Evaluation of anti-fatigue property of the extruded product of cereal grains mixed with Cordyceps militaris on mice

Physical fatigue is commonly defined as an inability to sustain or maintain voluntary activities, which is considered to be associated with the decline of physiology [1, 2]. Regular exercise, a balanced diet, and complementary and alternative medicine (CAM) are reported to effectively relieve fatigue, including providing energy substrates and enhancing elimination of metabolites. Among these approaches, CAM dietary therapy has been widely used in medical treatment and health care due to its minor side effects [3, 4].

Whole cereal grains as healthy foods have gained popularity due to their underlying protection against diet-related disorders, such as fatigue, obesity, diabetes and even cancers [5‐8]. In addition, the limited amino acids of cereal and legume were lysine and methionine respectively, while mixed cereal grains are able to compensate for deficiencies in the content and constitution of essential amino acids from a single source. Currently, mixed cereal grains have been applied to design and produce nutritional foods that are rich in dietary fiber and protein [9].

In recent years, research on extruded products including edible mushrooms has received increased attention in order to obtain an improved nutritional value, texture, flavor and digestion of food under conditions of thermal force and ultra-high pressure [10‐13]. C. militaris is a fungus renowned for its health-promoting properties, as well as for its medicinal use in Southeast Asia [14]. In a previous study, C. militaris was demonstrated to be a high quality protein and rich amino acid, which contains 29.43% protein, 2.51% fat, 1.20% dietary fiber, 37.52% carbohydrate and 0.051% cordycepin etc. Moreover, its bioactive components such as cordycepin, polysaccharides, ergosterol, and adenosine, play a key role in health care, and are used for various medicinal purposes such as anti-aging, anti-tumor, anti-cancer and anti-leukemic etc. [15‐17]. C. militaris was also often used in the formulation of functional foods. Inclusion of C. militaris into chicken essence products could be nutritional and healthy for consumers [18]. Recently, the aqueous extract of C. militaris had been demonstrated to possess the significant effect on resisting oxidation and relieving fatigue in mouse model [19]. Cordyceps sinensis (C. sinensis), widely accepted as tonic and healthy foods in Asia, has received more interests due to its biological activity. Many studies reported that C. sinensis has the prominent impacts on analgesic, antitumor, free radical scavenging, and immune modulation [20]. Compared with C. sinensis, the contents of cordycepin, adenine, guanosine and uracil were even higher in C.militaris, while cordycepic acid, amino acid, polysaccharides and inorganic elements levels were lower. Natural C. sinensis is expensive and rare in nature. C. militaris has medicinal properties similar to those of C. sinensis and could potentially be the alternatives for C. sinensis in health supplements. Due to high cost in the development of pharmaceutical drugs, more and more research interests have been put on natural products, which are proved to be safer and more efficacious with less side effects on body. Thus, C. militaris could be used as a high-quality raw material of functional food [21].

Weight-loaded swimming test is the most widely used method to evaluate anti-fatigue properties of compounds through assessing the reduction of muscular force or the exhaustion of contractile function [22]. On the basis of the test, the resistant fatigue mechanisms of compounds could be investigated through a series of biochemical parameters. Several theories have been proposed to explain sports fatigue, including the “exhaustion theory [23]”, “radical theory [24]”, “clogging theory”, “homeo-stasis disturbance theory”, “mutation theory” and “protective inhibition theory [25]”. Among these theories, the “exhaustion theory”, “clogging theory” and “radical theory” have drawn particular attention, which could explain the anti-fatigue effect of the extruded products by evaluating the values of biochemical criteria. The “exhaustion theory” proposed that depletion of muscle and liver glycogen could lead to the ocurrence of fatigue. However, “clogging theory”, the excessive accumulation of BLA and BUN would cause metabolic disorders, which eventually result in fatigue. “Radical theory” shows that intense exercise could produce an imbalance between the body’s oxidation system and its anti-oxidation system. Muscle cells have the defense mechanisms to scavenge reactive oxygen species, such as SOD, GSH-Px and CAT, and to protect cells against exercise-induced oxidative injury. Currently, research on healthy foods, such as C. militaris, Panax quinquefolium and fermented rice bran, have achieved great progress in relieving fatigue by reducing the CK and BLA contents and increasing glycogen and GSH-Px concentrations [4, 26, 27]. In our previous study, the proportions of cereal grains in a mixed powder were optimized by linear programming. Moreover, ECC was previously shown to possess profound characteristic features, such as the improvement in texture, nutrition and flavors. However, the physiological effects or health functions of ECC remain unclear.

In the previous study, C. militaris and blended powders from five cereal grains were prepared; a twin screw extruder was used to produce the extruded products. We hypothesized that EC and ECC could alleviate fatigue in mice and adding C. militaris could enhance the anti-fatigue effect of EC. To test the hypothesis, we established the mouse model of sports fatigue and studied the changes in anti-fatigue and anti-oxidant activities of EC and ECC. The swimming endurance time was determined as an indicator of athletic capability in mice, which could intuitively reflect the ability of resistance to fatigue. Following swimming test, mice were subject to a series of biochemical analysis in order to further explore the anti-fatigue mechanisms of EC and ECC such as measuring LDH and CK as well as the levels of BUN, BLA, MDA, muscle and liver glycogen. In addition, the anti-oxidant enzymes including SOD, CAT and GSH-Px were also measured.

The present study was designed to evaluate the effects of EC and ECC on endurance exercise in mice. The major findings are listed below:

The ratio of organ to body by weight was regarded as the organ index, which has been commonly used in toxicity tests. Organ damage could be evaluated in terms of the brain-body index [31]. Additionally, the interactions of boron and calcium were observed in the spleen-body index [32]. In our study, EC and ECC had no obvious effects on body weights or on the organ index among the groups, suggesting that all mice showed normal growth and that the extruded products had no toxic side effects on the mice during the experiment. In addition, the reason that the lower body weights of mice may be due to the fact that training can be helpful for the digestion of mice.

The fatigue tests including the weight-loaded swimming test, rolling-bar test, pole-climbing test and mouse tail suspension test can be performed to assess athletic ability [33, 34]. Changes of biochemical markers caused by loaded exercise were not completely consistent with sports intensity and not all of markers were suitable for evaluating the anti-fatigue effect. Among these tests, the anti-fatigue effect of EC and ECC can be evaluated by the weight-loaded swimming test, combining with fatigue related indicators. This method is standard as well as accurate, and it has been adopted widely by most of the researchers and China Food and Drug Administration [4, 30, 35, 36]. However, rolling-bar test, pole-climbing test and mouse tail suspension test require the technical equipment and it is not easy for experimenters to operate them expertly. Lactate test is also often used to evaluate the anti-fatigue effect of compounds on mice after exhaustive exercise, while the test requires the blood sample in inner canthus of each mouse before and after exercise. The procedure may be cumbersome and time consuming to test the lactate level of mice. However, in the weight-loaded swimming test, the workload could be standardized by adding the known amount of load to the tail of the animal to reduce the swimming time, and the endurance time was then recorded accurately, which would be practical to achieve accurate measurements in time [29]. The average of exhaustive swimming time for 4-week-old C57BL/6 mice is 42.2 min (flow rate is 7 l/min) [37]. In our study, after the weight-loaded swimming test, the remaining mice in each group were exhausted in weight-unloaded swimming within 90 min following the standard of exhaustion, which could lessen the effects of stimulation and interference on mice and accord with the generating mechanisms of fatigue. In addition, the concentrations of relevant indicators such as serum CK, BLA, BUN and glycogen, could be raised in exhaustive exercise and it will help improving the quality of detection [38].

For anti-fatigue experiments, the weight-loaded swimming test has been widely used to provide a more informative evaluation of exercise tolerance in animals [39]. Thus, the improvement in exercise endurance will be strong evidence for the anti-fatigue effect [29]. In our study, the results demonstrated that mice in the EC- and ECC-supplemented groups had prolonged swimming time, suggesting that EC/ECC could resist fatigue and ECC supplementation had the most significantly efficient effect, especially at the dose of 20 g/kg. These evidence collectively suggested that ECC-H group had the greatest (P < 0.05) anti-fatigue effect among the groups and C. militaris could effectively enhance the stamina of mice and delay the emerging fatigue [40]. Stress response represents the reaction of the body and disturbance of normal physiologic balance, along with physiologic changes including the increase of LDH, glucose, cholesterol levels. Moreover, the glucose level of mice could be increased in response to acute perturbations in the environment and it is helpful to increase exhaustive swimming time [41]. Thus, effects of EC and ECC on the recovery from exhaustion could be related to a resistance to the stress in exhaustive swimming. Most researchers choose Kunming, C57BL/6 and BALB/c mice (6–8 weeks) to establish fatigue model [4, 42, 43]. In our study, all of the mice had been forced to swim for 20 min after each treatment so as to be accustomed to swimming, Thus, the ages of mice would make a little impact on stress response.

Glycogen was used for long-term energy storage and could be consumed quickly to satisfy the emergent need for glucose when fatigue occurred [44]. Increasing glycogen levels in the liver and muscle could enhance endurance during exhaustive exercise [45]. EC and ECC are rich in proteins and carbohydrates, which could contribute to the storage of glycogen [42, 46]. Muscle and liver glycogen level increased with the increasing EC and ECC concentrations, which showed that the anti-fatigue activity of EC and ECC might be related to the improvement in the metabolic control of exercise and to the activation of energy metabolism [41]. There is also evidence showing that increasing the availability of fatty acids is mediated by a deceleration of glycogen depletion [43, 47]. C. militaris in ECC may decrease carbohydrate utilization by increasing lipid utilization during exercise, which could delay the rate of glycogen consumption. In addition, during the exhaustive exercise, myocytes will consume continuous anaerobic ATP which can be generated via oxidative phosphorylation in the contractile muscle. Under the above conditions, glycogen will be converted into glucose to generate ATP [48]. The aqueous extracts of C. militaris administration could increase ATP levels of mice after exhaustive swimming [19]. In our study, C. militaris in ECC may reduce glycogen consumption by increasing the concentration of ATP which would be helpful for mice to increase endurance in exhaustive swimming.

LDH activity indicates the degree of lactate metabolism, and the anti-fatigue activity could be evaluated by measuring LDH levels [49]. LDH has the ability to quench BLA, which is derived from the anaerobic metabolism of glucose during high-strength exercise [45, 50]. The data suggested that extruded products, especially C. militaris in ECC, could be beneficial to accelerate the metabolism of BLA by effectively strengthening the activity of LDH, which would effectively delay the generation of fatigue [19]. The CK level plays an important role as an indicator of fatigue [38]. An increase in the CK level may be due to damage of the skeletal muscle cell membrane induced by exhaustive exercise [51]. In our study, the results indicated that a high dose of C. militaris in ECC could make great contributions to anti-fatigue activity by accelerating the elimination of lactic acid from cells and by efficiently utilizing energy with potential scavenging of free radicals [37].

BLA is the fermented product of carbohydrates without oxygen, and BLA levels increase during anaerobic exercise [26, 41]. In addition, BLA decreases pH and affects the function of cardiac circulation and skeletal muscle systems [52]. The results showed that mice in the ECC-supplemented groups, especially those mice in the ECC-H group, showed a reduction in muscular lactate levels and an increased relative contribution of aerobic metabolism to ATP production during an exercise session [51].

When the body cannot achieve enough energy by metabolizing sugar and fat after a long period of intensive exercise, protein may contribute to the energy supply, and urea nitrogen also increases at this time, which could decrease the strength of the muscle and eventually cause fatigue [52, 53]. C. militaris extracts showed a significant suppressive effect on BUN, creatinine and urine protein levels in diabetic mice [54]. In our study, mice supplemented with ECC-H (20 g/kg) had the lowest BUN concentration, demonstrating that the addition of C. militaris to the EC could relieve fatigue by slowing down the metabolism rate of protein effectively.

Some diseases are relevant to oxidative damage caused by lipid peroxidation, which may produce harmful metabolites [55]. MDA is a product of lipid peroxidation, and its elevation indicates oxidative damage to the cell membrane [56]. Mice that were administered C. militaris polysaccharides at a high dose (0.2 g/kg body weight) had a strong ability to reduce various oxygen free radicals and prevent oxidative stress [53]. In the present study, the rising BUN levels after swimming in the NCG and EC-supplemented group clearly indicated that ECC possessed an ability to lower or retard MDA formation after exercise.

The primary antioxidant enzymes include SOD, CAT and GSH-Px. SOD catalyzes superoxide radicals to produce H₂O₂ and O₂. CAT and GSH-Px catalyze the breakdown of H₂O₂ to form water and O₂ [45]. These antioxidant defense mechanisms become weaker during chronic fatigue and other diseases [57]. Hence, an increase in these defense mechanisms can help to recover from fatigue. In our study, the data showed that ECC-H had the most significant effects on promoting increases in the activities of these antioxidant enzymes. At present, C. militaris polysaccharides have been found to protect tissues against oxidative damage in an experiment with mice [53]. Mice in the ECC-supplemented groups possessed a higher capability to prevent lipid oxidation and to preserve cell function by promoting SOD, CAT and GSH-Px levels, indicating that ECC had a better anti-fatigue effect on mice by improving the function of anti-oxidation [58].

In the present study, EC and ECC had prominent anti-fatigue properties. Our results showed that mice in the ECC-supplemented groups, especially in the ECC-H group (20 g/kg), had more significant anti-fatigue effects. It suggested that adding C. militaris could enhance the anti-fatigue effect of EC. ECC enhanced the metabolic control of exercise and activated energy metabolism by increasing muscle and hepatic glycogen levels. In addition, C. militaris in ECC may decrease the depletion of glycogen by increasing the availability of fatty acids and ATP level of mice. ECC also efficiently eliminated BLA and protected cells by increasing LDH levels and decreasing CK levels. ECC contributed to the reduction in protein consumption by decreasing BUN levels. Additionally, ECC improved endogenous cellular antioxidant enzymes in ECC-supplemented mice by decreasing MDA levels and increasing CAT, SOD and GSH-Px activities.

In summary, the current study demonstrated that supplementing mice with EC or ECC could promote the capacity for endurance exercise and accelerate recovery from fatigue. ECC had shown the most dramatic anti-fatigue effect, especially with ECC-H (20 g/kg), suggesting that ECC could potentially be included in functional foods for fighting fatigue.