neurological, psychiatric, and systemic diseases justed before enzymatic hydrolysis. The mixture was incu- (Huang et al. 2014). Recently, many researchers have pre- bated for 24 h at the optimal temperature for each sented the results on the anti-fatigue activity of natural homogenate, with stirring, and then boiled for 10 min at products (Yu et al. 2008; Zhang et al. 2006). Especially, 100 °C to inactivate the enzyme. After filtration, all hydro- red ginseng has been mainly focused on its anti-fatigue ac- lysates were stored at −70 °C for further experiments. tivity with the ability mitigating exercise-related muscle damage, maintaining homeostasis of the body and enhan- Preparation of the H. abdominalis mixture cing vital energy (Kim et al. 2013; Kim et al. 2016). On the The H. abdominalis mixtures were prepared by adding other hand, anti-fatigue activity of seahorse has not been different concentrations of red ginseng (RG). The lyophi- scientifically proven although seahorse is a well-known lized H. abdominalis powder and RG powder were traditional Chinese medicine. mixed as indicated in Table 1, and these seven mixtures Oxidative stress is caused by an imbalance between re- were labeled as % of RG. active oxygen species (ROS) and antioxidant molecules. Excess accumulation of ROS causes oxidative damage by Cell culture reacting with biomolecules including DNA, membrane The C2C12 myoblasts obtained from American Type lipids, cellular proteins, and diverse pathological states Culture Collection (ATCC, Manassas, VA, USA) were (Kang et al. 2013). Oxidative stress and ROS are the most cultured in Dulbecco’s Modified Eagle Medium (DMEM) important causes of exercise-induced disturbances (Fan supplemented with 10% heat-inactivated fetal bovine et al. 2016). In particular, an oxidative imbalance in the serum (FBS), streptomycin (100 mg/mL), and penicillin skeletal muscle results in increased muscle fatigability. (100 u/mL) at 37 °C in a 5% CO humidified incubator. Thus, antioxidants can be used to alleviate fatigue by To induce differentiation, 80% confluent cultures were counteracting the oxidative stress (Nam et al. 2016). switched to DMEM containing 2% horse serum (HS) for In this study, the effect of H. abdominalis on muscles 6 days with medium changes every other day. was investigated to scientifically verify its potential bio- activity. Also, the anti-fatigue activity of a mixture com- Cell viability prising H. abdominalis and red ginseng was investigated The cytotoxicity of the samples on C2C12 myoblasts to evaluate the synergy effect and to utilize H. abdominalis was determined by colorimetric MTT assays. The cells in the food market. The anti-fatigue activity of H. abdomi- nalis and a mixture was evaluated by measuring the levels Table 1 Percentage of HH and RG to prepare the H. abdominalis of physical fatigue-related biomarkers such as serum mixtures glycogen and ATP contents. HH (%) RG (%) The mixture (%) 80 20 100 Methods 70 30 100 Materials 60 40 100 H. abdominalis was kindly donated by Corea Center of Ornamental Reef & Aquariums CCORA (Jeju, Korea) and 50 50 100 lyophilized at −70 °C using a freeze dryer. The lyophilized 40 60 100 H. abdominalis powder was stored at −80 °C until use. 30 70 100 Red ginseng extract containing 30% saponin was pur- 20 80 100 chased from ILHWA Co., LTD. (Gyeonggi, Korea) and Kang et al. Fisheries and Aquatic Sciences (2017) 20:3 Page 3 of 8 were seeded at 5 × 10 cells per well into 48-well plates. content in the cells was measured via glycogen assay After the induction of differentiation, the cells were (Abcam, Cambridge, MA, USA). Glucoamylase hydrolyzes treated with various concentrations of the sample and glycogen to glucose, which was then specifically oxidized incubated for an additional 24 h at 37 °C. MTT stock so- to form an intermediate product that reacts with OxiRed lution (100 μL; 2 mg/mL in PBS) was then added to each probe to generate color. The color was detected by meas- well. After incubating for 4 h, the plate was centrifuged uring the absorbance at 450 nm. To determine ATP con- at 500 g for 10 min, and the supernatant was aspirated. tents, the cell lysates were deproteinized with 4 M The formazan crystals in each well were dissolved in di- perchloric acid (PCA) and 2 M KOH, and the supernatant methyl sulfoxide (DMSO). The amount of purple forma- was assessed using ATP assay kits (Abcam, Cambridge, zan was determined by measuring the absorbance at MA, USA). 540 nm. Statistical analysis Cell proliferation assay All measurements were made in triplicate, and all values The cell proliferation effect of the samples on C2C12 were represented as means ± SE. The results were sub- myoblasts was determined by using 5-bromo-2′-deox- jected to analysis of variance using Tukey’s test to yuridine (BrdU) assay (Millipore, Billerica, MA, USA). analyze the differences. p < 0.05 and p < 0.01 were con- The cells were seeded at 1 × 10 cells per well into 48- sidered significant. well plates. After the induction of differentiation by switching media, the cells were treated with various con- centrations of the sample and incubated for an add- Results and discussion itional 72 h at 37 °C. Then, the cell proliferation was Cytotoxicity of HH determined by BrdU reagent following manufacture Cell viability was estimated using the MTT assay, which protocol. In briefly, 10 μL of BrdU reagent was added to is a test of metabolic competence predicated upon the each well and the cells were incubated for 2 h. After in- assessment of mitochondrial performance. It is a colori- cubation, the cells were fixed using 100 μL fixing solu- metric assay, which is dependent on the conversion of tion. Then, the cells were washed using wash buffer and yellow tetrazolium bromide to its purple formazan de- 50 μL of anti-BrdU monoclonal was added to each well rivative by mitochondrial succinate dehydrogenase in the and the cells were incubated for 1 h at RT. The cells viable cells (Kang et al. 2012). The viabilities of C2C12 were washed using wash buffer and 50 μL of goat anti- myoblasts treated with different concentrations of HH mouse IgG was added to each well, and the cells were (50, 100, 150, and 200 μg/mL) were expressed to repre- incubated for 30 min at RT. Also, 50 μL of TMB sub- sent 100% viability (the viability of control cells; Fig. 1). strate was added to each well and then, 50 μL of stop so- In a preliminary experiment, HH concentrations up to lution was added to each well. Finally, the cell 200 μg/mL showed no significant cytotoxicity for 24 h. proliferation was calculated by comparison with the ab- sorbance at 450 nm of standard solutions of BrdU in the Effect of HH on cell proliferation non-treated cells. HH significantly enhanced cell proliferation in C2C12 myo- blasts compared with the control cells (Fig. 2). In particular, Anti-fatigue activity in oxidative stress-induced C2C12 HH treatment induced cell proliferation in a concentration- myoblasts dependent manner in the range of 100–200 μg/mL. The The anti-fatigue activity was determined by measuring cell numbers increased approximately 1.8-fold by HH treat- cell proliferation as well as the glycogen, ATP contents ment at 200 μg/mL concentration (Fig. 2). in H O -treated C2C12 myoblasts. The cells were 2 2 seeded into 48-well plates. Then, they were treated with various concentrations of the sample during the differen- Effect of HH on glycogen contents tiation period. After differentiation, fatigue was induced The skeletal muscles are the major site of glycogen stor- by adding H O to each well at a concentration of age in the body (Deshmukh et al. 2015). The glycogen 2 2 100 μM; then, the cells were incubated for an additional content in C2C12 myoblasts was increased by HH treat- 24 h at 37 °C. ment at concentrations of 50 and 100 μg/mL (Fig. 3). In C2C12 myoblasts, HH (100 μg/mL) increased glycogen Measurement of fatigue-related biochemical parameters content by 1.5-fold compared with that in the control To investigate the effect of the samples on muscle growth, cells. However, HH treatment at higher concentrations we determined several factors such as glycogen and ATP (150 and 200 μg/mL) decreased glycogen content. Thus, contents in C2C12 myoblasts. For analysis of the effects of it can be suggested that high concentrations of HH sup- the sample on glycogen accumulation, the glycogen pressed glycogen content. Kang et al. Fisheries and Aquatic Sciences (2017) 20:3 Page 4 of 8 1.5 0.5 CON 50 100 150 200 Seahorse hydrolysate (µg/mL) CON 50 100 150 200 Fig. 1 Cytotoxicity of seahorse hydrolysate on C2C12 myoblasts. The Seahorse hydrolysate (µg/mL) cell was treated with various concentrations of seahorse hydrolysate Fig. 3 Effect of seahorse hydrolysate on glycogen content in C2C12 (50, 100, 150, and 200 μg/mL) and incubated for 24 h. The cytotoxicity myoblasts. The cells were incubated with various concentrations of was assessed by MTT assay. The experiment was performed in seahorse hydrolysate (50, 100, 150, and 200 μg/mL) for 24 h. The triplicate. Each value indicates the mean ± standard error from three glycogen contents were assessed. The experiment was performed in independent experiments triplicate. Each value indicates the mean ± standard error from three independent experiments. *p < 0.05, **p < 0.01 Cytotoxicity of the mixture of HH and RG Although H. abdominalis was validated for use in food by the Ministry of Food and Drug Safety in February 2016, the validation was restricted to 20% of the entire composition. To use H. abdominalis as a food ingredient, we should pre- pare an H. abdominalis mixture by adding other materials. RG has been frequently used in traditional Asian medi- cine to treat many disorders, such as debility, aging, stress, diabetes, and insomnia (Tang et al. 2008). Especially, RG has been mainly focused on its anti-fatigue activity with the ability mitigating exercise-related muscle damage, maintaining homeostasis of the body, and enhancing vital energy (Kim et al. 2013; Kim et al. 2016). Thus, H. abdo- minalis mixtures were prepared by adding different con- centrations of RG (20, 30, 40, 50, 60, 70, and 80% of RG) to investigate the synergy effect between H. abdominalis and RG on anti-fatigue activity. Effect of these mixtures on cell viability was estimated using the MTT assay. The viabilities of C2C12 myoblasts treated with the mixtures at different concentrations (50, CON 50 100 150 200 100, 200, 250, and 500 μg/mL) were expressed to represent over 90% viability, which was similar to that of the control Seahorse hydrolysate (µg/mL) cells (Fig. 4). Thus, mixtures up to 500 μg/mL concentra- Fig. 2 Cell proliferation of seahorse hydrolysate on C2C12 myoblast. The tion did not show any significant cytotoxicity for 24 h. cell was treated with various concentrations of seahorse hydrolysate (50, 100, 150, and 200 μg/mL) and incubated for 72 h. The cell proliferation was assessed by BrdU assay. The experiment was performed in triplicate. Effect of the mixture of HH and RG on cell proliferation Each value indicates the mean ± standard error from three independent To assess the effect of HH (0% of RG) and RG mixture experiments. *p < 0.05, **p <0.01 on muscle growth, cell proliferation was measured as Cell proliferation ( ) Cell viability ( ) Glycogen contents (of control) Kang et al. Fisheries and Aquatic Sciences (2017) 20:3 Page 5 of 8 50 µg/mL 100 µg/mL 200 µg/mL 250 µg/mL 500 µg/mL CON 20304050607080 Mixture of Seahorse hydrolysate and RG ( of RG) Fig. 4 Cytotoxicity of the six mixtures of seahorse hydrolysate and RG on C2C12 myoblasts. The cell was treated with various concentrations of the six mixtures (50, 100, 150, 200, 250, and 500 μg/mL) and incubated for 24 h. The cytotoxicity was assessed by MTT assay. The experiment was performed in triplicate. Each value indicates the mean ± standard error from three independent experiments shown in Fig. 5. HH and RG mixtures showed the sig- The anti-fatigue activity of the mixtures of HH and RG nificant effect on cell proliferation. Especially, at the low was assessed in H O -treated C2C12 myoblasts by 2 2 concentrations (200 and 250 μg/mL), HH significantly measuring cell proliferation as well as the glycogen and enhanced proliferation of C2C12 myoblasts compared ATP contents. Severe and continuous exercise may ele- with that of mixtures as well as that of the control cells. vate the formation of ROS, thereby increasing oxidative However, at the high concentrations (400 and 500 μg/ stress. A sustained elevated oxidative stress can hamper mL), HH and RG mixtures did not show any significant mitochondrial function resulting in low ATP synthesis effects on cell proliferation to each other. and increased lactic acid in the muscles, consequently decreasing the physical efficiency. These observations Anti-fatigue activity of the mixtures of HH and RG suggest that improving the antioxidant status may en- Several biomarkers such as lactate dehydrogenase hance the overall physical performance by maintaining (LDH), glycogen, aspartate transaminase (AST), and ala- the pro-oxidant/antioxidant balance (Swamy et al. 2011). nine transaminase (ALT) have been used to investigate To induce oxidative stress in C2C12 myoblasts, the cells muscle injury during exhaustive exercise (Huang et al. were incubated with H O at a concentration of 2 2 2015). Also, fatigue is related to mitochondrial dysfunc- 100 μM. After H O treatment, the viability of C2C12 2 2 tion and diminished ATP levels (Singh and Singh 2014). myoblasts decreased to less than 60% compared to that 200 µg/mL 250 µg/mL 300 µg/mL 400 µg/mL 500 µg/mL CON 0 20 40 60 80 100 Mixture of Seahorse hydrolysate and RG ( of RG) Fig. 5 Cell proliferation of the six mixtures of seahorse hydrolysate and RG on C2C12 myoblast. The cell was treated with various concentrations of the six mixtures (50, 100, 150, and 200 μg/mL) and incubated for 72 h. The cell proliferation was assessed by BrdU assay. Effect of different mixtures of seahorse hydrolysate and RG on C2C12 myoblast proliferation. The experiment was performed in triplicate. Each value indicates the mean ± standard error from three independent experiments Cell proliferation ( ) Cell viability ( ) Kang et al. Fisheries and Aquatic Sciences (2017) 20:3 Page 6 of 8 300 µg/mL 500 µg/mL CON 0 20 40 60 80 100 Mixture of Seahorse hydrolysate and RG ( of RG) + H O 100 µM 2 2 Fig. 6 Protective effect of the six mixtures of seahorse hydrolysate and RG against H O -treated C2C12 myoblasts. The treatment of H O induced 2 2 2 2 a decrease in cell viability. All mixtures showed protective effect on H O -induced oxidative stress in C2C12 myoblasts. The experiment was 2 2 performed in triplicate. Each value indicates the mean ± standard error from three independent experiments in the control cells (Fig. 6). However, C2C12 myoblasts treated cells was lower than that of the control cells. treated with HH and RG mixtures showed increased via- However, in C2C12 myoblasts, the treatment with HH bility compared with that reported for the control cells. and RG mixtures increased the glycogen content to Notably, at a sample concentration of 500 μg/mL, cell more than double as compared to the values reported viability increased steadily with increasing percentage of for the control cells. In particular, RG60, RG80, and RG except at 100% RG, where only RG was present in RG100 showed increased glycogen content at a sample the mixture. concentration of 300 and 500 μg/mL (Fig. 7). Glycogen contents ATP contents Energy expenditure during exercise leads to physical fa- Muscular exercise causes rapid ATP consumption, and tigue, which is mainly caused by energy consumption energy deficiency is a critical reason for physical fatigue. and deficiency. Catabolized fat and carbohydrates are Therefore, compounds that promote ATP production considered the main sources of energy in the skeletal could be candidates for alleviating physical fatigue. The muscles during exercise, and glycogen is the predomin- skeletal muscle mainly catabolizes fat and carbohydrates ant source of glycolysis for energy production. There- as sources of energy during exercise (Nozawa et al. fore, glycogen storage directly affects exercise ability 2009). ATP content in the H O -treated cells was lower 2 2 (Wu et al. 2013). The glycogen content of the H O - than that in the control cells (Fig. 8). Although HH and 2 2 3.5 300 µg/mL 500 µg/mL 2.5 1.5 0.5 CON 0 20 40 60 80 100 Mixture of Seahorse hydrolysate and RG ( of RG) + H O 100 µM 2 2 Fig. 7 Effect of the six mixtures of seahorse hydrolysate and RG on glycogen content in H O -treated C2C12 myoblasts. H O treatment induced 2 2 2 2 a decrease in glycogen contents. All mixtures showed protective effect on H O -induced oxidative stress in C2C12 myoblasts. The experiment 2 2 was performed in triplicate. Each value indicates the mean ± standard error from three independent experiments Glycogen contents (of contorol) Cell viability ( ) Kang et al. Fisheries and Aquatic Sciences (2017) 20:3 Page 7 of 8 1.2 300 µg/mL 500 µg/mL 0.8 0.6 0.4 0.2 CON 0 20 40 60 80 100 Mixture of Seahorse hydrolysate and RG ( of RG) + H O 100 µM 2 2 Fig. 8 Effect of the six mixtures of seahorse hydrolysate and RG on ATP synthesis in H O -treated C2C12 myoblasts. The treatment of H O 2 2 2 2 induced a decrease in ATP contents. All mixtures showed a protective effect against H O -induced oxidative stress in C2C12 myoblasts. The 2 2 experiment was performed in triplicate. Each value indicates the mean ± standard error from three independent experiments RG mixtures did not increase the ATP content, RG80 contents. In particular, the 80% RG mixture showed an relatively increased the ATP content at 300 and 500 μg/ optimum effect on cell viability and ATP synthesis activ- mL concentrations of the mixture. ity. These results indicated that HH had anti-fatigue ac- Exercise-induced oxidative stress can cause the in- tivity at concentrations approved for use in food by the creased muscle fatigability. Thus, antioxidants can de- law in Korea. Especially, an 80% RG to HH mixture has crease the oxidative stress and improve the physiological the potential to ameliorate fatigue condition induced by condition (You et al. 2011). Some reports showed that a oxidative stress by increasing the fatigue-related bio- loach peptide has not only antioxidant activities but also chemical parameters such as glycogen and ATP contents an anti-fatigue effect in mice (You et al. 2011). Actually, in C2C12 myoblasts. Therefore, 80% RG to HH mixture the peptide showing in vitro antioxidant activity pos- can be used in food for ameliorating fatigue in Korea. sesses the in vivo anti-fatigue activity. The peptide acts Abbreviations as the scavenger for DPPH and hydroxyl radicals. Also, HH: Hippocampus abdominalis; RG: Red ginseng the anthocyanins of mulberry fruit have been assessed Acknowledgements in vitro antioxidant activity and in vivo anti-fatigue ac- This research was financially supported by the Ministry of Trade, Industry, and tivity (Jiang et al. 2013). These studies showed values of Energy (MOTIE), Korea, under the “Regional Specialized Industry Development in vitro study to evaluate the potential anti-fatigue activ- Program” supervised by the Korea Institute for Advancement of Technology (KIAT). ity through in vivo study. In the present study, the mix- Funding tures of HH and RG acted as the antioxidant for This study was funded from the Ministry of Trade, Industry, and Energy hydrogen peroxide and showed the anti-fatigue activity (MOTIE), Korea. on C2C12 myoblast. Furthermore, the mixtures have Availability of data and materials valuable needs to be investigated through in vivo animal The datasets supporting the conclusions of the article are included within study. the article. There is no additional data and materials to disclose. Authors’ contributions Conclusions NK contributed to conduct the research and prepare the draft manuscript. In this study, the effect of H. abdominalis on the mus- SYK contributed to conduct the cell experiments and analyze the biomarker levels. SR managed aquaculture of the seahorse and supported it for this cles was investigated to scientifically verify its potential study. JYK contributed to design the study and conduct the experiments. YJJ bioactivity. Also, the anti-fatigue activity of a mixture contributed to monitor the experiments and finalize the manuscript. All comprising HH and RG was evaluated to commercially authors read and approved the final manuscript. utilize H. abdominalis in food industry. The treatment Competing interests of HH to C2C12 myoblast induced the cell proliferation The authors declare that they have no competing interests. and glycogen contents. These results indicated that H. abdominalis had anti-fatigue activity on C2C12 myo- Consent for publication The manuscript has been read and approved by the authors, and none of its blast. Moreover, the treatment of the mixture compris- parts have been submitted and published elsewhere. The authors also ing HH and RG increased cell viability and the content declare that nobody who qualifies for authorship has been excluded from of fatigue-related biomarkers such as glycogen and ATP the list of authors. ATP contents (of control) Kang et al. Fisheries and Aquatic Sciences (2017) 20:3 Page 8 of 8 Ethics approval and consent to participate Wu R-E, Huang W-C, Liao C-C, Chang Y-K, Kan N-W, Huang C-C. Resveratrol protects Not applicable. against physical fatigue and improves exercise performance in mice. Molecules. 2013;18:4689–702. Author details You L, Zhao M, Regenstein J-M, Ren J. In vitro antioxidant activity and in vivo Department of Marine Life Sciences, Jeju National University, Jeju 63243, anti-fatigue effect of loach (Misgurnus anguillicaudatus) peptides prepared by Korea. Center of Ornamental Reefs and Aquariums, Jeju 63354, Korea. papain digestion. 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Segade Á, Robaina L, Otero-Ferrer F, García Romero J, Molina Domínguez L. � Our selector tool helps you to ﬁnd the most relevant journal Effects of the diet on seahorse (Hippocampus hippocampus) growth, body � We provide round the clock customer support colour and biochemical composition. Aquacult Nutr. 2015;21:807–13. � Convenient online submission Singh T, Singh K. Mitochondrial dysfunction and chronic fatigue syndromes: issues in clinical care (modified version). IOSR J Dental Med Sci. 2014;13:30–3. � Thorough peer review Swamy M, Naveen S, Singsit D, Naika M, Khanum F. Anti-fatigue effects of � Inclusion in PubMed and all major indexing services polyphenols extracted from pomegranate peel. Int J Integr Biol. 2011;11:69–72. � Maximum visibility for your research Tang W, Zhang Y, Gao J, Ding X, Gao S. The anti-fatigue effect of 20 (R)- ginsenoside Rg3 in mice by intranasally administration. Biol Pharm Bull. 2008; Submit your manuscript at 31:2024–7. www.biomedcentral.com/submit
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