Effects of intragastric administration of five oyster components on endurance exercise performance in mice

Abstract

Context: Oysters [Crassostrea plicatula Gmelin (Ostreidae)] are widely used for food in coastal areas. It is reported to have several qualities such as improving sexual and immune function. They has been approved by Chinese Ministry of Health as a functional food.

Objective: The effects of five types of oyster components (oyster meat, oyster glycogen, oyster protein, cooked liquid components, and water-insoluble components) on the swimming endurance of mice were investigated.

Materials and methods: First, the amino acid composition and sugar content of the five oyster components were analyzed by a physicochemical test. In the in vivo test, the control group was administered distilled water, and the five intervention groups were treated with various samples for 15 consecutive days [0.8 mg protein/(g BW·d) or 0.2 mg glycogen/(g BW·d)]. The swimming time was recorded through the exhaustive swimming test. The levels of serum lactic acid, blood urea nitrogen (BUN), liver glycogen, and gastrocnemius muscle glycogen were determined.

Results: Oyster protein had a minimum F-value (the mole ratio of branched-chain amino acids to aromatic amino acids) (2.68), contained 1.85 mmol/mL taurine and no sugar. The components (except for oyster protein) significantly improved endurance capacity of mice and increased the liver and muscle glycogen contents (p < 0.05), and reduced the lactic acid and BUN levels (p < 0.05). Oyster protein had little effect.

Discussion and conclusion: The effects of oyster components on the swimming endurance of mice may be attributed to the high ratio of the branched-chain amino acid composition, bioactivity of taurine, and glycogen.

• Biochemical analysis
• fatigue
• nutritional ingredient

Introduction

Fatigue is best defined as difficulty initiating or sustaining voluntary activities (You et al., 2011). Fatigue is accompanied by a feeling of extreme physical or mental tiredness, resulting from severe stress and hard physical or mental work (Huang et al., 2011). This common condition impairs the daily functions in human life and is manifested in various diseases such as hypertension, diabetes, and chronic heart failure (Shimizu et al., 2010). Fatigue may represent the final pathway to which many factors may contribute; therefore, its pathophysiology is multifactorial (Raison et al., 2009). Recent studies have reported that the main methods to eliminate fatigue include the reduction of free radicals (Smith & Reid, 2006; Zheng et al., 2012), administration of energy substances (Nozaki et al., 2009), and acceleration of the elimination of metabolic products (Ma et al., 2008). Pharmacologists have developed several drugs to resolve this condition (Wang et al., 2008). The report documenting vitamin E reduced both resting and exercise-induced pentane production in humans was reported by Dillard et al. (1978). Many kinds of liquid nutritive and tonic crude drugs (NTDs) including vitamins and amino acids are widely used in Japan (Tadano et al., 2003). However, these drugs often have several negative effects (Zlott & Byrne, 2010). The best drug candidates improve exercise capacity, postpone fatigue, and accelerate the elimination of fatigue in human beings, but also have few side effects (Akazawa et al., 2010). Thus, nutritionists and athletic physiologists are dedicated in looking for bioactive products from natural resources. Recently, many bioactive materials with potent antifatigue activity were extracted from natural resources (Huang et al., 2011). Although these bioactive materials are less potent than the synthetic ones, they do not exhibit side effects.

Marine creatures are rich sources of diverse bioactive compounds (Kim & Wijesekara, 2010). Given that marine species comprise approximately half of the total global biodiversity, the development of marine creatures as a source of novel bioactive substances is growing rapidly (Aneiros & Garateix, 2004). Functional materials from the marine environment include polyunsaturated fatty acids, polysaccharides, minerals, vitamins, antioxidants, enzymes, and bioactive peptides (Kim et al., 2002). Fatigue can be reduced by several bioactive materials from marine creatures, such as jellyfish collagen hydrolysate (Ding et al., 2011), grass carp protein or peptide (Ren et al., 2008), loach peptides (You et al., 2011), etc.

Oyster farming is the primary aquaculture process in the coastal waters of China, with a production of approximately 350 000 tons annually. Thus, China is the leading oyster producer in the world. In many parts of the world, people have fresh oyster consumption habits. Some people processed oyster into kinds of foods. Oyster sauce is the main product, and it is widely used in southeast and east Asian countries because of increased consumer interest in their taste and flavor (Je et al., 2005a). Some are processed into dried oyster and oyster glycogen. The nutritive value of oysters is very high. The nutritional value (dry flesh weight basis) of oysters are as follows: fat, 7.8–8.7%; glycogen, 21.6–38.9%; and protein, 39.1–53.1% (Linehan et al., 1999).

Herbal medicines and natural compounds have been investigated as an important resource for postponing fatigue, accelerating the elimination of fatigue-related metabolites, and improving athletic ability (Huang et al., 2012), such as Atriplex lentiformis (Torr.) S. Wats (Chenopodiaceae) (Awaad et al., 2012) and Cordyceps sinensis Berkeley (Ascomycetes) (Kumar et al., 2011). Compared with them, oyster is a food material, and it is used widely to treat fatigue by Chinese coastal residents as a traditional Chinese medicine. Oyster has long been considered a potent remedy with few adverse effects. However, the source of the bioactivity has not been fully understood. In the current study, we prepared minced oyster meat, oyster glycogen, oyster protein, cooked liquid component, and water-insoluble component. The oyster sauce was made from cooked liquid component, and the production of dried oyster was similar to that of the water-insoluble component. This study evaluates the antifatigue action of the oral administration of these samples on mice through exhaustive swimming time and on some biochemical parameters including liver glycogen, gastrocnemius muscle glycogen, serum lactic acid, and blood urea nitrogen (BUN).

Materials and methods

Materials

Edible parts of the flesh oyster [Crassostrea plicatula Gmelin (Ostreidae)] were purchased from a local market in Xiamen, China, and minced using a chopper (Braun Co., Frankfurt, Germany).

Preparation of samples

The oyster glycogen was prepared according to the method of Kanoh et al. (2004) and Nunesa et al. (2008) with some modifications. Minced oyster meat (100 g) was mixed with 100 mL deionized water. The mixture was digested for 30 min with 100 mL of 30% (w/v) KOH solution at 100 °C. After cooling to room temperature, 150 mL of pure ethanol was added to ensure total glycogen precipitation. The precipitate was washed with ethanol. The aqueous glycogen was subsequently vacuum dried.

Minced oyster meat was mixed with a triple volume of organic solvent mixture (v_{trichloromethane}:v_methanol = 1 : 1). Extraction was conducted for 1 h at 50 °C, then cooled and filtered. This procedure was repeated three-times. The residue was dried in air. Then, α-amylase and diastatic enzyme were added to hydrolyze the polysaccharide until no sugar was detected. The solution was centrifuged for 10 min at 4000 rpm. The sediment was dried at 60 °C and stored in a desiccator for further use.

Minced oyster meat was mixed with a 10-fold volume of deionized water and boiled for 20 min. Then, the mixture was centrifuged for 20 min at 4000 rpm. The supernatant was cooked and served as the liquid component, and the sediment was the water-insoluble component.

Amino acid determination and total sugar assay

The amino acid profile was determined according to the method of Cao et al. (2009), using the amino acid analyzer with a C18 high-performance liquid chromatography column. Total sugar was quantified photometrically using the phenol–sulfuric acid method, with dextrose used as a standard (Cuesta et al., 2003).

Animal experiment

All procedures with animal subjects have been approved by The Ethics Committee of the Jimei University (SYXK (Min) 2012-0005). A total of 120 male Kunming mice [specific pathogen-free grade, Approval No. SCXK-(Min) 2008-B001], with an average weight of 20 ± 2 g, were used in the experiment. The mice (five per cage) were housed in an air-conditioned specific pathogen-free grade level laboratory (25 ± 2 °C). The mice were grown under moderate humidity (50 ± 10%) with a 12 h/12 h light/dark cycle. Noise was less than 60 dB. The mice were acclimatized to their environment for 3 d before starting the experiments. They had free access to water and a balanced murine diet. After adaptation, the mice were randomly assigned to six groups with 20 mice each (Table 1). Samples were administered to the mice by intragastric administration 0.5 h before swimming training every day at 9:00 a.m.

Table 1. Assigned mouse groups.

Group name	No. of mice	Diets
C (Control group)	20	Basal diet^a+ distilled water
O (Oyster group)	20	Basal diet + 0.8 mg protein /(g BW·d)^b
G (Glycogen group)	20	Basal diet + 0.2 mg glycogen /(g BW·d)^c
P (Protein group)	20	Basal diet + 0.8 mg protein /(g BW·d)^b
L (Cooked liquid  components group)	20	Basal diet + 0.8 mg protein /(g BW·d)^b
I (Water-insoluble  components group)	20	Basal diet + 0.8 mg protein /(g BW·d)^b

^aA balanced murine basal diet can be obtained freely at any time.

^bAccording to the crude protein content of each sample, 0.8 mg protein/(g BW·d) meant one mouse can get 0.8 mg crude protein supplementation per g body weight daily.

^cAbout 0.2 mg glycogen/(g BW·d) meant 0.2 mg oyster glycogen supplementation per g body weight daily.

Training program

A special swim-training model was used. Mice were placed in a swimming tank (50 cm × 50 cm × 40 cm) with 30 cm deep water and subjected to swim-training at 30 °C water after the sample administration. During the exercise protocol, the mice in the control group were kept in a plastic cage containing approximately 3 cm of water maintained at the same temperature to exclude potential stress and other potential confounding effects (Kumar et al., 2011). The training lasted for 14 d. During the first 2 d, the mice swam for 20 min. Then, the swimming time was extended by 10 min every 2 d. On the 15th day, the final body weight was measured. The 20 mice housed in each group were randomly divided into two subgroups with 10 mice each. One subgroup was loaded with a lead block weighing approximately 2% of their bodyweight attached to the tail (Huang et al., 2011). Then, the mice were subjected to the exhaustive swimming test (time to exhaustion was defined as the time when the mouse failed to rise to the surface to breathe). After the exhaustive swimming test, blood was collected from the mice for further research. The other subgroup was used for collecting liver and gastrocnemius muscle after forced unloading swimming for 80 min.

Biochemical analysis related to fatigue

At the end of the exhaustive swimming test, blood was collected from the orbital sinus to determine serum lactic acid and BUN levels (Jung et al., 2007). The assay of these biochemical parameters was performed using commercially available kits (Product nos. A019 and C013-2) from the Institute of Biological Engineering of Nanjing Jiancheng (Nanjing, China).

Mouse livers were collected to determine the liver glycogen content. The liver was dissected immediately after it was removed, washed with 0.9% saline, blotted dry with filter papers, quick-frozen in liquid nitrogen, and stored at −80 °C (Giesel et al., 2009; Pederson et al., 2005). Liver samples were accurately weighed and homogenized in 8 mL of homogenization buffer for liver glycogen analysis. The gastrocnemius muscle was also removed and weighed for muscle glycogen analysis. The liver/muscle glycogen content analysis was performed using the Assay Kit A043 (Institute of Biological Engineering of Nanjing Jiancheng, Nanjing, China). All the assays were conducted by strictly following the recommended procedures.

Statistical analysis

All the tests were conducted in triplicate. The experimental data were expressed as mean ± standard deviation. The results were subjected to one-way ANOVA. Duncan and Dunnett’s T3 tests were performed to determine the significant difference between the samples within the 95% confidence interval using SPSS 12.0 software (SPSS Inc., Chicago, IL).

Results

Amino acid and sugar contents of the five oyster components

In this study, the mole ratio of branched-chain amino acids (BCAA) to aromatic amino acids was defined as the F-value. Based on the amino acid analysis, the cooked liquid component had the highest F-value, followed by oyster meat, water-insoluble component, and protein. Among the samples, the cooked liquid component exhibited the highest taurine content, followed by oyster meat, and then protein. Taurine was untested in the water-insoluble component (Table 2).

Table 2. Amino acid composition of samples (mmol/mL).

Amino acid	Oyster meat	Oyster protein	Cooked liquid components	Water-insoluble components
Valine (Val)	29.58	35	19.47	35.63
Isoleucine (Ile)	25.57	29.81	12.63	29.33
Leucine (Leu)	36.34	46.77	19.89	45
Tyrosine (Tyr)	13.93	18.2	7.4	17.91
Phenylalanine (Phe)	17.88	23.41	16.16	22.78
Taurine (Tau)	62.4	1.85	177.94	–
Branched-chain  amino acids  (BCAA)	91.48	111.57	52	109.95
Aromatic amino  acids (AAA)	31.8	41.62	23.56	40.69
F-value^a	2.88	2.68	4.16	2.70

^aF-value was defined as a mole ratio of BCAA to AAA.

Among the samples, glycogen had the highest sugar content, followed by oyster meat, cooked liquid component, and water-insoluble component. Sugar was not tested in oyster protein (Table 3).

Table 3. Total sugar content of samples (mg/mL).

	Oyster meat	Glycogen	Oyster protein	Cooked liquid components	Water-insoluble components
Total sugar	12.1	20.0	0	9.2	2.3

Effect on the body weight of mice

At the beginning and end of the exercise protocol, changes in the mice body weights were determined. The results are shown in Table 4. The body weights did not differ across the groups at day 0 or 15 (p > 0.05), which illustrates that the intragastric administration of samples had no effect on the body weight. The result also verified that the swim-training model of mice is credible.

Table 4. Effects on mouse body weight.

		Body weight(g)^a
Group name	No. of mice	Initial (day 0)	Final (day 15)
C (Control group)	20	19.4 ± 1.3	35.2 ± 2.0
O (Oyster group)	20	19.2 ± 1.0	34.4 ± 2.1
G (Glycogen group)	20	19.2 ± 1.4	33.7 ± 2.1
P (Protein group)	20	19.3 ± 1.3	34.6 ± 1.9
L (Cooked liquid components group)	20	19.2 ± 1.2	34.4 ± 1.6
I (Water-insoluble components group)	20	19.0 ± 1.3	35.2 ± 2.1

^aValues were expressed as means ± SD. Values among groups had no significant difference based on Duncan’s multiple range test (p < 0.05, n = 20).

Effect on the swimming capacity of mice

We measured the swimming duration of mice administered with the five oyster components. The results are shown in Figure 1. Compared with the control group, all treatment groups have significantly increased endurance time (p < 0.05).

Figure 1. Effects on swimming endurance time of mice. Values were expressed as means ± SD of mice per group. Values with different superscripts indicate a significant difference among groups based on Duncan’s multiple range test (p < 0.05, n = 10).

Effect on the serum lactic acid content of mice

In the present study, no significant difference in the levels of serum lactic acid between the five intervention and the control group was found before the exhaustive swimming test (p > 0.05) (Table 5). After the test, the level of serum lactic acid was significantly lower in mice treated with oyster meat, glycogen, cooked liquid component, and water-insoluble component compared with that in the control group (p < 0.05). The level of serum lactic acid of the protein group was lower than that of the control group but not significantly (p > 0.05).

Table 5. Effect on the contents of serum lactic acid of mice.

		Serum lactic acid (mmol/L)
Group name	No. of mice	Before exhaustive swimming test*	After exhaustive swimming test§
C (Control group)	10	5.8 ± 0.8^a	12.1 ± 0.9^a
O (Oyster group)	10	5.0 ± 1.7^a	9.1 ± 0.7^b
G (Glycogen group)	10	4.1 ± 0.4^a	9.8 ± 0.5^b
P (Protein group)	10	5.8 ± 1.0^a	11.0 ± 1.1^a
L (Cooked liquid  components group)	10	5.5 ± 1.3^a	9.0 ± 0.9^b
I (Water-insoluble  components group)	10	4.6 ± 0.5^a	9.6 ± 1.3^b

*Values were expressed as means ± SD of mice per group. Values among groups had no significant difference based on Duncan’s multiple range test (p < 0.05, n = 10).

§Values with different superscripts indicate a significant difference among groups based on Duncan’s multiple range test (p < 0.05, n = 10).

Effect on the BUN content of mice

As shown in Table 6, no significant difference in the levels of BUN between the five intervention and the control groups was found before the exhaustive swimming test (p > 0.05). After the test, a significant decrease in the BUN level of fatigued mice was observed in all treatments (p < 0.05).

Table 6. Effect on the contents of serum BUN of mice.

		BUN (mmol/L)
Group name	No. of mice	Before exhaustive swimming test*	After exhaustive swimming test§
C (Control group)	10	6.5 ± 0.3^a	16.1 ± 0.5^a
O (Oyster group)	10	6.9 ± 0.4^a	13.5 ± 1.2^b
G (Glycogen group)	10	6.9 ± 0.3^a	14.2 ± 1.1^b
P (Protein group)	10	7.2 ± 0.3^a	14.4 ± 0.7^b
L (Cooked liquid  components group)	10	7.0 ± 0.2^a	14.1 ± 1.9^b
I (Water-insoluble  components group)	10	6.3 ± 1.4^a	14.1 ± 1.4^b

*Values were expressed as means ± SD of mice per group. Values among groups had no significant difference based on Duncan’s multiple range test (p < 0.05, n = 10).

§Values with different superscripts indicate a significant difference among groups based on Duncan’s multiple range test (p < 0.05, n = 10).

Effect on the glycogen contents in the liver and gastrocnemius muscle of mice

In the present study, a significant increase in liver glycogen content was observed in mice treated with oyster meat, glycogen, protein, cooked liquid component, and water-insoluble component compared with the control group (p < 0.05), as shown in Table 7. The gastrocnemius muscle glycogen content was also enhanced by oyster component ingestion. However, no significant difference between the protein and control groups was found (p > 0.05).

Table 7. Effect on the contents of liver and gastrocnemius muscle glycogen of mice.

		Glycogen (mg/g)
Group name	No. of mice	Liver	Gastrocnemius muscle
C (Control group)	10	6.7 ± 1.5^a	1.35 ± 0.5^a
O (Oyster group)	10	13.1 ± 1.8^bc	2.2 ± 0.2^b
G (Glycogen group)	10	13.9 ± 1.1^c	2.1 ± 0.2^b
P (Protein group)	10	11.2 ± 1.0^b	1.4 ± 0.2^a
L (Cooked liquid  components group)	10	13.5 ± 1.2^bc	2.2 ± 0.2^b
I (Water-insoluble  components group)	10	12.2 ± 1.5^bc	1.9 ± 0.3^b

Values were expressed as means ± SD of mice per group. Values with different superscripts indicate a significant difference among groups based on Duncan’s multiple range test (p < 0.05, n = 10).

Discussion

Administration of protein or amino acids is critical to facilitate recovery from fatigue (Jin et al., 2009). The effect of BCAA supplementation on the performance during extensive exercise has been well studied (Shimomura et al., 2004; Volpi et al., 2003). BCAA contributes to energy metabolism during exercise by acting as energy substrates to expand the pool of the intermediates in the tricarboxylic acid cycle and gluconeogenesis. The rate of BCAA uptake by the muscle is increased during sustained exercise (Monteiro et al., 2009). BCAA and aromatic amino acids in the plasma compete against each other for binding to the carrier and hence for transport into the brain. The increase in aromatic amino acids causes the increase of tryptophan in the brain, indirectly resulting in fatigue (Acworth et al., 1986). Thus, a high ratio of BCAA composition plays an ergogenic role in the maintenance of exercise performance. Taurine supplementation can increase exercise capacity (Beyranvand et al., 2011). The mechanism may be through the modulation of intracellular Ca²⁺ levels. Taurine may also have a potent antioxidant role (El Zahraa et al., 2012). Oysters have high taurine content (Je et al., 2005b), which may contribute to delaying fatigue.

Glycogen is the main form of glucose reserve in shellfish (Gao et al., 2008). It can be ingested as an energy material in vivo and is advantageous for delaying fatigue and improving performance (Maughan, 1991).

When doing high-intensity exercise, lactic acid can be produced through anaerobic glycolysis. The accumulation of blood lactic acid during high-intensity exercise might reduce pH value in muscle tissue and blood, and then cause acidosis which is harmful to the body performance (Gobatto et al., 2001; Wang et al., 2008). The main pathways to remove excess lactic acid is the conversion of lactate to glucose via gluconeogenesis. So, serum lactic acid was measured as an index of anaerobic glucose metabolism. If medicine could inhibit the accumulation of lactic acid or accelerate the clearance of lactic acid, it will also have antifatigue effect (Ding et al., 2011). Our results suggested that reducing the level of lactic acid may be a pathway of these components’ antifatigue effect except for oyster protein. This also illustrated that pure protein had little contribution to supply glucose via gluconeogenesis.

BUN, the metabolism product of protein and amino acid, is a sensitive index for evaluating the bearing capability when human bodies suffer from a physical load (Huang et al., 2011). Thus, the more the body is adapted for exercise tolerance, the more significantly the BUN level increases (Tsopanakis & Tsopanakis, 1998). Reduced BUN levels also reflect the reduction in protein metabolism, indicative of enhanced endurance. The results suggested that the different components could decrease the level of BUN of mice after swimming, which indicated that they could reduce protein metabolism and ameliorates fatigue.

Energy for exercise is initially derived from the breakdown of glycogen and later from circulating glucose released by the liver (Suh et al., 2007). The contribution of glycogen to energy production during exhaustive exercise is necessary because glycogen can be degraded rapidly to produce adenosine triphosphate both aerobically and anaerobically (Andreeva et al., 2001). Fatigue happens when most of the glycogen is already consumed. Thus, liver and muscle glycogen are sensitive parameters related to fatigue (Ding et al., 2011). Enhancement of exercise capacity could be accounted for by a reduced rate of liver and muscle glycogen breakdown (Jung et al., 2004). In the present study, the remarkably increased liver and muscle glycogen levels seem to be a great contributor for the increase in swimming time. This result supports the hypothesis that glycogen resynthesis is of high metabolic priority during high-intensity exercise (Kimber et al., 2003).

In conclusion, oyster meat enhanced the swimming capacity of mice through the utilization of taurine and glycogen. Oyster glycogen provided energy for exercise and delayed fatigue. The cooked liquid component enhanced the swimming capacity because of its high ratio of BCAA composition as well as high taurine and glycogen content. The water-insoluble component affected the endurance of mice mainly because of its glycogen content. Oyster protein had little effect on reducing fatigue. The result also illustrated that many of the proteins that occur naturally could not exert their physiological action directly (Korhonen & Pihlanto, 2006; Ren et al., 2008), which may be attributed to the protein-bound amino acids that were difficult to utilize. Furthermore, the antifatigue mechanisms of oyster components in vivo need further exploration.

Declaration of interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript.

Acknowledgements

This project was supported by the Earmarked Fund for Modern Agro-industry Technology Research System of China (CARS-48) and the Li Shangda Fund of Jimei University (ZC2011016).