Anti-fatigue activity of a mixture of seahorse (Hippocampus abdominalis) hydrolysate and red ginseng
© The Author(s) 2017
Received: 22 December 2016
Accepted: 14 February 2017
Published: 17 March 2017
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, H2O2-induced oxidative stress caused a 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.
KeywordsHippocampus abdominalis Anti-fatigue activity C2C12 myoblast Mixture of Hippocampus abdominalis hydrolysate and red ginseng
Seahorse is a well-known ingredient in traditional Chinese medicine and is used as an invigorant for the treatment of erectile dysfunction, impotence, wheezing, and nocturnal enuresis. Modern scientific research has proven the pharmaceutical effects of seahorse. Hippocampus kuda has various bioactivities such as anti-tumor, anti-aging, and anti-fatigue as well as Ca2+ channel blocking properties (Kumaravel et al. 2010). A peptide derived from H. kuda has been shown to be effective in chondrocytes and inflammatory arthritis (Kumaravel et al. 2012). In addition, seahorses have a putative free radical scavenging effect in controlling aging process (Kumaravel et al. 2012). However, the natural source of seahorse has dramatically reduced owing to overfishing, unsustainable trade, and habitat destruction (Qian et al. 2012). Therefore, seahorses became the first commercially valuable marine genus to be protected and included in Appendix II of the Convention on International Trade in Endangered Species (CITES) in 2004 (Segade et al. 2015).
Hippocampus abdominalis is one of the largest seahorse species growing up to 35 cm in length (Perera et al. 2016). It was validated for use as a food ingredient by the Ministry of Food and Drug Safety in February 2016. However, the validation was restricted to 50% of the entire composition. For use in food, we should try to prepare a mixture of H. abdominalis by adding other materials. In addition, biological activities of H. abdominalis have rarely been reported so far.
Fatigue is a common distressing condition accompanied by a feeling of extreme physical or mental tiredness that often results in diverse disorders such as anemia, thyroid dysfunction, premature aging, and depression. It could also have adverse effects on work efficiency, physical activities, life quality, and social relationships (Huang et al. 2011). Fatigue is caused by sleep deprivation, inadequate rest, low mood, stress, nutritional imbalance, insufficient exercise, as well as side effects of medications. Chronic fatigue is a persistent unexplainable fatigue lasting for more than 6 months, and it is considered a complex symptom of various neurological, psychiatric, and systemic diseases (Huang et al. 2014). Recently, many researchers have presented the results on the anti-fatigue activity of natural products (Yu et al. 2008; Zhang et al. 2006). Especially, red ginseng 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). On the other hand, anti-fatigue activity of seahorse has not been scientifically proven although seahorse is a well-known traditional Chinese medicine.
Oxidative stress is caused by an imbalance between reactive oxygen species (ROS) and antioxidant molecules. Excess accumulation of ROS causes oxidative damage by reacting with biomolecules including DNA, membrane lipids, cellular proteins, and diverse pathological states (Kang et al. 2013). Oxidative stress and ROS are the most important causes of exercise-induced disturbances (Fan et al. 2016). In particular, an oxidative imbalance in the skeletal muscle results in increased muscle fatigability. Thus, antioxidants can be used to alleviate fatigue by counteracting the oxidative stress (Nam et al. 2016).
In this study, the effect of H. abdominalis on muscles was investigated to scientifically verify its potential bioactivity. Also, the anti-fatigue activity of a mixture comprising H. abdominalis and red ginseng was investigated to evaluate the synergy effect and to utilize H. abdominalis in the food market. The anti-fatigue activity of H. abdominalis and a mixture was evaluated by measuring the levels of physical fatigue-related biomarkers such as serum glycogen and ATP contents.
H. abdominalis was kindly donated by Corea Center of Ornamental Reef & Aquariums CCORA (Jeju, Korea) and lyophilized at −70 °C using a freeze dryer. The lyophilized H. abdominalis powder was stored at −80 °C until use. Red ginseng extract containing 30% saponin was purchased from ILHWA Co., LTD. (Gyeonggi, Korea) and lyophilized at −70 °C using a freeze dryer. The lyophilized red ginseng powder was stored at −80 °C until use. Alcalase, a commercial food-grade protease, was purchased from Novozyme Co. (Novozyme Nordisk, Bagsvaerd, Denmark). The other chemicals and reagents used were of analytical grade.
Preparation of H. abdominalis hydrolysate (HH)
The enzymatic hydrolysis of H. abdominalis was performed using Alcalase under optimal conditions (50 °C and pH 8). The dried H. abdominalis powder was homogenized in distilled water and hydrolyzed using the enzyme at an enzyme/substrate (E/S) ratio of 1:100 for enzymatic reactions. The optimal pH of the homogenates was adjusted before enzymatic hydrolysis. The mixture was incubated for 24 h at the optimal temperature for each homogenate, with stirring, and then boiled for 10 min at 100 °C to inactivate the enzyme. After filtration, all hydrolysates were stored at −70 °C for further experiments.
Preparation of the H. abdominalis mixture
Percentage of HH and RG to prepare the H. abdominalis mixtures
The mixture (%)
The C2C12 myoblasts obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), streptomycin (100 mg/mL), and penicillin (100 u/mL) at 37 °C in a 5% CO2 humidified incubator. To induce differentiation, 80% confluent cultures were switched to DMEM containing 2% horse serum (HS) for 6 days with medium changes every other day.
The cytotoxicity of the samples on C2C12 myoblasts was determined by colorimetric MTT assays. The cells were seeded at 5 × 104 cells per well into 48-well plates. After the induction of differentiation, the cells were treated with various concentrations of the sample and incubated for an additional 24 h at 37 °C. MTT stock solution (100 μL; 2 mg/mL in PBS) was then added to each well. After incubating for 4 h, the plate was centrifuged at 500 g for 10 min, and the supernatant was aspirated. The formazan crystals in each well were dissolved in dimethyl sulfoxide (DMSO). The amount of purple formazan was determined by measuring the absorbance at 540 nm.
Cell proliferation assay
The cell proliferation effect of the samples on C2C12 myoblasts was determined by using 5-bromo-2′-deoxyuridine (BrdU) assay (Millipore, Billerica, MA, USA). The cells were seeded at 1 × 104 cells per well into 48-well plates. After the induction of differentiation by switching media, the cells were treated with various concentrations of the sample and incubated for an additional 72 h at 37 °C. Then, the cell proliferation was determined by BrdU reagent following manufacture protocol. In briefly, 10 μL of BrdU reagent was added to each well and the cells were incubated for 2 h. After incubation, the cells were fixed using 100 μL fixing solution. Then, the cells were washed using wash buffer and 50 μL of anti-BrdU monoclonal was added to each well and the cells were incubated for 1 h at RT. The cells were washed using wash buffer and 50 μL of goat anti-mouse IgG was added to each well, and the cells were incubated for 30 min at RT. Also, 50 μL of TMB substrate was added to each well and then, 50 μL of stop solution was added to each well. Finally, the cell proliferation was calculated by comparison with the absorbance at 450 nm of standard solutions of BrdU in the non-treated cells.
Anti-fatigue activity in oxidative stress-induced C2C12 myoblasts
The anti-fatigue activity was determined by measuring cell proliferation as well as the glycogen, ATP contents in H2O2-treated C2C12 myoblasts. The cells were seeded into 48-well plates. Then, they were treated with various concentrations of the sample during the differentiation period. After differentiation, fatigue was induced by adding H2O2 to each well at a concentration of 100 μM; then, the cells were incubated for an additional 24 h at 37 °C.
Measurement of fatigue-related biochemical parameters
To investigate the effect of the samples on muscle growth, we determined several factors such as glycogen and ATP contents in C2C12 myoblasts. For analysis of the effects of the sample on glycogen accumulation, the glycogen content in the cells was measured via glycogen assay (Abcam, Cambridge, MA, USA). Glucoamylase hydrolyzes glycogen to glucose, which was then specifically oxidized to form an intermediate product that reacts with OxiRed probe to generate color. The color was detected by measuring the absorbance at 450 nm. To determine ATP contents, the cell lysates were deproteinized with 4 M perchloric acid (PCA) and 2 M KOH, and the supernatant was assessed using ATP assay kits (Abcam, Cambridge, MA, USA).
All measurements were made in triplicate, and all values were represented as means ± SE. The results were subjected to analysis of variance using Tukey’s test to analyze the differences. p < 0.05 and p < 0.01 were considered significant.
Results and discussion
Cytotoxicity of HH
Effect of HH on cell proliferation
Effect of HH on glycogen contents
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 prepare an H. abdominalis mixture by adding other materials.
RG has been frequently used in traditional Asian medicine 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. abdominalis mixtures were prepared by adding different concentrations 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 the mixture of HH and RG on cell proliferation
Anti-fatigue activity of the mixtures of HH and RG
Exercise-induced oxidative stress can cause the increased muscle fatigability. Thus, antioxidants can decrease the oxidative stress and improve the physiological condition (You et al. 2011). Some reports showed that a loach peptide has not only antioxidant activities but also an anti-fatigue effect in mice (You et al. 2011). Actually, the peptide showing in vitro antioxidant activity possesses the in vivo anti-fatigue activity. The peptide acts as the scavenger for DPPH and hydroxyl radicals. Also, the anthocyanins of mulberry fruit have been assessed in vitro antioxidant activity and in vivo anti-fatigue activity (Jiang et al. 2013). These studies showed values of in vitro study to evaluate the potential anti-fatigue activity through in vivo study. In the present study, the mixtures of HH and RG acted as the antioxidant for hydrogen peroxide and showed the anti-fatigue activity on C2C12 myoblast. Furthermore, the mixtures have valuable needs to be investigated through in vivo animal study.
In this study, the effect of H. abdominalis on the muscles was investigated to scientifically verify its potential bioactivity. Also, the anti-fatigue activity of a mixture comprising HH and RG was evaluated to commercially utilize H. abdominalis in food industry. The treatment of HH to C2C12 myoblast induced the cell proliferation and glycogen contents. These results indicated that H. abdominalis had anti-fatigue activity on C2C12 myoblast. Moreover, the treatment of the mixture comprising HH and RG increased cell viability and the content of fatigue-related biomarkers such as glycogen and ATP contents. In particular, the 80% RG mixture showed an optimum effect on cell viability and ATP synthesis activity. These 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 has the potential to ameliorate fatigue condition induced by oxidative stress by increasing the fatigue-related biochemical parameters such as glycogen and ATP contents in C2C12 myoblasts. Therefore, 80% RG to HH mixture can be used in food for ameliorating fatigue in Korea.
This research was financially supported by the Ministry of Trade, Industry, and Energy (MOTIE), Korea, under the “Regional Specialized Industry Development Program” supervised by the Korea Institute for Advancement of Technology (KIAT).
This study was funded from the Ministry of Trade, Industry, and Energy (MOTIE), Korea.
Availability of data and materials
The datasets supporting the conclusions of the article are included within the article. There is no additional data and materials to disclose.
NK contributed to conduct the research and prepare the draft manuscript. SYK contributed to conduct the cell experiments and analyze the biomarker levels. SR managed aquaculture of the seahorse and supported it for this study. JYK contributed to design the study and conduct the experiments. YJJ contributed to monitor the experiments and finalize the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The manuscript has been read and approved by the authors, and none of its parts have been submitted and published elsewhere. The authors also declare that nobody who qualifies for authorship has been excluded from the list of authors.
Ethics approval and consent to participate
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