Anti-fatigue property of Cordyceps guangdongensis and the underlying mechanisms

Abstract

Context: Cordyceps guangdongensis T.H. Li, Q.Y. Lin & B. Song (Cordycipitaceae) is a nontoxic folk medicine and can be cultivated, with noticeable effects of anti-H₉N₂, life-prolonging and treating chronic renal failure.

Objective: The anti-fatigue effect of C. guangdongensis, possible mechanism and active constituent were investigated.

Materials and methods: Treatment mice were treated with C. guangdongensis powder (0.455, 0.91 and 1.82 g/kg bw daily for low, middle and high doses, respectively); treatment rats were fed, respectively, with ethanol, petroleum ether, ethyl acetate, n-butanol, aqueous phase and hot water extract fractions, for 30 d. Forced swimming time to exhaustion, blood urea nitrogen (BUN) and hepatic glycogen (HG) levels of mice and blood lactic acid (BLC) levels of rats were determined.

Results: The swimming times to exhaustion of mice were very significantly (p < 0.01) longer in low-, middle- and high-dose groups (respectively 1.87-, 1.94- and 1.88-times), and significantly (p < 0.05) longer in the n-butanol fraction group (1.52-times), hot water extract group (1.88- times) and refined polysaccharide group (2.66-times) than in blank control; the BLC levels of rats were significantly (p < 0.05) lower in the ethyl alcohol partition group (84.8%), the n-butanol fraction group (84.0%) and the hot water extract group (84.4 %) than in blank. The BUN and HG levels were not significantly different.

Discussion and conclusion: Cordyceps guangdongensis can potently alleviate fatigue through reducing the accumulation of BLC; a functional constituent was the refined polysaccharide. This might become a new functional food for fatigue resistance.

• Fatigue resistance
• functional food
• fungus
• polysaccharide

Introduction

Fatigue is known to be accompanied by a feeling of physical or mental tiredness, usually resulting from severe stress of hard physical or mental work (Akazawa et al., 2010; Chen et al., 2009). For several decades, health experts and athletic physiologists have been industriously looking for natural active products that not only can improve athletic ability, postpone fatigue and accelerate the elimination of fatigue in human beings, but also have few side effects (Kim et al., 2002).

Some species of Cordyceps (Cordycipitaceae) are the members of traditional Chinese functional food or traditional Chinese herbs which have many functional effects such as relieving the stress for humans living in technologically developed societies by stimulating basic and secondary responses of the immune system (Lakhanpal & Rana, 2005). The most famous species is Cordyceps sinensis (Berk.) Sacc. (Cordycipitaceae), which has long been used to treat multitude of ailments, promote longevity, increase athletic power, etc. (Paterson, 2008; Zhou et al., 2009). Studies have suggested that supplementation of cultured C. sinensis mycelial powder to healthy young volunteers during exhaustive running could significantly augment the energy generation and anti-fatigue ability as compared to placebo controls (Nagata et al., 2002, 2006); supplementation of extract of cultured C. sinensis mycelium enhanced the performance of long distance runners (Hiyoshi et al., 1996) and oral administration of hot water fraction of C. sinensis mycelia (Koh et al., 2003) and polysaccharides (Li & Li, 2009) could increase the swimming time to exhaustion, alleviate fatigue and stress in mouse-forced swimming tests. Although C. sinensis has an obvious effect of anti-fatigue, its expense and shortage of resources have restricted its application (Sharma, 2004). Therefore, it is essential to exploit new alternative resources with similar functions.

Cordyceps guangdongensis Li, Lin & Song (Cordycipitaceae) is potentially a good alternative of C. sinensis in some respects. A Cordyceps species discovered in South China (Lin et al., 2008) has been successfully cultivated (Lin et al., 2010). Tests proved that the fungus possesses the same or similar components to those of C. sinensis (Lin et al., 2009). Previous research indicated that cultivated fruit bodies of C. guangdongensis were nontoxic (Yan et al., 2010b), and had noticeable effects of anti-H9N2 virus (Yan et al., 2010a), life-prolonging activities (Yan et al., 2011) and therapeutic effects on chronic renal failure (Yan et al., 2012). However, the knowledge about its anti-fatigue activity, the bioactive components or extraction fractions for fatigue resistance and the underlying mechanisms, is rather limited.

The present study was designed to evaluate the anti-fatigue activities of cultivated fruit bodies of C. guangdongensis and their ethanol, petroleum ether, ethyl acetate, n-butanol, aqueous phase and hot water extract fractions; the active components and possible mechanism were also to be investigated.

Materials and methods

Drugs and chemicals

Anshen Bunao Ye (a popular Chinese patent medicine used in fatigue resistance; Jilin Aodong Yanbian Pharmacuetical Co. Ltd., Jilin, China, batch no 0903066). EtOH (AR), petroleum ether (AR), ethyl acetate (AR) and n-butanol (AR; Guangdong Huankai Microbial Sci. & Tech. Co. Ltd., Guangzhou, China). Blood urea nitrogen (BUN) kit (Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co. Ltd., Shanghai, China, batch no. 091201); hepatic glycogen (HG) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China, batch no. 20100518) and blood lactic acid (BLC) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China, batch no. 20100618).

Test substance

Crushed powder (80 mesh and finer) of cultivated fruit bodies of C. guangdongensis was provided by Guangdong Institute of Microbiology. The strain source and cultivation conditions were as mentioned in earlier reports (Lin et al., 2008, 2010).

Preparation of the ethanol extract and the fractions

Ethanol extract and the fractions of cultivated fruit bodies of C. guangdongensis were prepared with crushed powder. The powder (1.5 kg) was immersed in aqueous ethanol (7500 mL) for 24 h, and then extracted for 2 h. The solvent was evaporated under vacuum to collect 300 g crude ethanol extract (yield, 20%). The crude ethanol extract was then suspended in water and partitioned successively with petroleum ether, ethyl acetate and n-butanol. Each fraction was evaporated in vacuum to yield the residues of petroleum ether 24 g (1.6%), ethyl acetate 9 g (0.6%), n-butanol 24 g (1.6%) and aqueous phase 36 g (2.4%). The extracts were concentrated under reduced pressure at 45–55 °C, lyophilized to obtain powder, and then used as test samples. For pharmacological studies, fractions were suspended with 0.05 ml aqueous solution of Tween 80. The doses employed were expressed as milligram of the dried extract per kilogram body weight (bw). The residue of n-butanol fraction was dissolved into distilled water.

Preparation of the hot water extract and the fractions

The dried powder of cultivated fruit bodies of C. guangdongensis was extracted twice with water at 100 °C for 2 h. The water extract was concentrated under reduced pressure to a certain volume (65%).

The crude polysaccharide fraction was obtained through water extraction and alcohol precipitation; and the refined polysaccharide was obtained by being deproteinized with Sevag reagent and depigmented with hydrogen peroxide solution.

Animals

Specific pathogen free male and female mice (weighing 16–18 g each), Sprague–Dawley male rats (weighing 160–180 g each) were housed in cages under automatically controlled conditions of temperature (25 ± 1 °C) and relative humidity (40–70%). They were provided with free access to laboratory standard diet and water. The room lights were on for 12 h/day starting at 7:00 am. The care and treatment of experimental animals conformed to the guidelines for the ethical treatment of laboratory animals. All animals have been approved by Guangdong Laboratory Animals Monitoring Institute, and license nos were 0064089 for male mice, 0063644 and 0063726 for female mice and 0066048 for male rats, respectively.

Assaying the anti-fatigue effects of the cultivated fruit bodies in mice

After 1 week of adaptation, 50 female mice were randomly divided into five groups, respectively, as a blank control group, a positive group and three treatment groups, with 10 in each group. The animals in the treatment groups were gavaged, respectively, with three doses of cultivated fruit bodies of C. guangdongensis (0.455 g/kg bw for low dose, 0.91 g/kg bw for middle dose and 1.82 g/kg bw for high dose) and the positive group was treated with Anshen Bunao Ye (10 ml/kg bw), while the blank control group received the same volume of distilled water. All the animals were treated orally daily (9:00 am) for consecutive 30 d.

Thirty minutes after the last oral administration, five groups of mice were individually forced to swim in the acrylic plastic pool (50 × 50 × 40 cm) containing water at 25 ± 1 °C (30 cm deep). Every mouse was forced to swim with a lead block weighting approximately 5% of its body weight attached to the tail. The swimming time to exhaustion was used as the index of the forced swimming capacity. The animals were assessed to be exhausted when they failed to rise to the surface of water to breathe within a 7 s period.

Assaying the anti-fatigue effects of different extracts of the fruit bodies in animals

After 1 week of adaptation, 80 female mice for assaying forced swimming capacity, 80 female mice for assaying HG level, 80 male mice for assaying BUN level and 80 male rats for assaying BLC level were, respectively, randomly divided into eight groups, respectively, as a blank control group, a positive group and six treatment groups, with 10 in each group. The animals in the treatment groups were gavaged, respectively, with ethanol, petroleum ether, ethyl acetate, n-butanol, aqueous phase and hot water extracts. The blank control group was treated with the similar volume of vehicle, and the positive group was treated with Anshen Bunao Ye (20 mL/kg bw for mice and 10 mL/kg bw for rats). All the animals were treated orally daily (9:00 am) for consecutive 30 d. The dose of each group is presented in Table 1.

Table 1. Dose design of experiment groups for assaying the anti-fatigue effects of different partitions of the fruit bodies in animals.

	Doses (g/kg bw)
Groups	Mice	Rats
①	–	–
②	20	10
③	0.364	0.26
④	0.029	0.021
⑤	0.011	0.008
⑥	0.029	0.021
⑦	0.044	0.031
⑧	1.183	0.845

①, Blank control group; ②, positive control group; ③, ethanol fraction group; ④, petroleum ether fraction group; ⑤, ethyl acetate fraction group; ⑥, n-butanol fraction group; ⑦, aqueous phase group and ⑧, hot water extract group.

Dose unit for ② positive control group is ml/kg bw, which is approximately the same as g/kg bw for Anshen Bunao Ye.

Assaying the forced swimming capacity of mice

Thirty minutes after the last oral administration, eight groups of female mice were individually forced to swim in the acrylic plastic pool (50 × 50 × 40 cm) containing water at 25 ± 1 °C (30 cm deep). Every mouse was forced to swim with a lead block weighting approximately 5% of its body weight attached to the tail. The swimming time to exhaustion was used as the index of the forced swimming capacity. The mice were assessed to be exhausted when they failed to rise to the surface of water to breathe within a 7 s period.

Assaying the HG level of mice

Thirty minutes after the last oral administration, each mouse was anesthetized to death with a high concentration ether in an acrylic plastic immobilizer and liver was collected and made into 10% homogenates with normal saline at −80 °C as soon as possible. The livers were washed three times with physiological saline solution and weighed. HG was tested according to the procedures provided with the HG detection kits. The HG content expressed as milligram HG per gram hepatic tissue.

Assaying the BUN level of mice

Thirty minutes after the last oral administration, the mice were forced to swim for 90 min without a load. After a 60 min rest, 0.5 mL of blood from the left eyeball of mice was collected in the tube. Blood sample was cooled for about 3 h at (2–8) °C, the serum was prepared by centrifugation at a speed of 2000 × g, 4 °C for 15 min and the levels of BUN were determined by automatic biochemical analyzer with BUN kits. The BUN content was expressed as mol nitrogen per liter blood serum.

Assaying the BLC level of rats

Thirty minutes after the last oral administration, 0.5 ml of blood from each rat was collected for the first collection. After the rats were forced to swim for 10 min without a load, 0.5 mL of blood was collected for the second collection. After 20 min of rest, 0.5 ml of blood was collected for the third collection. BLC level was determined according to the procedures provided with BLC kit. The BLC level expressed as millimole BLC per liter blood. The level of BLC was calculated as the area under the curve of blood lactate = 5 × (blood lactate before swimming + 3 × swimming 0 min blood lactate + 2 × blood lactate after a 20 min break).

Assaying the anti-fatigue effects of water-soluble polysaccharides in mice

After one week of adaptation, 40 female mice were randomly divided into four groups, respectively, as a blank control group, a positive group, a crude polysaccharide group (gavaged with crude polysaccharide, 0.2077 g/kg bw) and a refined polysaccharide group (gavaged with refined polysaccharide, 0.182 g/kg bw). The positive group was treated with Anshen Bunao Ye (20 ml/kg bw), while the blank control group received the same volume of distilled water. All the animals were treated orally daily (9:00 am) for consecutive 30 d.

Thirty minutes after the last oral administration, four groups of mice were individually forced to swim in the acrylic plastic pool (50 × 50 × 40 cm) containing water at 25 ± 1 °C (30 cm deep). Every mouse was forced to swim with a lead block weighting approximately 5% of its body weight attached to the tail. The swimming time to exhaustion was used as the index of the forced swimming capacity. The animals were assessed to be exhausted when they failed to rise to the surface of water to breathe within a 7 s period.

Clinical observations and body-weight measurements of the animals

During all the tests above, clinical observations were made once daily following the treatment at approximately the same time each day. Body weights were measured at the initiation of the experiments, and then once a week.

Statistical analysis

All the statistical analyses were performed using SPSS version 11.0 (Chicago, IL), the significance of the mean difference was determined by one-way ANOVA. All data were recorded as mean ± SD. Significance was assumed when p < 0.05, high significance was assumed when p < 0.01.

Results

Effects of the fruit bodies on swimming time and body weight in mice

Changes of the weight-loaded swimming time to exhaustion and body weight during the experimental period are presented in Table 2. There are significant differences in the swimming time to exhaustion between the blank control group and each treatment group. The swimming times to exhaustion of all groups treated with fruit bodies of C. guangdongensis were significantly longer than that of the blank control group (p < 0.01). The swimming times were, respectively, 1.87-times in low-dose group, 1.94-times in middle-dose group and 1.88-times in high-dose group as long as that in the blank control group.

Table 2. Effects of fruit bodies of C. guangdongensis on swimming time to exhaustion and body weights of mice.

		Body weight (g)
Groups	Swimming time to exhaustion (min)	Initial	Week 1	Week 2	Week 3	Week 4
①	13.79 ± 4.10	26.4 ± 1.6	28.5 ± 1.3	29.9 ± 1.6	31.5 ± 2.0	32.3 ± 2.8
②	26.96 ± 10.87**	26.4 ± 1.6	27.8 ± 2.0	29.4 ± 2.8	30.9 ± 3.2	31.9 ± 3.4
③	25.73 ± 8.55**	26.5 ± 1.8	28.1 ± 2.2	29.2 ± 2.7	30.7 ± 2.9	31.3 ± 3.4
④	26.73 ± 8.75**	26.8 ± 1.9	28.5 ± 3.2	29.0 ± 3.0	30.3 ± 3.0	30.9 ± 3.5
⑤	25.96 ± 11.31**	26.7 ± 1.8	27.0 ± 1.6*	28.1 ± 1.8*	28.6 ± 2.5**	29.5 ± 2.7*

①, Blank control group; ②, positive control group; ③, low-dose C. guangdongensis group; ④, middle-dose C. guangdongensis group and ⑤, high-dose C. guangdongensis group.

Values are expressed as mean ± SD of 10 mice per group.

*p < 0.05 versus blank control group and **p < 0.01 versus blank control group.

The mean body weights of the mice in high-dose C. guangdongensis group were slightly higher than those of the blank control group from week 1 to week 4, but the results were not considered related to the test substance. There were no statistically significant changes in body weights of other groups, compared with the blank control group during initial and terminal stages.

Effects of different partitions of the fruit bodies on anti-fatigue ability

Results of the forced swimming capacity

Changes of the weight-loaded swimming times to exhaustion and body weight during the assay of HG experimental period are presented in Table 3. The swimming times to exhaustion of the n-butanol fraction group and hot water extract group of mice were significantly longer than that of the blank control group (p < 0.05). They were 1.52-times in n-butanol fraction group, 1.88-times in hot water extract group and 2.66-times in refined polysaccharide group as long as that in blank control group.

Table 3. Effects of different partitions of fruit bodies of C. guangdongensis on swimming time to exhaustion and body weights of mice.

		Body weight (g)
Groups	Swimming time to exhaustion (min)	Initial	Week 1	Week 2	Week 3	Week 4
①	23.3 ± 10.9	20.8 ± 0.9	28.3 ± 1.6	30.2 ± 1.6	32.6 ± 2.5	35.2 ± 2.0
②	22.3 ± 13.8	20.7 ± 0.9	29.0 ± 1.5	31.5 ± 2.0	34.6 ± 2.9	35.8 ± 2.6
③	42.0 ± 32.7	20.8 ± 0.9	28.2 ± 1.4	30.2 ± 2.0	32.2 ± 2.1	33.9 ± 2.5
④	42.1 ± 33.0	20.9 ± 1.0	27.6 ± 0.9	29.4 ± 1.9	31.1 ± 2.5	32.8 ± 2.8*
⑤	33.0 ± 26.3	20.7 ± 0.9	27.8 ± 1.8	29.9 ± 2.2	32.2 ± 3.0	33.9 ± 2.9
⑥	35.4 ± 14.2*	20.7 ± 0.9	27.5 ± 1.7	30.9 ± 2.1	33.3 ± 1.9	34.8 ± 1.7
⑦	30.3 ± 19.1	20.7 ± 0.9	27.4 ± 1.8	28.8 ± 3.2	31.4 ± 2.2	33.4 ± 1.9*
⑧	43.9 ± 24.8*	20.7 ± 1.0	28.4 ± 1.0	30.2 ± 1.4	31.9 ± 1.9	34.3 ± 1.7

①, Blank control group; ②, positive control group; ③, ethanol fraction group; ④, petroleum ether fraction group; ⑤, ethyl acetate fraction group; ⑥, n-butanol fraction group; ⑦, aqueous phase group and ⑧, hot water extract group.

Values are expressed as mean ± SD of 10 mice per group.

*p < 0.05 versus blank control group.

The mean body weights of the mice in petroleum ether fraction group and aqueous phase fraction group were slightly lower than that of the blank control group at week 4, but it was not considered related to the test substance. There were no statistically significant changes in body weights of other groups, compared with the blank control group during initial and terminal stages.

Effects on HG levels and body weight changes of mice

Changes of HG levels and body weight during the assay of HG experimental period are presented in Table 4. There were no statistically significant changes in HG levels of all treatment groups compared with the blank control group.

Table 4. Effects of different partitions of fruit bodies of C. guangdongensis on HG levels and body weight changes.

		Body weight (g)
Groups	HG levels (mg/g)	Initial	Week 1	Week 2	Week 3	Week 4
①	31.0 ± 10.6	21.2 ± 0.7	27.7 ± 1.2	30.7 ± 1.3	32.3 ± 2.0	34.0 ± 1.6
②	27.8 ± 11.7	21.3 ± 0.8	28.3 ± 1.9	32.1 ± 2.7	33.4 ± 2.3	35.2 ± 3.5
③	26.3 ± 7.7	21.2 ± 0.7	26.6 ± 2.1	28.6 ± 2.8*	30.1 ± 2.1*	31.6 ± 2.0**
④	24.5 ± 11.4	21.1 ± 1.2	25.9 ± 1.0**	29.3 ± 1.2*	30.6 ± 1.5*	32.5 ± 2.6
⑤	24.4 ± 13.5	21.2 ± 0.7	26.0 ± 2.6	29.0 ± 1.9*	29.7 ± 2.3*	31.9 ± 2.7*
⑥	24.5 ± 8.8	21.3 ± 0.8	27.5 ± 1.0	30.3 ± 1.6	32.3 ± 2.0	33.6 ± 2.5
⑦	25.3 ± 11.1	21.2 ± 0.8	27.3 ± 1.1	29.3 ± 1.2*	31.2 ± 1.0	32.6 ± 1.4*
⑧	31.8 ± 14.8	21.2 ± 0.7	27.7 ± 2.1	29.8 ± 2.3	32.3 ± 2.8	33.5 ± 4.0

①, Blank control group; ②, positive control group; ③, ethanol fraction group; ④, petroleum ether fraction group; ⑤, ethyl acetate fraction group; ⑥, n-butanol fraction group; ⑦, aqueous phase group and ⑧, hot water extract group.

Values are expressed as mean ± SD of 10 mice per group.

*p < 0.05 versus blank control group and **p < 0.01 versus blank control group.

The mean body weights of the mice in ethanol fraction group and ethyl acetate fraction group at weeks 2, 3 and 4, petroleum ether fraction group at weeks 1, 2 and 3 and aqueous phase fraction group at weeks 2 and 4 were slightly lower than that of the blank control group, but the differences were not considered related to the test substance. There were no statistically significant changes in body weights of other groups, compared with the blank control group during initial and terminal stages.

Effects on BUN levels and body weight changes of mice

Changes of BUN levels and body weight during the assay of BUN experimental period are presented in Table 5. There were no statistically significant changes in BUN levels of all treatment groups except the positive control group, compared with the blank control group.

Table 5. Effects of different partitions of fruit bodies of C. guangdongensis on BUN levels and body weight changes of mice.

		Body weight (g)
Groups	BUN levels (mol/l)	Initial	Week 1	Week 2	Week 3	Week 4
①	9.4 ± 2.8	22.3 ± 1.6	34.9 ± 2.2	38.6 ± 2.3	41.3 ± 2.5	43.1 ± 3.2
②	6.6 ± 1.4*	22.2 ± 1.2	36.6 ± 1.5	41.0 ± 1.5*	43.5 ± 1.4*	45.6 ± 1.3*
③	8.4 ± 2.2	21.6 ± 1.5	35.3 ± 2.3	37.8 ± 2.6	40.8 ± 2.7	43.1 ± 2.6
④	10.0 ± 3.1	21.6 ± 1.1	34.2 ± 2.2	37.7 ± 1.9	41.0 ± 2.1	43.7 ± 2.5
⑤	8.8 ± 2.1	21.3 ± 0.9	33.1 ± 1.8	36.3 ± 1.9*	37.5 ± 2.4**	41.4 ± 3.1
⑥	9.3 ± 2.4	22.2 ± 1.1	34.3 ± 1.5	40.0 ± 0.8	43.3 ± 1.8	45.9 ± 1.7*
⑦	8.6 ± 1.7	21.5 ± 0.7	35.5 ± 1.9	39.3 ± 2.4	42.0 ± 3.1	44.5 ± 3.3
⑧	9.4 ± 1.8	21.6 ± 1.2	34.8 ± 2.0	39.4 ± 2.7	42.1 ± 3.2	44.2 ± 4.1

①, Blank control group; ②, positive control group; ③, ethanol fraction group; ④, petroleum ether fraction group; ⑤, ethyl acetate fraction group; ⑥, n-butanol fraction group; ⑦, aqueous phase group and ⑧, hot water extract group.

Values are expressed as mean ± SD of 10 mice per group.

*p < 0.05 versus blank control group and **p < 0.01 versus blank control group.

The mean body weights of the mice in the positive control group at weeks 2, 3 and 4, ethyl acetate fraction group at weeks 2, 3, and n-butanol fraction group at week 4, were slightly different with that of the blank control group, but it was not considered related to the test substance. There were no statistically significant changes in body weights of other groups, compared with the blank control group during initial and terminal stages.

Effects on BLC levels and body weight changes of rats

Changes of BLC levels and body weight during the assay of BLC experimental period are presented in Table 6. The changes in BLC levels were significantly decreased in the positive group, ethyl alcohol partition group, n-butanol partition group and hot water extracts group in comparison with that in blank control group (p < 0.05). The BLC levels were 84.8% in ethyl alcohol partition group, 84.0% in n-butanol fraction group, and 84.4% in hot water extract group, as high as that in the blank control group.

Table 6. Effects of different partitions of fruit bodies of C. guangdongensis on BLC levels and body weight changes of rats.

		Body weight (g)
Groups	BLC levels (mmol/l)	Initial	Week 1	Week 2	Week 3	Week 4
①	86.3 ± 15.5	239.3 ± 9.3	306.0 ± 13.9	356.4 ± 19.0	379.5 ± 21.3	418.1 ± 26.6
②	73.1 ± 11.9*	239.1 ± 9.5	302.7 ± 10.3	345.0 ± 10.1	366.8 ± 9.5	402.7 ± 13.7
③	73.2 ± 9.6*	238.9 ± 9.0	297.3 ± 13.5	340.8 ± 21.9	355.3 ± 25.5*	382.4 ± 32.1*
④	71.5 ± 19.7	239.5 ± 10.4	297.4 ± 11.9	337.6 ± 16.2*	355.9 ± 16.3*	386.0 ± 22.7*
⑤	83.1 ± 15.3	238.7 ± 9.1	303.0 ± 12.6	351.2 ± 19.2	363.8 ± 19.3	398.1 ± 26.9
⑥	72.5 ± 11.1*	239.0 ± 11.5	302.3 ± 19.0	346.1 ± 23.9	366.2 ± 26.8	399.1 ± 33.0
⑦	84.8 ± 15.7	240.3 ± 13.3	299.3 ± 18.7	339.7 ± 27.1	368.0 ± 32.4	396.0 ± 37.1
⑧	72.8 ± 11.2*	240.1 ± 12.8	304.5 ± 11.5	350.4 ± 9.9	376.5 ± 11.0	398.0 ± 15.2

①, Blank control group; ②, positive control group; ③, ethanol fraction group; ④, petroleum ether fraction group; ⑤, ethyl acetate fraction group; ⑥, n-butanol fraction group; ⑦, aqueous phase group and ⑧, hot water extract group.

Values are expressed as mean ± SD of 10 rats per group.

*p < 0.05 versus blank control group.

The mean body weights of the rats in ethanol fraction group at weeks 3 and 4, and petroleum ether fraction group at weeks 2, 3 and 4, were slightly different than that of the blank control group, but it was not considered to be related to the test substance. There were no statistically significant changes in body weights of other groups, compared with the blank control group during initial and terminal stages.

Effects of water-soluble polysaccharides on weight-loaded swimming time and body weight changes of mice

Changes of the swimming times to exhaustion and body weight are presented in Table 7. The results showed that the crude polysaccharide and refined polysaccharide significantly improved the mouse swimming time to exhaustion compared with the blank control group (p < 0.05), and the swimming times were, respectively, 1.23-times in crude polysaccharide group and 2.31-times in the refined polysaccharide group, as long as that in the blank control group. There were no significant differences in body weights of mice among the treatment groups, compared with the control group during initial and terminal stages.

Table 7. Effects of polysaccharides on swimming time to exhaustion and body weights of mice.

		Body weight (g)
Groups	Swimming time to exhaustion (min)	Initial	Week 1	Week 2	Week 3	Week 4
①	26.7 ± 13.9	20.3 ± 0.8	25.6 ± 2.0	29.5 ± 2.7	31.4 ± 3.0	32.3 ± 2.7
②	40.3 ± 14.8*	20.4 ± 1.3	24.8 ± 1.6	28.2 ± 2.0	30.4 ± 2.8	31.2 ± 3.2
③	32.9 ± 13.5	20.1 ± 0.8	25.6 ± 1.6	29.1 ± 2.1	31.1 ± 2.6	31.8 ± 2.8
④	61.9 ± 36.8*	20.7 ± 1.1	25.8 ± 2.0	29.4 ± 1.6	31.3 ± 1.6	31.9 ± 1.6

①, Blank control group; ②, positive control group; ③, crude polysaccharide group and ④, refined polysaccharide group.

Values are expressed as mean ± SD of 10 mice per group.

*p < 0.05 versus blank control group.

Discussion

Fatigue is a normal phenomenon which is caused by prolonged exercise or work; great consumption of glycogen and largely accumulation of metabolic such as lactic acid could induce fatigue. Previous research indicated that cultivated fruit bodies of C. guangdongensis had noticeable effects of anti-oxidation (Zeng et al., 2009) and life-prolonging activities (Yan et al., 2011), suggesting that cultivated fruit bodies of C. guangdongensis could enhance the tolerance of anti-fatigue.

It is well accepted that the most important physiological effect of fatigue is on the energy metabolism of muscular activity (Belluardo et al., 2001), the swimming exercise is known to induce blood biochemical changes (Moriura et al., 1996) and the improvement of exercise endurance is the most powerful macro representation of anti-fatigue enhancement. So, the weight-loaded forced swimming test was conducted to evaluate its anti-fatigue activity, the bioactive components or extraction fractions for fatigue resistance and the underlying mechanisms in the present study. The results demonstrated that the swimming times to exhaustion of mice were very significantly (p < 0.01) longer in low-, middle- and high-dose groups (respectively 1.87-, 1.94- and 1.88-times), and significantly (p < 0.05) longer in n-butanol fraction group (1.52-times), hot water extract group (1.88-times) and refined polysaccharide group (2.66-times), compared with the blank control, which indicated that C. guangdongensis has an obvious anti-fatigue ability.

Meanwhile, the muscle produces high lactic acid when doing high-intensity exercise, and the increased level of lactic acid will induce many side effects of various biochemical and physiological processes, which were harmful to the body performance. Rapid removal of lactic is beneficial for fatigue resistance (Huang et al., 2011). The present study demonstrated that the BLC levels of rats were significantly (p < 0.05) lower in the ethyl alcohol partition group (84.8%), n-butanol fraction group (84.0%) and hot water extract group (84.4%) compared with the blank control group, which could demonstrate that the hot water extracts and n-butanol partition of fruit bodies of C. guangdongensis could quickly remove the BLC and then possess anti-fatigue properties.

It has been known that the polysaccharides of C. sinesis are contained in the n-butanol and water extracts and possess anti-tumor, anti-oxidation and stimulation of the immune system activities (Chen et al., 1997); the anti-fatigue effects of fruit bodies and polysaccharides of C. sinensis were related to their anti-oxidant properties (Holliday & Cleaver, 2008; Paterson, 2008; Wang et al., 2003; Zhou et al., 2009). The water extract of fruit bodies of C. guangdongensis could significantly inhibit peroxidation of linoleic acid with a high rate over 95% at low concentration (Zeng et al., 2009). It could be now hypothesized that the polysaccharide-derived anti-oxidant property of fruit bodies of C. guangdongensis might limit the oxidative stress and promote the anti-fatigue capacity. According to the result of this study, the anti-fatigue effects of the crude and refined polysaccharides from fruit bodies of C. guangdongensis were obviously positive, indicating that the functional constituent for fatigue resistance was the polysaccharide from the fruit bodies. The result is consistent with those from the studies on other Cordyceps (Li & Li, 2009), reporting that oral supplementation of Cordyceps polysaccharides could increase exhaustion time, alleviate fatigue and stress in mouse swim tests. However, the molecular mechanism underlying the anti-fatigue function of C. guangdongensis is still unknown. Further study should be focused on the molecular mechanism and anti-fatigue-related gene regulation.

Conclusion

Based on the above tests, the cultivated fruit bodies of C. guangdongensis possess potent abilities to alleviate fatigue, the possible mechanisms were to reduce the accumulation of BLC and then protect the muscle tissue and the active constituent was a refined polysaccharide from the fruit bodies. This research suggests that the cultivated fruit bodies of C. guangdongensis have potential as a new functional food for anti-fatigue.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by Guangdong academy of science funds for Young Scholar, China (No. qnjj201202), Science and Technology Planning Project of Guangdong Province, China (No. 2012B091100258, No. 2012B091100072).

Acknowledgements

The authors sincerely thank the Department of Comparative Medicine of Guangdong Medical Laboratory Animal Center for the substantial assistance in the process of the experiments.